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Department of Pediatrics, Oregon Health Sciences University, Portland, Oregon 97201
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Abstract |
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 Top Abstract I. IntroductionII. IGFBP FamilyIII. Mac25IV. CCN FamilyV. L56VI. ESM-1VII. Structural Relationships...VIII. Functional Relationships...IX. Evolutionary Relationships...X. SummaryReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. IGFBP FamilyIII. Mac25IV. CCN FamilyV. L56VI. ESM-1VII. Structural Relationships...VIII. Functional Relationships...IX. Evolutionary Relationships...X. SummaryReferences |
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) is well defined, with profound effects
on the growth and differentiation of normal and malignant cells. The
established components of the IGF system include IGFs (IGF-I and
IGF-II), type I and type II IGF receptors, IGF-binding proteins
(IGFBPs), and IGFBP proteases. IGF-I and IGF-II, which are structurally
similar to insulin, are two highly homologous small hormone peptides of
approximately 7 kDa molecular mass. First identified in 1957, they were
originally named sulfation factors (1), nonsuppressible insulin-like
activity (2), and multiplication-stimulating activity (3). They
were renamed somatomedins (4) and subsequently IGFs (5). IGFs are
ubiquitously expressed and are important mitogens that affect cell
growth and metabolism. In addition to endocrine effects exerted by
circulating IGFs (6, 7), locally produced IGFs exert paracrine, as well
as autocrine, effects on cell proliferation (8, 9, 10). The IGFs interact
with specific cell surface receptors, designated type I and type II IGF
receptors, and can also interact with the insulin receptor. The
mitogenic effects of IGF are mediated mainly through interactions with
the type I IGF receptor, which, like the insulin receptor, is a
receptor with tyrosine kinase activity. The type II IGF receptor is
structurally distinct, binds primarily IGF-II, but also serves as a
receptor for mannose-6-phosphate-containing ligands (11). The role(s)
of the type II receptor in mediating IGF action is less well defined
(12).
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10-10 M) than do the
type I IGF receptors (kd
10-8–10-9 M). Therefore, IGFBPs
act not only as carriers of IGFs, thereby prolonging the half-life of
the IGFs, but also function as modulators of IGF availability and
activity (see review in Ref. 10). In the past several years, knowledge
of the biological roles of IGFBPs has expanded, with a steady
accumulation of data indicating that, in addition to modulating IGF
bioactivity, IGFBPs are capable of important biological actions
independent of their abilities to bind IGFs (13). Evidence implicates
the direct association of IGFBPs with a variety of extracellular and
cell surface molecules (14, 15, 16), with consequent effects upon important
biological processes such as modulation of bone cell proliferation (17)
and growth arrest of breast and prostate cancer cells (15, 18, 19, 20).
There are numerous data, in vitro and in vivo,
supporting the importance of IGFBPs for cell growth by both
IGF-dependent and IGF-independent mechanisms. Of particular interest is the recent discovery of several groups of cysteine-rich proteins with discrete, but striking, structural and functional similarities to the IGFBPs (21, 22, 23, 24). This has led to the proposal of an IGFBP superfamily, comprised of the IGFBPs and these IGFBP-related proteins (IGFBP-rPs) (23). Since several comprehensive reviews on IGFBPs are available (10, 25, 26), the present review will include only the most recent information on IGFBP structure and function and will focus on the IGFBP-rPs and their structural, functional, and evolutionary relationships with the conventional IGFBPs.
A. Concept of an IGFBP superfamily
First coined by Dayhoff in 1978, the term "superfamily" was
used to discriminate between closely related and distantly related
proteins (27). The relatedness of proteins was based solely on
similarities between the primary protein structures, with amino acid
similarities set arbitrarily at equal to or greater than 50% for
closely related proteins (considered a family), and less than 50% for
those more distantly related (considered a superfamily). With the
wealth of information now available on protein structures from
different organisms, the more acceptable classification of proteins
into families and superfamilies is determined not only by amino acid
similarities, but also by considering ancillary features such as
tertiary structures (conformational similarities), functional
similarities, and even tissue specificity (28). Furthermore,
establishing relatedness among proteins requires that the evolutionary
relationships be considered. A current definition of a superfamily,
therefore, is a number of families who share some structural and
functional characteristics that have been conserved through evolution.
The list of superfamilies of genes is long, and includes the globins,
collagens, actins, immunoglobulins, serine proteases, and, more
recently, the transforming growth factor-ß and the nuclear
receptor superfamilies.
The existence of proteins able to bind IGFs with high affinity had been
suspected since the late 1960s [see review (29)]. The first IGFBP to
be purified and its cDNA cloned was IGFBP-1 (30, 31, 32, 33, 34, 35). Development of
the Western ligand blot techniques, using 125I-IGF ligands
to probe for proteins immobilized on nitrocellulose filters (36)
facilitated elucidation of the IGFBPs. By 1991, six IGFBPs (IGFBP-1 to
IGFBP-6) demonstrating high IGF binding affinity had been identified
from a variety of biological fluids, mammalian and nonmammalian, and,
in many cases, their respective cDNAs and genes had been cloned and
characterized. The structural characteristics of the human IGFBPs are
summarized in Table 1.
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The discovery of cysteine-rich proteins sharing similarities with the
IGFBPs led to the proposal of a new superfamily, an IGFBP superfamily
(23), an hypothesis consistent with the current definition for a
superfamily. The six established IGFBPs were classified as a family
based on two key features. First, the IGFBPs are cysteine-rich proteins
(16–20 cysteines in the pre-peptides) sharing high similarity in their
primary amino acid sequences. Structurally, the cysteines are clustered
at the conserved N-terminal third (12 cysteines in IGFBP-1 to -5;
10 in IGFBP-6) and at the conserved C-terminal third (6 cysteines) of
the proteins (Fig. 2). The N and C
domains are separated by a midregion of little similarity among the
IGFBPs. The second key feature of the IGFBPs is their unique ability to
bind IGFs with high affinity, presumably as a result of the N and C
domains forming the correct tertiary configuration for high-affinity
IGF binding. These two criteria, used to distinguish and classify
conventional IGFBPs, were recently reevaluated in light of the
identification of additional cysteine-rich proteins that share
structural similarities with the IGFBPs: they carry the N-terminal
domain of IGFBPs, but deviate from the common IGFBP structure in the
midregion and C terminus. Functionally, at least four of these proteins
are able to bind IGFs in in vitro assays, albeit at 100-fold
or lower affinity than that observed with IGFBPs (22, 23, 37, 38, 39).
These results, thus, substantiated that these proteins, while not
falling into the classical definition of IGFBPs, are certainly related
to the IGFBPs.
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B. Superfamily nomenclature
The various IGFBP-rPs were discovered by a number of different
groups and designated accordingly (Table 2). Although both mammalian and
nonmammalian IGFBP-rPs have been described, only the human IGFBP-rPs
are presented in Table 2. At present, there are four proteins/families
that are related to the IGFBPs. Mac25 was originally identified as a
cDNA derived from leptomeninges (40); the mac25 cDNA was
subsequently expressed in a baculovirus system, and the synthesized
protein was shown to bind IGFs and was renamed IGFBP-7 (22).
Independently, the same protein has been purified from human diploid
fibroblast cells and named prostacyclin-stimulating factor [PSF (41)]
and from human bladder carcinoma cells [tumor adhesion factor (TAF)
(42)]. The CCN family of proteins consists of a human growth
factor-inducible, immediate-early gene [cyr61 (43),
connective tissue growth factor (CTGF) (44), and the human
nephroblastoma overexpression gene (novH) (45)]. Recently,
three new members of the CCN family have been identified in Wnt-1
(cysteine-rich glycosylated signaling proteins that are oncogenic)
transformed cells: WISP-2 (46) and its rat counterpart, rCop-1 (47);
WISP-1 (46) and the mouse orthologue, Elm-1 (48); and WISP-3 (46).
Independently, WISP-2 was identified in primary human osteoblast cells
and designated CTGF-L [CTGF-like (39)]. Two other proteins related to
the IGFBPs are L56 (49), also named HtrA (50), a potential serine
protease of IGFBPs, and endothelial cell-specific molecule, ESM-1 (51).
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and include 1) retention of the multiple published names; 2) naming the
proteins IGFBP-7 through -12 (and higher); and 3) naming the proteins
IGFBP-rPs (IGFBP-rP)-1 to -6 (and higher). The latter nomenclature is
recommended (52) since these IGFBP-rPs do not fall into the
conventional definition of IGFBPs, and this nomenclature will be
employed in this review. The IGFBP-rPs will be discussed in depth in Sections III–VI. Their structural and functional relationships with the IGFBPs will be presented in Sections VII and VIII, respectively.
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II. IGFBP Family |
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TopAbstractI. IntroductionII. IGFBP FamilyIII. Mac25IV. CCN FamilyV. L56VI. ESM-1VII. Structural Relationships...VIII. Functional Relationships...IX. Evolutionary Relationships...X. SummaryReferences |
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In this section, an analysis of IGFBP structure is summarized, based on information gathered from studies with mammalian IGFBPs. Correlation between IGFBP structure and function at the molecular level has only recently begun to be elucidated. Particular attention has been paid to the unique attributes of the N-terminal domain, alone and in combination with other IGFBP domains, since it is this portion of the molecule that is conserved exclusively in the IGFBP-rPs.
A. Structure of IGFBPs
The primary structures of mammalian IGFBPs appear to contain three
distinct domains of roughly equivalent sizes: the conserved N-terminal
domain, the highly variable midregion, and the conserved C-terminal
domain. Alignment [Clustral method (59), DNA Star program] of the
human IGFBPs indicates that, overall, the human IGFBPs share
approximately 36% similarity (defined as "the direct comparisons of
amino acid sequence without accounting for phylogenetic
relationships", DNA Star), although as presented below, alignment of
the conserved N and C domains shows significantly higher similarities.
Between mammalian species, each IGFBP is highly conserved.
1. N-terminal domain. In the mature IGFBP peptides, the N
terminus third of the proteins contains 80–93 amino acid residues
after the signal peptides (Fig. 2) and shares approximately 58%
similarity. Ten to 12 of the 16–20 cysteines found in the prepeptides
are located within this domain. In IGFBP-1 to -5, these 12 cysteines
are fully conserved, whereas in IGFBP-6, 10 of the 12 cysteines are
invariant. Interestingly, rat IGFBP-6 is missing an additional 2
cysteines (the first 2 cysteines in the primary sequence) in the
N-terminal domain (60). The high number of cysteines within such a
small domain suggests that this domain is highly structured, with a
maximum of 6 disulfide bonds formed (5 in the case of IGFBP-6). The
even number of cysteines suggests that intradomain disulfide bond
formation is more likely than interdomain disulfide linkages with
cysteines in the C-terminal domain. Indeed, two recent studies have
provided data supporting the hypothesis that the N-terminal and
C-terminal domains are not linked by disulfide linkages (61, 62).
Furthermore, analysis of tryptic digested fragments of human
IGFBP-6 indicated that the native disulfide linkages in IGFBP-6
occur between cysteines that are close together in the primary
sequence, forming sequential subdomains with at least 2
disulfide-linked subdomains in the N-terminal domain (61, 63). In the
N-terminal domain, the first 6 cysteines form the first subdomain, and
the next 4 cysteines form the second subdomain. Interestingly, in rat
IGFBP-3 (64) and human IGFBP-5 (65), it was similarly demonstrated that
the last 4 cysteines of the N-terminal domain formed overlapping
disulfide linkages (Cys56-Cys69 and
C63-C89 for rat IGFBP-3 (64), consistent
with the second subdomain structure proposed by Neumann et
al. (61). These results have been recently supported by nuclear
magnetic resonance (NMR) spectroscopy of this subdomain from human
IGFBP-5, which shows a rigid, globular structure stabilized by the two
disulfide bridges (65).
Within the N-terminal domain, a local motif (GCGCCxxC) is well conserved among the IGFBPs. The exception is in IGFBP-6, which substitutes a GCAEAEGC sequence, thereby accounting for the two "missing" cysteines in IGFBP-6. The significance of this motif is as yet unknown, but, as indicated below, is also highly conserved in the IGFBP-rPs. One hypothesis is that the motif may be important in interactions with IGFs. However, IGFBP-6 has retained the high affinity for IGFs (in particular IGF-II), although the internal GCC amino acids are replaced with AEA. A search of the protein data bank (BLAST search, SwissProt) revealed several other proteins containing the GCGCCxxC motif, including a cysteine-rich protein found in the eggshell of the silk moth [chorion protein (66)]. Variants of this motif are also found in GP40, a small undefined protein found in Mycobacteriophage 15 (67), in the peplomer protein [a viral spike glycoprotein (68)], and in metallothionein-like protein 1 (69). The role(s) of the GCGCCxxC motif, or its close variants, are not known. Clearly, the GCGCCxxC motif appears to be highly conserved in, but not unique to, IGFBPs.
2. Midregion. For the human IGFBPs, the midprotein segment, ranging in size from 55 amino acid residues to 95 amino acids, separates the N-terminal domain from the C-terminal domain. The amino acid sequence for each midsegment appears to be unique to the protein, with shared similarity less than 15%. The belief is that this region acts structurally as a hinge between the N- and C-terminal domains.
Intriguingly, posttranslational modifications (glycosylation, phosphorylation) of the IGFBPs have been found so far in the midregion, but not in the N- or C-terminal domains. There has been no clear evidence to date that IGFBP-1 or -2 are glycosylated (an early paper suggests that IGFBP-1 was glycosylated (70), whereas IGFBP-3 and -4 are N-glycosylated, and IGFBP-5 and -6 are O-glycosylated. N-glycosylation occurs only on an asparagine that is part of the consensus sequence Asn-X-Ser/Thr, where X is any amino acid except proline. Consistent with this prediction, three N-glycosylation sites in the mature protein, Asn89, Asn109 and Asn172 (corresponding to prepeptide, Asn116, Asn136, and Asn199) have been characterized in IGFBP-3 (71), and one, Asn104 (Asn125 of prepeptide), in IGFBP-4 (72). Although there is one potential N-glycosylation site in the C terminus of IGFBP-6, this site does not appear to be glycosylated (73). In contrast to the N-glycosylation sites, there are no consensus sequences for predicting O-glycosylation sites. Nevertheless, it has been demonstrated that IGFBP-5 (74) and IGFBP-6 (61, 73) are both O-glycosylated. Very recently, the O-glycosylation sites in human IGFBP-6 were determined to be within the midregion at 5 residues, Thr126, Ser144, Thr145, Thr146, and Ser152 (61). The O-glycosylation states of other mammalian IGFBP-6 indicate that in the rat, mouse (75, 76), and pig (53) IGFBP-6 is glycosylated to a lesser extent than human IGFBP-6 (61); bovine IGFBP-6 may be similarly glycosylated as human IGFBP-6. The ability to bind IGFs with high affinity appears not be influenced by N- or O-glycosylation, although there may be effects on other function(s) of IGFBPs, such as resistance to proteolysis (61).
Three of the six IGFBPs, IGFBP-1, -3, and -5, are posttranslationally modified by phosphorylation (77). Phosphorylation of proteins is an important and critical posttranslational modification mechanism that is used by cells to stringently regulate the activities of numerous intracellular proteins, including proteins involved in the signal transduction pathways, in the cell cycle, and in gene expression. The purpose of phosphorylating secreted proteins like IGFBPs is unclear, but there is evidence that, at least for human IGFBP-1, phosphorylation enhances the affinity of hIGFBP-1 for IGFs by 5-fold (78, 79). Phosphorylation in all three IGFBPs is predominantly at serine residues found in the midregion of the IGFBPs. Phosphorylation of hIGFBP-1, first described by Frost and Tseng (80) and by Jones et al. (78), is at three serine residues of the mature peptide: Ser101 and Ser119, both in the midregion of the protein, and Ser169 (located at the amino-terminal end of the hIGFBP-1 C-terminal domain) (81). Frost and Tseng (80) demonstrated that, under in vitro conditions, casein kinase II and cAMP-dependent protein kinase are able to phosphorylate IGFBP-1. Recently, phosphorylated rat IGFBP-1 was described (82). Unlike hIGFBP-1, however, only two serine residues were phosphorylated (Ser107 and Ser132 in the nonconserved midregion), and more importantly, phosphorylation did not appear to affect IGF binding.
The phosphorylation status of human IGFBP-3 was analyzed by Hoeck and Mukku (83), who showed that Ser111 and Ser113 (Ser138 and Ser140 of prepeptide), which are within the consensus sequences for protein kinase CKII, were phosphorylated. Both serines are also in close proximity to one of the N-glycosylation sites, Asn109 (Asn136 of prepeptide). Further, they demonstrated that phosphorylation did not appear to affect IGF binding by IGFBP-3. Interestingly, phosphorylation of IGFBP-3 can be up-regulated by IGFs, through a mechanism involving IGF-I-type I IGF receptor interaction (84). The significance of phosphorylated IGFBP-3, and the significance of IGF regulation of phosphorylation, are unclear but may affect IGFBP-3 interactions with acid-labile subunit (ALS) or with the cell surface (84).
Evidence for the phosphorylation of IGFBP-5 at serine and threonine residues is limited to one report (85), although, like all the IGFBPs, there are several potential phosphorylation sites (84). The biological significance of IGFBP-5 phosphorylation is unknown.
3. C-terminal region. The C-termini of IGFBPs, like the
N-terminal domain, are highly conserved and, among the human IGFBPs,
share a similarity of approximately 34%. The remaining 6 cysteines of
the total 16–20 cysteines are found in the C terminus and are strictly
conserved (Fig. 2). Evidence from two independent studies indicated
that the 6 cysteines are involved in intradomain disulfide bond
formation (58, 59, 63). Neumann and Bach (63), in their studies of
human IGFBP-6, and Forbes et al. (62), in their studies
of bovine IGFBP-2, deduced that the disulfide bonding pattern of the
C-terminal region was between adjacent cysteines.
The primary sequence of all members of the IGFBP family surrounding the
last 5 cysteines is strikingly similar (40%), implying that the
tertiary structure of the C-terminal domain should be almost identical.
Interestingly, the amino acid sequences encompassing these last 5
cysteines share 37% similarity with the thyroglobulin-type-I domain
(86). The thyroglobulin-type I domain consists of about 65 amino acid
residues, which are repeated 10 times in the N-terminal part of
thyroglobulin (86). Its function(s) is unknown, but the domain is found
in a number of proteins with varying physiological functions in
different organisms (87). These include the major histocompatibility
complex class II-associated p41 invariant chain fragment (88), nidogen
(89), saxiphilin (90, 91), a tumor-associated cell surface antigen known
also as GA733 (92), a cysteine protease inhibitor from the egg of Chum
salmon (93), equistatin, a new inhibitor of cysteine proteinases from
sea anemones (94), and entactin-2, a new basement membrane protein
(95). In IGFBPs, the role of this domain has yet to be determined, but
is likely to affect binding to IGFs, and perhaps participate in the
binding of IGFBPs to cell surfaces and/or to the extracellular matrix
(ECM) proteins via heparin-binding sites. Consistent with this is the
observation that IGFBP-1 and -2 contain RGD motifs, which are known to
be involved in binding integrins (96). Similarly, heparin binding
motifs (xBBBxxBx, where B is a basic residue, Arg, Lys, or His, and X
is any residue) are found within the C-terminal domains of IGFBP-3, -5,
and -6, and, for IGFBP-3 and -5, are involved in binding to cell
surface and/or the ECM (16, 97, 98, 99, 100).
B. Correlations between structure and function
1. IGF-IGFBP interactions. The IGFBPs were so designated
because of their abilities to bind IGFs with high affinity
(Kd 10-10 M). However, the
precise molecular interactions between IGFBPs and IGFs are still
unclear. It has also become apparent that IGFBPs can interact with
proteins other than IGFs, including the ALS from serum, insulin,
components of the cell surface, ECM proteins, and, potentially,
intracellular components. These additional interactions may result in
biological consequences not directly related to IGF action.
Correlations between IGFBP structure and function have recently begun
to emerge and will be summarized here.
It is worth noting here that the methods used to detect and study IGF-IGFBP interactions have become increasingly sensitive, permitting better assessment of both high and low IGF-affinity binding. Although the ligand blotting technique is the preferred and most commonly used method for detecting IGF-IGFBP interactions, more sensitive methods include affinity cross-linking assays, charcoal solution binding, solid-phase binding, and, recently, BIACORE analysis. Since the methods used vary among research groups, discrepancies in IGF-binding affinities among different studies are likely to arise, and it is often not obvious whether such differences reflect technical variations or underlying biology.
The high-affinity binding of IGFs by IGFBPs has long been hypothesized to involve interactions between the conserved N-terminal and C-terminal domains. Support for this hypothesis initially came from in vivo observations that in biological fluids, IGFBPs can be proteolysed resulting in diminished affinities for IGFs. Proteolysis was, thus, proposed as a mechanism for modulating IGF bioavailability (101, 102, 103, 104, 105). Proteolysis of IGFBPs was first observed in serum from pregnant women, where it was demonstrated that IGFBP-3 was proteolytically cleaved to yield a predominant 29–30 kDa form that was still capable of binding IGFs, but with reduced affinity (101, 106). Since those observations, proteolysis of IGFBP-2 to -6 has been described in numerous studies of various biological fluids from different organisms, generating IGFBP fragments with decreased or no apparent affinity for IGFs.
In vitro generation of IGFBP fragments by limited proteolysis supports the in vivo data. For example, limited proteolysis of recombinant human IGFBP-3 (nonglycosylated and glycosylated) with the serine protease prostate-specific antigen (PSA) (102, 107) or with plasmin (108, 109) generated a 22/25 kDa fragment with weak affinity for IGFs (residues 1–160) and a 16-kDa fragment (residues 1–95 that includes the N-terminal domain) with no detectable affinity for IGFs by affinity cross-linking assays. Vorwerk et al. similarly generated an approximately 15-kDa plasmin-digested IGFBP-3 fragment, corresponding to the N-terminal domain and part of the midregion (amino acid residues 1–97), capable of weakly binding to IGF-I, and detectable by both Western ligand blot and affinity cross-linking assays (110). Proteolytically modified IGFBP-4 generated a 16-kDa fragment that also could be affinity cross-linked specifically to IGF-I and IGF-II, although with a 20-fold lower affinity compared with intact IGFBP-4 (111). This 16-kDa IGFBP-4 fragment, like the 16-kDa N terminus IGFBP-3 fragment, corresponds to the N-terminal region and a small portion of the midregion (112). Similarly, a 23-kDa IGFBP-5 fragment from osteoblast-like cells that is carboxy truncated (113, 114) and a 10-kDa fragment (residues 1–94 of mature peptide) corresponding to the N-terminal fragment of endoproteinase Asp N-digested IGFBP-5 (65), demonstrated decreased binding affinity for IGF-I and IGF-II. Thus it appears that the N-terminal domain, with perhaps part of the midregion, can bind IGFs, but high-affinity IGF-binding also requires the added presence of the C-terminal domain.
The ability of IGFBP proteolytic fragments to bind IGFs, albeit with reduced affinity, has been further investigated using in vitro generated recombinant IGFBP peptides. Spencer and Chan (115) generated IGFBP-3 fragments that essentially corresponded to the N-terminal half (residues 1–147) and the C-terminal half (residues 151–263) of the IGFBP and showed that each of these fragments bound IGFs, but with less affinity than intact IGFBPs. Only a handful of studies have examined in depth the ability of the N-terminal domain to interact with IGFs, and results have been mixed. Baxter and Firth synthesized IGFBP-3 fragments that correlated to the N-terminal domain, the N-terminal domain plus the midregion, and a mutant IGFBP-3 in which the midregion was deleted (116). None of the fragments, however, was detectable by ligand blotting, although recent data indicate the N-terminal domain bound 125I-labeled IGF-II in solution binding assays (117). In contrast, recombinant N-terminal domain fragments generated by Yamanaka et al. (118) and by Vorwerk et al. (110), were detectable by ligand blotting, as well as by the more sensitive affinity cross-linking assay.
Further delineation of specific regions and subdomains of IGFBPs involved in IGF binding has come from limited chimera studies, as well as regional mutagenesis of the N and C domains. In the IGFBP N-terminal domain, the conserved GCGCCxxC motif was thought to be important for interactions with IGFs. The fact that the motif is incompletely conserved in IGFBP-6 (GCAEAxxC), however, would suggest that the role of this motif in the binding of IGFs may be subtle, or that there may be other explanations for its conservation in the IGFBPs and IGFBP-rPs.
In a recent study of human IGFBP-4 deletion mutants, Qin et al. (119) concluded that Leu72-Ser91 is important for IGF-II binding, as deletion of this region rendered the N-terminal peptide undetectable by ligand blot. Further, within this segment, a structural disruption generated by a His74 (a basic amino acid conserved in IGFBP-4 from different species, but not conserved among the IGFBPs) to Pro74 point mutation reduced the affinity of full-length IGFBP-4 for IGF-II by 50-fold. The N-terminal domain of rat IGFBP-3 demonstrated a reduction in IGF-II binding to less than 12% relative to full-length IGFBP-3 as determined by a sensitive solid-phase binding assay (64). A smaller fragment of the rat IGFBP-3 corresponding to the last 4 cysteines of the N-terminal domain [i.e., the second N-terminal subdomain described by Neumann et al. (61) and Kalus et al. (65)] dramatically reduced IGF-II binding by four-logs. The same subdomain in recombinant human IGFBP-5 demonstrated 10- to 200-fold reduced affinity for IGFs by BIACORE analysis (65). The remaining N-terminal region, i.e., the N-terminal subdomain encompassing the first 6 (IGFBP-6) or 8 cysteines (IGFBP-1–5), has not been tested for IGF affinity. Interestingly, Hobba et al. (120, 121) showed that in bovine IGFBP-2, Tyr60, which is within the second subdomain and highly conserved among the IGFBPs and across species, substitution by Ala60 or Phe60 reduced, but did not abolish, affinity for IGF-I (4-fold and 8.4-fold, respectively) and for IGF-II (3.5-fold and 4-fold, respectively). These results were consistent by both charcoal binding assays and BIACORE analysis (120, 121). Mutations of adjacent residues, which are well conserved, did not reduce affinity. From these results, it can be deduced that Tyr60 is probably one of the many contact points with IGFs. This is supported by NMR studies of IGFBP-5-IGF-II complexes, in which the analogous Tyr (Tyr50) is proposed to interact with IGF-II, as are residues Val49, Pro62, and Lys68-Leu74 (65). Based on this handful of studies, it can be inferred that the two N-terminal subdomains proposed by Neumann et al. (61) are important for the integrity of the (partial) IGF binding pocket. Further studies are required to elucidate the precise points of contact with IGFs, which may vary from IGFBP to IGFBP.
The C-terminal domain of IGFBPs, without question, is essential for high affinity IGF binding, although more data are available regarding their non-IGF binding properties than their IGF binding characteristics (see below). Chimeras constructed between rat IGFBP-3 and IGFBP-2 indicated, not surprisingly, that the C-terminal domain from IGFBP-3 can be exchanged for the C-terminal domain of IGFBP-2 with no loss of IGF-II binding (64). However, replacement of the IGFBP-3 midregion with the IGFBP-2 midregion reduced the relative affinity of the resultant chimera for IGF-II by at least 37%, suggesting that the midregion of each IGFBP may maximize high-affinity IGF binding by the specific IGFBP.
Mutagenesis of the carboxy end of the IGFBP-1 cDNA (122) showed that
deletion of the C-terminal 20 amino acids resulted in loss of IGF
binding by ligand blotting. In contrast to IGFBP-1, deletion of a
similar region in human IGFBP-4 (C-terminal
Lys215-Glu237) had no effect on relative IGF
binding (bands in ligand blot assessed by radioactivity), but an
additional deletion of 10 amino acids (removal included the highly
conserved Cys-Trp-Cys-Val motif) reduced relative binding to less than
15% of wild-type IGFBP-4 (119). Similar sequential C-terminal deletion
studies, with recombinant bovine IGFBP-2 and using charcoal-binding
assays, suggested that loss of the region spanning the last four
cysteines reduced IGF binding (62). In particular, results suggest that
residues Lys222–Asn236 may be in close
proximity to the N-terminal domain, to allow both domains to interact
with IGF. Consistent with this proposal, recent site-specific
mutagenesis of the strictly conserved amino acids, Gly203
or Gln209, within the corresponding region in rat IGFBP-5,
reduced IGF-I binding affinity by 8- and 6-fold, respectively (123). In
contrast, mutagenesis of adjacent basic amino acid residues in the
equivalent region of human IGFBP-5 (amino acids 201–218) did not alter
IGF-I binding affinity, although ability to interact with ECM was
affected (see below and Ref. 124). The IGF binding properties of the
C-terminal domain, itself, have yet to be tested thoroughly, although a
recent study indicated that a natural C-terminal fragment of human
IGFBP-2 retained partial IGF-binding activity (125). This
observation is consistent with an earlier study where it was observed
that a proteolyzed rat IGFBP-2 fragment containing half the
midregion and the C-terminal domain showed similar reduced IGF
binding compared with full-length rat IGFBP-2 (126). In contrast,
a synthetic peptide corresponding to half the midregion and C-terminal
domain of IGFBP-4 (as defined in this review; see Fig. 2),
His121-Glu237, did not show detectable IGF
binding (radioactivity in the bands from ligand blots were quantitated)
(119); neither did an analogous C-terminal peptide,
Asp135-Phe246, from IGFBP-5 demonstrate IGF
binding (65). A comparable region in IGFBP-3, on the other hand, had
demonstrable IGF binding capabilities by solution assays (115) and by
ligand blot (G. R. Devi, D.-H. Yang, R. G. Rosenfeld, and Y. Oh,
unpublished).
The midregion of the IGFBPs does not appear to bind IGFs; its contribution to the high-affinity binding of IGFs is likely to relate to its ability to promote a tertiary structure, which permits optimal relationships between the N-terminal and C-terminal domains.
2. Effect of posttranslational modification on IGF binding. Limited data are available on the effects of posttranslational modification of IGFBPs on IGF binding. Results so far indicate that neither glycosylation nor phosphorylation appear to have much influence on the IGF binding affinities of IGFBPs (83, 84). The exception is the phosphorylation of human IGFBP-1, where it has been shown that phosphorylation enhances IGF binding by at least 5-fold (78). Similar results were not observed with rat IGFBP-1 (82).
3. Other structure-function associations of IGFBPs. The regions of IGFBPs that are involved in functions unrelated to IGF binding appear to be predominantly in the mid- and C-terminal domains. To date, the only function clearly associated with the N-terminal domain is IGF binding, and more recently, insulin binding (110, 118). This does not rule out other potential functions for the N-terminal domain, either alone or in concert with other IGFBP domains. Lalou et al. (108), for example, have reported that IGFBP-3 (residues 1–95) inhibits cell replication. For the midregions, aside from simply acting as a "hinge" between the N-terminal and C-terminal domains, the fact that these regions are posttranslationally modified suggests that specific functions, as yet undefined, may be associated with this region. For IGFBP-3, the midregion appears to be involved in specific membrane association (127). Interestingly, the proteolytic sites for a number of IGFBP proteases are found in the midregion. It is possible that individual characteristics of each IGFBP reside in these nonconserved regions.
In the C-terminal domain, more information is available about functions
other than IGF binding. The C-terminal domain of IGFBP-3,
irrespective of its ability to bind IGF-II, has been shown to be
essential for interactions with the acid-labile subunit (64, 116), most
likely through the IGFBP-3 basic region,
Lys228-Arg232 (116). A recent report suggested
that in addition to ALS, IGFBP-3 can interact with other high mol wt
proteins found in human serum (128); whether these interactions are
through the C-terminal region or midregion is unknown. Interestingly,
IGFBP-5 also forms a ternary complex with ALS and IGFs (129). Since the
C-terminal domain between IGFBP-3 and -5 is highly similar (54%),
particularly in the sequences spanning the basic region (see Fig. 2),
IGFBP-5 presumably also interacts wth ALS through this domain.
The other notable motifs in the C-terminal domains are the RGD sequence found in analogous positions in IGFBP-1 (amino acid residues 221–223) and IGFBP-2 and the highly basic heparin-binding sequences found in the thyroglobulin type I domain in IGFBP-3, -5, and -6. The RGD motif in IGFBP-1 was shown by Jones et al. (14) to interact with integrins, which are a large family of heterodimeric cell adhesion receptors involved in both cell-cell and cell-ECM interactions (130). It has been hypothesized that interactions of IGFBPs with the ECM, via the integrins, may allow the IGFBPs to provide a reservoir of IGFs (26). In IGFBP-3, the heparin-binding motif can associate with glycosaminoglycan-containing molecules, like proteoglycans found on cell surfaces and in ECM (116, 131). While the consequences of IGFBP-3 interacting with glycosaminoglycan are unclear, these interactions may enhance localization of IGFBP-3 to the cell suface and, perhaps, the ECM (131). The same motif in IGFBP-5 and IGFBP-6 may have similar functions and, in fact, there is strong evidence that the highly basic region surrounding the heparin-binding motif (Arg201-Arg218) mediates binding of IGFBP-5 to osteoblast cells (16), to ECM (124, 132, 133), and to mesangial cell surface (134). The highly basic regions from all three IGFBPs (IGFBP-3, -5, and -6) are capable of specifically inhibiting IGFBP-4 degradation, and the inhibition of IGFBP-4 degradation is abrogated by IGFs (98). The mechanisms by which this inhibition is mediated are not understood, but since IGFBP-3, -5, and -6 are not themselves substrates for the IGFBP-4 protease, one hypothesis is that the highly basic region in these IGFBPs may act as a protease inhibitor (98). The physiological ramifications of inhibiting IGFBP-4 degradation are unclear, although IGFBP-4 is known to be a potent inhibitor of IGF actions and proteolysis of IGFBP-4 could, therefore, potentiate IGF actions.
One of the most intriguing observations made within recent years has been evidence for the targeting of IGFBP-3 and IGFBP-5, but not IGFBP-1 or -2, to the nucleus. Although there are no definitive consensus amino acid sequences for nuclear localization signals (NLS) (135), many proteins do contain sequences rich in basic amino acids similar to the NLS (PKKKRKV) of SV40 large T antigen. Potential NLS sequences in IGFBP-3 and in IGFBP-5 were first noted in 1994 by Radulescu (136). It was not until 1997, however, that the evidence supporting nuclear IGFBP-3 was published (137, 138). Not only was endogenous IGFBP-3 clearly found in the nucleus of lung cancer cells (138), but labeled recombinant IGFBP-3 added exogenously to wounded opossum kidney cells was transported into the nucleus, whereas in resting cells, IGFBP-3 was internalized and accumulated in the endosomal compartment (137). Intriguingly, IGFs bound to IGFBP-3 can also localize to the nucleus. In human keratinocytes, nuclear IGFBP-3 was detected in cells undergoing division (139). Recent in vitro studies have demonstrated that both recombinant IGFBP-3 and IGFBP-5, but not IGFBP-1 or -2, can translocate from the extracelluar compartment to the nucleus in rapidly dividing human breast cancer cells (140). Site-specific mutagenesis confirmed that the putative NLS in IGFBP-3 is the predicted basic sequence in the C-terminal domain (Lys215-Arg232) (139). The biological significance of translocating IGFBPs into the nucleus is unclear at present but is consistent with potential IGF-independent actions of some IGFBPs (see below).
C. Biological functions of IGFBPs
The detailed biological functions of IGFBPs have been well
reviewed in recent years (10, 13, 24, 25) and will not be reiterated in
this review. The aim in this section is to put into perspective the
correlations made between the structure of IGFBPs and their functions
in the context of IGF-dependent vs. IGF-independent actions
of IGFBPs.
1. IGF-dependent actions of IGFBPs. The term "IGF-dependent" functions of IGFBPs has been used to define functions of IGFBPs, both positive and negative, that are directly linked with IGF bioactivities (10, 13, 25, 26). Since IGFBPs are well established secreted proteins, this inevitably meant that the focus has been on the extracellular sequestration of IGFs by IGFBPs, and the effects this sequestration has on the consequent loss of interactions between IGFs and the type I IGF receptor. There is a plethora of in vivo and in vitro studies describing and supporting this mechanism of IGFBP action. Most recently, the approach taken has been to directly test this hypothesis by generating recombinant mutated forms of IGFBPs with reduced affinities for IGFs, and subsequently testing whether these mutants have effects on IGF bioactivities. This was most clearly demonstrated in the case of IGFBP-4, an IGFBP known to inhibit the mitogenic effect of IGFs on bone cell growth. Mutations in human IGFBP-4 that greatly reduced its affinity for IGF-II resulted in an inability of the mutant IGFBP-4 to inhibit IGF-II-induced human osteoblast proliferation (119).
An extension of the IGF-dependent actions of IGFBPs is investigations into the mechanisms of IGF release from IGFBPs. Reducing affinity for IGFs is an obvious mechanism for the release of IGFs and is achieved by proteolysis of IGFBPs, alteration in phosphorylation status of IGFBP-1 (78), and perhaps also by IGFBP conformational changes, such as via binding of the IGFBPs to ECM and/or to the cell surface. Molecular evidence for the importance of IGFBP proteolysis to IGF-dependent actions was provided recently by site-specific mutagenesis of the proteolytic site, resulting in enhanced IGFBP growth-inhibitory effects (141, 142). In contrast, there is yet to be evidence for release of IGFs by conformational change of IGFBP-IGF interactions, although interactions between IGFBPs and ECM and/or cell surface (see above) would support this postulate. An alternative hypothesis, suggested in a recent study, implicate a physical occlusion effect based on the observation that, in IGFBP-5, the regions involved in IGF and ECM interactions overlap (123).
In contrast to the extracellular effects of IGFBP on IGF-type-I IGF receptor complexes, intracellular effects of IGFBPs, particularly any effects on type-I IGF receptor signaling pathway, have yet to be addressed. In light of very recent data indicating the internalization and nuclear localization of IGFBP-3 and of IGFBP-5, it may be necessary to redefine "IGF-dependent" actions.
2. IGF-independent actions of IGFBPs. Given the classical definition of IGF-dependent actions of IGFBPs, IGF-independent actions of IGFBPs are defined as biological effects exerted by IGFBPs that involved neither binding of IGFs nor activation or inhibition of the type I IGF receptor. There has been a steady accumulation of data supporting the existence of IGF-independent actions for IGFBP-3 and IGFBP-5 (13) and limited data for IGFBP-1 (14). The recent demonstration of IGFBP-3 and -5 translocation to, and localization in, the nucleus support the concept that these two IGFBPs have functions unrelated to direct IGF actions. Presumably, these IGF-independent functions are through the C-terminal domains and perhaps also the midregions of the IGFBPs (see Section II.B.3. above).
As initially demonstrated by Oh et al. (18) in breast cancer cells, the epithelial growth-inhibitory actions of IGFBP-3 are mediated through specific binding of IGFBP-3 to cell surface molecules that are not type I IGF receptors. The purification and cDNA cloning of a specific IGFBP-3 receptor, however, remains elusive, but, using the yeast two-hybrid system, cDNAs encoding IGFBP-3 interacting proteins have been obtained (Y. Oh, unpublished). Interestingly, a recent report suggests that the type V TGFß receptor could be the putative IGFBP-3 receptor (143, 144) and that IGFBP-4 and IGFBP-5 may also interact with this receptor (144). Presence of this receptor, however, has not been convincingly demonstrated in breast cancer cells (Y. Oh, unpublished).
A similar sequence of events has led to the conclusion that IGFBP-5 also has biological actions that are IGF independent (16). Supporting this hypothesis, a novel, putative IGFBP-5 membrane receptor, a 420-kDa membrane protein, was very recently purified from osteoblast cells (145). Although not fully characterized and the cDNA not cloned, it would appear, at least in vitro, that the binding of IGFBP-5, through the basic region in its C-terminal domain, to the receptor stimulated phosphorylation of the receptor.
D. Genomics of IGFBPs
1. Chromosomal locations of IGFBPs. The genomic locations of
all human IGFBPs are known and are summarized in Table 1.
Interestingly, the genes for IGFBP-1 and IGFBP-3 not only reside on the
same chromosome, at the locus 7p14-p12, but are only 20 kb apart, with
transcription orientated in a tail-to-tail configuration (146). IGFBP-2
and IGFBP-5 constitute another gene pair, located 20–40 kb apart on
chromosome 2q. Based on amino acid similarity analysis, IGFBP-1 is more
closely related to IGFBP-2 than to IGFBP-3, which, in turn, is more
closely related to IGFBP-5. IGFBP-4, found on chromosome 17q12–21.1,
is more closely related to IGFBP-1 and -2, whereas IGFBP-6, located on
chromosome 12q13, appears to be the most divergent of the IGFBPs. The
similarity in configuration of the human IGFBP genes, especially the
gene pairs, is striking, and, together with analysis of the protein
sequences, has led to the hypothesis that a tandem gene duplication and
inversion occurred early in the evolution of the IGFBPs [one
suggestion is that IGFBP-6 is the proto-IGFBP, (58)], and subsequent
gene duplications primarily involved partial chromosome duplication
(see review in Ref. 58).
An intriguing observation that was made in the analysis of chromosomal locations of the human IGFBP genes is that the genes appear to co-map with genes encoding homeoboxes (HOX) and epidermal growth factor receptors [see review by Reinecke and Collet (58)]. Homeobox is a conserved element of 180 bp that is found in all homeotic (and also nonhomeotic) genes. The importance of homeotic genes is that they are the master control genes that regulate development of higher organisms. Thus, by association, the inference is that IGFBPs are important and fundamental proteins in development. The evolutionary implication is that there may be an association between the evolution of the vertebrate homeobox genes, the epidermal growth factor receptors, and the IGFBPs. Since this area is summarized in a very recent, comprehensive review on IGF phylogeny by Reinecke and Collet (58), readers are referred to that review for more details.
2. IGFBP gene structures. The gene structures of human
IGFBPs are highly similar, although the sizes of the genes vary
from 5.7 kb for IGFBP-1 to 33 kb for IGFBP-5 (Table 1), due to
variations in the sizes of the introns. All of the IGFBPs are encoded
by four exons, with the exception of IGFBP-3, which carries an extra
exon, exon 5, that is not translated. The corresponding exons among the
IGFBPs are equivalent in size, with exon 1 less than 600 bp, exons 2
and 3 both small exons of less than 230 bp, and exon 4 more variable in
size. There is a stiking correlation between these exons and the three
protein domains of IGFBPs. The N-terminal domain, as defined in Fig. 2, is encoded within exon 1 in all of the IGFBPs, as is the
5'-untranslated region and a few amino acids of the midregion. Exon 2
encodes for the nonconserved midregion. Both exon 3, which ends
precisely at the invariant Gln (Q) residue in the thyroglobulin domain,
and exon 4 encode for the conserved C-terminal domain. The fact that
the N-terminal domain is contained within one exon strongly supports
the concept of an IGFBP superfamily, as will be discussed in
Section IX.
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III. Mac25 |
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). The
gene for human IGFBP-rP1 has been localized to chromosome 4q12–13
(147). A mouse homolog, sharing 87.5% nucleotide identity and 94.4%
similarity with human IGFBP-rP1, has been described (148). Three groups
independently identified the human IGFBP-rP1 protein, and each has
continued to use its own designations. Without a doubt, however, they
are the same protein. The structural relationship to the IGFBPs was
initially noted by Murphy et al. (40), who were the first to
identify the putative protein, deduced from the cDNA clone,
mac25. Akaogi et al. (42) and Yamauchi et
al. (41) independently purified and characterized the protein,
which they designated TAF and PSF, respectively. Oh et al.
(22) synthesized the Mac25 protein in a baculovirus system,
demonstrated its ability to bind IGFs, and provisionally named the
protein IGFBP-7, later to be redesignated IGFBP-rP1. Structurally, the
region of similarity of IGFBP-rP1 (Mac25/TAF/PSF) to IGFBPs is confined
to the N-terminal domain (see Section VII). Functionally,
the protein appears to have multiple roles, including the ability to
bind IGFs and insulin (see Section VIII), but the
physiological significance of this protein is still largely unknown.
The structural and binding characteristics of IGFBP-rP1 will be
discussed in more detail in Sections VII–VIII. Below, a
historical perspective of IGFBP-rP1 will be presented. 1. Mac25. Murphy et al. (40) employed subtractive hybridization to search for genes whose expression were altered in meningioma cell lines, compared with normal leptomeningeal cells. The cDNA clone they designated mac25 was found to be preferentially expressed in normal leptomeningeal cells, compared with meningiomas. mac25 Expression in breast carcinomas has also been examined and it was noted that expression may be related to the estrogen receptor status of the cancer cells: that is, the presence of estrogen receptor (ER) mRNA appeared to be negatively correlated to expression of mac25 mRNA. A more extensive examination of mac25 expression between ER+ vs. ER- breast cancer cell lines indicated that some ER- cancer cells also did not express mac25 mRNA (147). mac25 cDNA was identified by differential display, as one of the genes overexpressed in senescent normal human mammary epithelial cells (HMEC) (147), and as one of the genes that was down-regulated in breast carcinomas (149). Furthermore, there appeared to be a significant (5/10 tumor tissues examined) loss of heterozygosity in the mac25 gene in breast tumors (149). Consistent with these observations was a recent in situ hybridization study of IGFBP-rP1 (mac25/TAF/PSF) expression in normal prostate tissue vs. prostate tumors, where a marked decrease in IGFBP-rP1 expression was associated with increasing malignancy (150). Interestingly, a malignant prostatic cell line stably transfected with IGFBP-rP1 cDNA was shown to be poorly tumorigenic in both in vitro and in vivo assays, when compared with cells stably transfected with empty vector, suggesting a potential tumor-suppressive function for IGFBP-rP1 (151).
Expression of IGFBP-rP1 is regulated by growth factors. In midpassage HMEC (147), breast cancer cells Hs578T (Y. Oh, unpublished), and in immortalized prostate epithelial cells (P69) (150), IGFBP-rP1 expression is up-regulated by retinoids. TGFß also up-regulates IGFBP-rP1 expression, both at the mRNA and protein levels, in Hs578T and P69 cells (150). Whether IGFBP-rP1 mediates the epithelial growth-inhibitory effects of TGFß and retinoic acids has yet to be determined.
One recent study indicated that mac25 mRNA expression is higher in dividing mouse myoblasts than in nondividing, undifferentiated myotubes (152), suggesting that IGFBP-rP1 may play a role in differentiation of muscles. IGFBP-rP1 may also play a role in differentiation of rat osteoblast cells, as PTH and the glucocorticoid, cortisol, both increase IGFBP-rP1 mRNA (153, 154).
2. TAF. TAF, a 30-kDa protein isolated from the conditioned media of a human bladder carcinoma cell line, was so named because it was tumor derived and promoted cell adhesion activity. Initial studies of the purified protein showed that it promoted the attachment and spreading of rat liver cells and human endothelial cells, but did not stimulate endothelial cell growth (42). Subsequent structural analysis of the purified protein and its cDNA indicated identity with PSF (Ref. 41 and see below) and close similarity with Mac25 (37, 42). The discrepancies in primary amino acid sequence between PSF and Mac25 will be discussed below. A monoclonal antibody generated against a C-terminal peptide of purified TAF was used to determine the distribution of TAF in various human cancer tissues (155). Results, based solely on immunohistochemical staining of tissues using this monoclonal antibody, indicated that TAF appears to specifically accumulate in new blood vessels in various human cancer tissues, but not in those of normal tissues, and also in capillary tube-like structures of cultured vascular endothelial cells. These observations, in conjunction with an affinity of TAF for type IV collagen, that was inhibitable by heparin, suggested that TAF may be involved in the formation of new capillary vessels by vascular endothelial cells. This led to the suggestion of renaming the protein "angiomodulin" (155). TAF, at high concentrations (1 µg/ml), also appears to be capable of stimulating and enhancing IGF and insulin-mediated fibroblast cell growth (37). The seemingly diverse functions of IGFBP-rP1 (TAF/Mac25) can be reconciled by the fact that expression and function of this protein are most likely cell type specific, but further studies are clearly necessary.
3. PSF. Yamauchi et al. (41) was the third group to purify "IGFBP-rP1." Their interest was in an activity found in plasma that stimulated prostacyclin production in endothelial cells but that was reduced in patients with diabetes mellitus (156, 157). Prostacyclin is a vasodilator and inhibitor of platelet adhesion and aggregation, whose synthesis is stimulated by many factors, including proteases such as thrombin (158). The prostacyclin-stimulating activity in serum was relatively heat stable, acid labile, and nondialyzable (156, 157). A similar activity was detected in the conditioned media of human diploid fibroblast cells (41). Purified PSF was approximately 31 kDa on SDS-PAGE and was able to stimulate prostacyclin production in endothelial cells at a concentration as low as 10 ng/ml (41). PSF was subsequently identified to be the same protein as Mac25 and TAF (159). An antibody generated to a synthetic PSF C-terminal peptide indicated that PSF is expressed in arterial endothelial cells and in smooth muscle cells of human tissues (160, 161).
Although PSF, Mac25, and TAF are the same protein, there are a few discrepancies in the published cDNA nucleotide sequences between PSF and Mac25. Four nucleotides differ in the signal peptide region, resulting in three amino acid substitutions; one nucleotide differs in the N-terminal domain of the molecules resulting in an Arg for Mac25, and Lys for PSF, both basic amino acids. Finally, the major difference is an extra nucleotide found near the C terminus of Mac25 that results in a stop codon within 5 amino acids of the insertion. In PSF, the lack of this one extra nucleotide generated a completely different sequence and extended the sequence by 10 amino acids. Of the two sequences, the PSF sequence is most likely the correct sequence, as it concurs with amino acid sequencing data (41), and with the cloned genomic IGFBP-rP1 gene (V. Hwa and R. G. Rosenfeld, unpublished and Ref. 159). In addition, TAF has an identical sequence with PSF (37). It is quite likely, therefore, that the discrepancy between the Mac25 and PSF cDNAs was the result of errors in the sequencing of mac25.
In summary, IGFBP-rP1 appears to be involved in diverse biological functions, from regulation of epithelial cell growth, to stimulation of fibroblast cell growth, to stimulation of prostacyclin production in endothelial cells. Further, it can associate with type IV collagen (37) and can bind IGFs and insulin (22, 37, 118). Its expression is regulated, not only by specific growth factors such as IGFs, PTH, cortisol, TGFß, and retinoic acid, but by unknown factors involved in the progression of tumorigenesis, in senescing epithelial cells, in diabetes, and in vascular development. IGFBP-rP1 has thus been hypothesized to have a significant biological role in senescence, tumor suppression, and vascular disease; these multiple effects will need to be substantiated.
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IV. CCN Family |
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A. IGFBP-rP2 (CTGF)
Connective tissue growth factor was the first human protein of the
CCN family to be described (44) and the second protein tested and shown
to be related to the IGFBPs (23). CTGF (IGFBP-rP2) was purified from
media conditioned by human umbilical vein endothelial cells (HUVEC) as
the major secreted protein that was immunoreactive with antibody
against platelet-derived growth factor (PDGF) (44). Interestingly, it
was subsequently shown that the two proteins did not share any regions
of amino acid sequence similarity (44). The purified protein is a
monomer of 36–38 kDa that demonstrated mitogenic activity and
chemotactic activities for fibroblastic cells. Furthermore, the protein
could bind to the cell surface of endothelial cells and was
competitively displaced by purified PDGF (44). Subsequent studies have
demonstrated that a 10-kDa proteolytic fragment of CTGF, corresponding
to the CT domain, can associate with the cell surface of fibroblasts
and is capable of inducing cell proliferation (166, 167, 168). These
activities are inhibitable by heparin and clearly do not involve the
PDGF receptor (168). The specific cell surface receptor for CTGF is not
known, although a recent study has suggested that, at least for human
chondrocytes, CTGF, which participates in endochondral ossification
(169), interacts with an uncharacterized 280-kDa membrane protein
(170).
The chromosomal location for the CTGF gene has been determined to be 6q23.1 and proximal to the oncogene, c-myb (45). The cDNA for CTGF encodes a 38-kDa protein with two potential glycosylation sites and hybridizes to a single 2.4- kb mRNA species in Northern blots (44). Expression of CTGF is regulated in a manner consistent with an immediate-early gene. In fibroblast cells, it is selectively up-regulated by TGFß, a potent stimulator of fibroblast cell proliferation and a critical factor in cell regeneration and wound repair, within an hour of exposure to TGFß (171). Unlike other immediate-early genes, however, short-term exposure to TGFß induces prolonged CTGF mRNA expression, for up to 36–48 h (171, 172). A novel TGFß response element found in both the human and murine CTGF promoters, but absent in other genes regulated by TGFß, suggests that regulation of CTGF gene expression may function by a mechanism distinct from other TGFß-regulated genes (172). Importantly, some of the biological effects of TGFß on fibroblast and endothelial cells appear to be mediated by the up-regulated CTGF protein (173, 174, 175, 176, 177).
Since the discovery and initial characterization of CTGF, there has been considerable research into the regulation, biology, and clinical implications of this protein, which will be briefly summarized here. The readers are referred to recent reviews for more comprehensive coverage (162, 174, 178, 179, 180). Because of clinical implications in fibrosis and mucosal repair, IGFBP-rP2 (CTGF) research has focused on its role(s) in fibroblast and endothelial systems. However, IGFBP-rP2 may also be important for epithelial growth, as recent data suggest that TGFß, which is inhibitory for epithelial cell proliferation, up-regulates IGFBP-rP2 expression (mRNA and protein) in mammary cells (181). Interestingly, in situ studies of mammary tumors have suggested that IGFBP-rP2 mRNA expression is exclusively in the fibrous stroma (182). The implications are unclear at present.
B. IGFBP-rP3 (NovH)
The gene nov was first discovered in
myeloblastosis-associated virus type I-induced avian
nephroblastomas (183). Expression of nov was elevated in
these nephroblastomas, compared with normal adult avian kidney cells,
suggesting that nov may be a protooncogene. Supporting this
concept, human novH (184) expression was shown to be
elevated in Wilms tumors of the stromal type, which histologically are
similar to avian nephroblastomas (45, 184). Of particular interest is
that the novH gene maps to chromosome 8q24.1, proximal to
c-myc (45), a region often involved in chromosomal
abnormalities associated with human tumors, including Wilms tumor. The
expression of novH appears to be inversely correlated with
the expression of the tumor suppressor gene, WT1 (185, 186), whose
inactivation is postulated to participate in the etiology of Wilms
tumors. Indeed, recent studies indicate that WT1 does transcriptionally
down-regulate novH expression (187).
Aside from its oncogenic potential, novH and nov are involved in other biological processes. For example, the effects of nov on chicken embryo fibroblast (CEF) cells are quite different: overexpression of nov inhibits fibroblast cell growth, although, interestingly, overexpression of an N-terminally truncated form of nov (which deleted the N-terminal domain) induced cellular transformation of the fibroblast cells (183). Consistent with the growth-inhibitory effects observed in CEF cells, it was demonstrated that nov was expressed only in quiescent CEF cells, and that transformation of CEF by p60v-src oncogene down-regulated expression of the nov gene (188). In humans, novH is associated with the developing kidney, where observations suggest that NovH protein is stably accumulated in embryonic kidney in glomerular podocytes undergoing differentiation, and, after birth, the persistence of high levels of NovH protein may be required for maintenance of podocyte structure and/or for specfic podocytic functions (186).
The structures of mammalian and nonmammalian Nov proteins are similar to that of Cef-10 (164), Cyr61 (165), Fisp-12 (189), and CTGF (44). The human NovH protein, deduced from the cloned cDNA, indicated that the cDNA encodes a putative 39-kDa secreted polypeptide (184, 186). Immunoblots of biological fluids and media conditioned by various cell lines indicate that NovH is at least 44 kDa and is N glycosylated (38, 186). Interestingly, intracellular isoforms of NovH were detected and appeared to be less stable than the extracellular form (186). Like all members of the CCN family, Nov/NovH consists of four domains (21), of which the first domain (after the signal peptide) is an IGFBP N-terminal domain, leading to the redesignation of NovH as IGFBP-rP3. Recently, Burren et al. (38) demonstrated that IGFBP-rP3 could bind IGF with low affinity, similar to that detected for IGFBP-rP1 and -rP2. More structural and functional information will be given in Sections VII and VIII below.
C. IGFBP-rP4 (Cyr61)
The cyr61 gene was originally identified in mouse 3T3
fibroblasts as an immediate-early gene that was rapidly activated by
serum, PDGF, fibroblast growth factor, and
12-O-tetradecanoylphorbol-13-acetate (165). Unlike other
immediate-early genes, but similar to CTGF, induced cyr61
mRNA persists for a considerable time after induction. The human
cyr61 gene, cloned recently, and mapped to chromosome
1p22-p31 (43), is similar to mouse cyr61 in both structure
[sharing 85% amino acid similarity (190)] and function. Both
cyr61 mRNAs are not detected in quiescent fibroblasts, but
are abundant in logarithmically growing cells and serum-stimulated
cells (165, 190). Human cyr61 mRNA was also recently shown
to be up-regulated by factors important for osteoblast function and
differentiation, such as 1
,25-dihydroxyvitamin D3, EGF,
tumor necrosis factor (TNF), and interleukin-1 (191).
Cyr61 protein, like the rest of the CCN family, is a secreted protein.
However, unlike the other members, it is not readily detected in
conditioned media of cell lines examined, apparently because it
associates with the ECM and cell surfaces, most likely through its
heparin binding regions (192, 193); (see recent review in Ref. 163). In
fact, it was recently demonstrated that Cyr61 protein adheres to HUVEC
cells through integrin vß3 (194). This
adherence of Cyr61 to HUVEC cells may be a mechanism by which Cyr61
promotes the attachment and spreading of endothelial cells (193).
Support for this hypothesis comes from recent studies in which purified
Cyr61 was shown to promote angiogenesis through an
vß3-dependent pathway (195). Cyr61 also
promotes the adhesion of fibroblasts and epithelial cells (193, 196),
induces chemotaxis of fibroblasts (193), enhances growth
factor-stimulated DNA synthesis in both fibroblast and endothelial
cells (176, 193, 196), and plays a role in chondrogenesis (197).
There is recent evidence that Cyr61 may also play a role in tumorigenesis. Stably cyr61-transfected gastric adenocarcinoma cells demonstrated increased tumor growth when tested in a nude mouse model, suggesting that Cyr61 promoted tumor growth (195). However, Cyr61 protein expression was down-regulated in prostate carcinomas (198). Although the role of Cyr61 in cancer is unclear, it is of interest to note that the human cyr61 gene is mapped to chromosome 1p22-p31 (43), as abnormalities of chromosome 1p have been shown to correlate with breast cancer (199), neuroblastoma (200), and pheochromocytoma (201).
The human cyr61 cDNA encodes a 381-amino acid protein rich in cysteine and proline residues (43, 190). There are two distinct mRNA species, a major one at 2.5 kb and a minor one at about 4.0 kb, which are believed to be either alternatively spliced transcripts or transcripts with different polyadenylation signals (43). Cyr61 is structurally consistent with other members of the CCN family, but has yet to be tested for its ability to bind IGFs. It is predicted, however, that it will prove capable of binding IGFs with low affinity, similar to that observed with CTGF and NovH.
D. New members
1. IGFBP-rP7 (rCOP-1/WISP-2/CTGF-L). The gene
rCop-1 was very recently identified by differential display
from rat embryo fibroblasts (REFs) as one of three genes whose
expression was lost specifically upon cell transformation (47). By
sequence comparison, it appears that rCop-1 belongs to the CCN family
of proteins, but, unlike the other CCN proteins, it only has the first
three conserved protein domains and lacks the last domain (the CT
domain). The cDNA encodes a unique 250-amino acid protein with a signal
peptide and is detectable as a single 1.7 kb transcript, thereby ruling
out the possibility that rCop-1 mRNA is a result of alternative
splicing of other CCN transcripts. However, until the gene for rCop-1
is fully characterized, this possibility cannot be completely ruled
out. Although rCop-1 has a signal peptide, it is not detectable in
conditioned media of fibroblast cells, nor is it associated with the
ECM, like Cyr61, perhaps due to loss of the CT domain. Rather, it seems
to be predominantly cell surface associated. Not only is the structure
of rCop-1 distinct from the other CCN proteins, but the pattern of
expression of rCop-1 mRNA indicates that its regulation may be through
different mechanisms than for the rest of the CCN family. It is not an
immediate-early gene, like the CTGF and cyr61
genes; it is not serum inducible, and, in fact, expression is inversely
related to that of cyr61 in normal fibroblast cells.
Overexpression of rCop-1 in transformed cells reduced tumorigenicity
and increased cell death. In primary cultures of rat and mouse
fibroblasts, the rCop-1 gene was detected only when cells became
senescent during passage in culture.
A human ortholog of the rCop-1 gene, WISP-2 (46) and CTGF-L (39), has been subsequently identified and redesignated IGFBP-rP7 in this review. The gene maps to human chromosome 20q12–20q13 (46) and appears to be linked to tumorigenesis (46) as well as to the modulation of bone turnover (39). WISP-2/IGFBP-rP7 mRNA expression was reduced in human colon tumors and is one of three WISP genes that are regulated by Wnt-1, a glycosylated signaling protein critical in developmental processes and linked to tumorigenesis (202, 203).
Kumar et al. (39) identified a CTGF-like cDNA by analysis of an expressed sequenced tag cDNA library derived from primary human osteoblasts. Expression of CTGF-L/IGFBP-rP7 appeared to be predominantly in osteoblasts at sites of high bone turnover. Functionally, recombinant CTGF-L/IGFBP-rP7 had a 10-fold higher affinity for IGF-II than for IGF-I by both Western ligand blotting and cross-linking assays. Further, in osteoblast cells, it inhibited osteocalcin (a marker of mineralizing osteoblasts) production and promoted cell adhesion and integrin binding. The implication is a role of CTGF-L/IGFBP-rP7 in bone turnover, through mechanisms yet to be determined.
2. IGFBP-rP8 (ELM1/WISP-1). The Elm1 gene ("expressed in low-metastatic type 1 cells") was cloned by differential display as a novel mouse gene that was preferentially expressed in low, but not high, metastatic type 1 murine melanoma cells (48). Functionally, overexpression by stable transfection indicates that it can suppress the in vivo growth and metastatic potential of murine melanoma cells. Elm1 cDNA encodes a predicted secreted protein of 367 amino acids, which conserves the four CCN protein domains. An alignment analysis with other members of the CCN family, including mouse Nov and mouse Fisp12, indicates it is not an ortholog of other CCN family members. Recently, the human ortholog, designated WISP-1, was identified (46). Unlike WISP-2 (CTGF-L/IGFBP-rP7), WISP-1 mRNA is overexpressed in human colon tumors. Interestingly, the WISP-1 gene is mapped to the human chromosome 8q24.1–8q24.3, in the same region as the novH (IGFBP-rP3) gene (chromosome 8q24.1).
3. IGFBP-rP9 (WISP-3). The third new member of the CCN family is WISP-3 (46). The WISP-3 gene is mapped to human chromosome 6q22–6q23, in close proximity to the CTGF (IGFBP-rP2) gene [chromosome 6q23.1 (45)]. The deduced amino acid sequence from the WISP-3 cDNA indicates that it contains the four typical protein domains of the CCN family. Like WISP-1, WISP-3 expression is up-regulated in human colon tumors (46).
In summary, it seems likely that the CCN family of proteins will continue to expand as new members are identified. The implication for the IGFBP superfamily is that the number of IGFBP-rPs will also continue to expand.
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V. L56 |
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L56 was independently identified by differential display analysis of transcripts expressed in osteoarthritic cartilage (50). HtrA (L56) expression (mRNA and protein) was up-regulated in osteoarthritis and demonstrated endoproteolytic activity (50). Mutagenesis of the putative active site (Ser328 to Ala) eliminated the enzymatic activity (50).
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VI. ESM-1 |
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, interleukin-1ß, and interferon d. The biological
actions of the protein are unknown. Structurally, the cDNA encodes a
small secreted protein of about 20 kDa, consisting of two potential
protein domains: a larger N-terminal domain that is similar to the
IGFBP N-terminal domain and a C terminus that does not appear to share
significant similarities with any known proteins. Of the proteins that
contain the IGFBP N-terminal domain, ESM-1 shows the least, but still
significant, similarity to the IGFBPs (see below). The ability to
interact with IGFs is unknown. |
VII. Structural Relationships Within the IGFBP Superfamily |
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TopAbstractI. IntroductionII. IGFBP FamilyIII. Mac25IV. CCN FamilyV. L56VI. ESM-1VII. Structural Relationships...VIII. Functional Relationships...IX. Evolutionary Relationships...X. SummaryReferences |
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A. Protein domains (modules)
Conceptually, a domain may be defined as a region of a protein
that can fold into a tertiary structure independent of
neighboring sequences. A domain found in two otherwise
nonhomologous proteins is considered to be evolutionarily mobile, and,
therefore, termed a module (206). Under these definitions, the
N-terminal domain of IGFBPs may properly be considered a module, as it
is found in the IGFBP-rPs, as well as in the IGFBPs. The primary
sequences of human IGFBPs and IGFBP-rPs (schematically presented in
Fig. 3) indicate that the only common
region among the present members of the IGFBP superfamily is the
N-terminal domain. An optimized alignment (Clustral method, DNA Star
program) of these N-terminal domains, and the (averaged) similarity
matrix based on this alignment, are shown in Fig. 4 and Table 3. The alignment also suggests that the
N-terminal domain extends up to the last conserved cysteine (xGxCx
sequence, where x is any amino acid residue). The N-terminal domain
(excluding the signal pepide) is, thus, fairly consistent in size,
ranging from 70 amino acids (IGFBP-rP2) to 93 amino acids (for
IGFBP-3). The exception is IGFBP-rP6 [ESM-1 (51)], which has the
largest N-terminal domain of the IGFBP superfamily (130 amino acid
residues, not counting the putative signal peptide), and carries all of
its 18 cysteines within its N-terminal domain.
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) is well conserved; as noted
above, the motif in IGFBP-6 shows the least homology.
Based on the alignment shown in Fig. 4, the similarity matrix
(Table 3) indicates that the N-terminal domains of the IGFBP-rPs have
significant similarities to that in the IGFBPs (40–57%). Among the
IGFBP-rPs, IGFBP-rP6 has the least similarity (37–41% similarity) to
members of the IGFBP superfamily. As expected, the N-terminal domains
among the IGFBP family show a high 68% relative similarity, as does
the N-terminal domains among the CCN family (66%). The N-terminal
domains of IGFBP-rP1 and IGFBP-rP5 also share a higher similarity
(61%) compared with their similarities with either the CCN family or
IGFBPs.
The similarity between the IGFBP family and the IGFBP-rPs significantly
decreases to less than 15% beyond the N-terminal domain. Unlike the
IGFBPs, the IGFBP-rPs do not contain the thyroglobulin-type I domain
(see above) at the C terminus. Analysis of the IGFBP-rP1 amino acid
sequence (24) using the Gapped BLAST database search program reveals
that immediately adjacent to the N-terminal domain is a stretch of
30–45 amino acid residues that has 30% similarity to the Kazal family
of serine proteinase inhibitors, including the (human) pancreatic
secretory trypsin inhibitor (207). This domain, known as a KI domain,
is also found in follistatin, leading to the hypothesis that Mac25 was
a follistatin-like protein (148). Interestingly, the KI domain can be
found in IGFBP-rP5, in the analogous position (Fig. 5). The remainder of the IGFBP-rP1
sequence shares (41%) similarity with the Ig-like domains found in
heparin sulfate proteoglycan (208, 209) and in the fibroblast growth
factor receptor (210). The functions of these regions with respect to
the biological activities of IGFBP-rP1 are not known. The C terminus of
IGFBP-rP5 is highly similar (58%) to the family of HtrA/Do serine
proteases found in bacteria (49).
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The striking feature of the IGFBP-rPs is the modular architecture
of the protein domains, first noted by Bork (21) for the CCN family.
Each of the domains noted above is modular, in that they are found in
combination with other domains, generating mosaic proteins (28, 206, 211). It is noteworthy, as suggested above, that the IGFBP N-terminal
domain is a bona fida module, and that the IGFBPs, like the
IGFBP-rPs, therefore conform to a mosaic protein structure. A schematic
presentation of the mosaic structures of members of the IGFBP
superfamily is shown in Fig. 5, where the N-terminal domain is the
conserved module.
B. Gene structure and correlation with protein domains
The gene locations for all members of the human IGFBP
superfamily were discussed in Sections II and
III, and are summarized in Table 1. IGFBP genes consist of
four exons, with the exception of IGFBP-3, which has an additional
untranslated fifth exon. The IGFBP-rP1, IGFBP-rP3 (novH),
and IGFBP-rP2 genes each contain five exons (174, 184, 212);
presumably, IGFBP-rP4 gene also has five exons, as the mouse IGFBP-rP4
gene has five exon-coding sequences (213).
Analysis of known gene structures for the IGFBPs and IGFBP-rPs
(IGFBP-rP1, -rP2, and -rP3) supports the concept of conservation of the
N-terminal domain (Fig. 6). The
N-terminal domain in the IGFBPs is encoded by exon 1. In IGFBP-rP1, the
N-terminal domain, like in the IGFBPs, is encoded within one exon, exon
1 (Fig. 6). Exon 1 of IGFBP-rP1, additionally, encodes the
5'-untranslated region, signal peptide, and the complete KI domain. The
remaining exons, 2–5, encode the Ig-like domain. Strikingly, for
IGFBP-rP2 and -rP3, each exon encodes one protein domain. Thus the
N-terminal domain (T1QR–TGICT76) is encoded by
exon 2, VWC by exon 3, thrombospondin type I repeat by exon 4, and CT
and 5'-untranslated region by exon 5.
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VIII. Functional Relationships Within the IGFBP Superfamily |
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3 x 10-8 M), as
does IGFBP-rP7 [CTGF-L (39)]. The binding of IGFs by the IGFBP-rPs,
although of lower affinity, was, nevertheless, specific, as PRL, GH 23
(118), and the C-peptide of proinsulin (38) have been shown to be
unable to bind to the IGFBP-rPs. Interestingly, the N terminus of
IGFBP-3 is capable of binding IGF, but with significantly reduced
affinity (110, 118). The decrease in affinity for IGFs by both the
IGFBP-rPs and the recombinant IGFBP-3 N terminus was attributed to the
lack of the appropriate C terminus, although it is certainly possible
that the N-terminal domain of the IGFBP-rPs is not "optimally"
configured. The fact that IGFBP-rPs appear to have at least 100-fold lower affinity for IGFs, compared with the IGFBPs, suggests that there are two distinct classes of IGF binders in the IGFBP superfamily: the low-affinity IGF binders (IGFBP-rPs and IGFBP proteolytic fragments) and the high-affinity IGF binders (IGFBP-1–6). The role(s) of the high-affinity IGF binders in modulating IGF activity is well established. In contrast, the role(s) of the low-affinity IGF binders in modulating IGF activity is less clear. It is likely, however, that regulation of IGF activity is not the major function of the IGFBP-rPs, and that the IGFBP-rPs may, in fact, be more involved in functions independent of their direct effects on IGFs. In this regard, it is worth pointing out that IGF-independent actions have also been demonstrated for some of the conventional IGFBPs (see Section II).
B. Insulin binding
An unexpected action of IGFBP-rP1, and most recently, IGFBP-rP3 is
their ability to bind insulin at an affinity at least equal to their
ability to bind IGFs (37, 38, 118). To date, no insulin-binding
proteins have been described, and none of the IGFBPs was believed to
bind insulin with any degree of significance. In retrospect, it was
only the lack of an appropriate detection technique that led to this
incorrect conclusion. Yamanaka et al. (118) have recently
demonstrated that, in a Western ligand blot using reduced, denaturing
conditions, IGFBP-1 to -6 are capable of binding
125I-labeled insulin, although with considerably lower
affinity than IGFBP-rP1 has for insulin.
Based on the ability of IGFBPs and IGFBP-rP1 to bind IGFs and insulin,
a model for the secondary/tertiary structure of the IGFBPs that would
accomodate IGF binding, as well as insulin binding, active sites was
proposed (Ref. 118 and Fig. 7). The model
suggests that the folding of IGFBPs is such that the N and C termini
together form a high-affinity IGF-binding site, which simultaneously
partially hides an insulin-binding site deep within the protein.
Disruption of this tertiary structure through proteolysis of the
IGFBPs, or by reducing the protein through the use of agents such as
dithiothreitol, not only disrupts the high-affinity IGF-binding site,
but also exposes the partially hidden insulin-binding site. The result
is an increase in affinity for insulin, with a concomitant decrease in
affinity for IGFs. Consistent with this hypothesis, in vitro
generation of the N terminus of IGFBP-3 (110, 118), as well as in
vivo proteolysed N-terminal fragments of IGFBP-3 from human urine,
demonstrate reduced affinity for IGFs and enhanced insulin binding
(110, 214). For IGFBP-rP1 and IGFBP-rP3, the presence of a different C
terminus from that of the IGFBPs results in essentially the same effect
and may explain both the decrease in affinity of IGFBP-rP1 for IGFs and
its ability to bind insulin (118). Additionally, it has been shown that
the synthetic N terminus [amino acid residues 1–97 (110)], as well
as IGFBP-rP1 (118), can effectively inhibit the autophosphorylation of
the ß-subunit of the insulin receptor in insulin
receptor-overexpressing NIH3T3 cells, indicating a biological effect of
the N terminus in the inhibition of insulin action. It is thus
suggested that the N terminus of IGFBP-3 (and of the IGFBP-rPs) may
have important biological roles in the modulation of both IGF and
insulin actions.
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Given their relatively low affinity for IGFs, the IGFBP-rPs, as suggested above, are likely to be involved primarily in functions independent of their effects on IGF and insulin binding. Their initial discoveries were in systems unrelated to IGF or insulin actions. For example, IGFBP-rP1 was purified as a PSF (41) and as a TAF (42); IGFBP-rP2 was shown to be a mitogen and to be chemotatic for fibroblast cells (44); IGFBP-rP3 was proposed to be a potential oncogene (183). Subsequent studies have suggested that the IGFBP-rPs have multiple functions (see Section III), with no evidence for the direct involvement of IGFs or insulin, although Akaogi et al. (37) reported that IGFBP-rP1 enhanced the mitogenic actions of IGF and insulin. The domains responsible for these functions are not known, although for IGFBP-rP2, it has been demonstrated that the CT domain mediates IGFBP-rP2 binding to fibroblast cells, resulting in cell proliferation (166). Interestingly, a truncated form of chicken IGFBP-rP3 (Nov) lacking the N-terminal domain was capable of inducing cellular transformation of CEF cells (183). It is thus predicted that the IGF/insulin-independent actions of the IGFBP-rPs most probably are mediated through the CT domains. Whether the N-terminal domain may participate in these actions has yet to be elucidated and is an area that requires further investigation.
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IX. Evolutionary Relationships Within the IGFBP Superfamily |
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), with the
corresponding similarity matrix (Table 3), indicates that the Tsg
N-terminal domain shares 44% and 34%–50% similarity to the N
termini of IGFBPs and IGFBP-rPs, respectively.
Since arthropods (like Drosophila) evolved well before
vertebrates, it is clear that the N-terminal domain is ancient. As the
amino acid residues of the N terminus share a significant similarity, a
phylogenetic tree of the N-terminal domains can be generated
(Cladogram, DNA Star program), that indicates the number of amino acid
substitution events leading to divergence (Fig. 8). IGFBP-rP6 diverged from the rest of
the proteins early on the evolutionary scale, whereas the point of
divergence for the CCN proteins (IGFBP-rP2-rP4), Tsg, IGFBP-rP5, and
the IGFBPs probably occurred later, and at relatively close points in
evolutionary time, based on an estimated 52 substitution events for
each branch in total.
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The IGFBP-rPs clearly have very different C-terminal domains from the
IGFBPs and from each other (see Section II above and Fig. 5). In IGFBP-rP1, the Ig-like domain at the C terminus is one of the
most common of rudimentary protein modules, found not only in
antibodies and T cell receptors, but also in hundreds of other animal
proteins involved in cell-cell attachment and communications, and even
in several bacterial proteins (206). Proteins containing the Ig domain
are considered part of the Ig superfamily (206). In the CCN family
(IGFBP-rP2 to IGFBP-rP4), the three other protein domains are conserved
in the frog IGFBP-rP2 (CTGF), and each domain can also be found in many
other unrelated proteins (21). Interestingly, the thrombospondin domain
is part of the TRAP protein from Plasmodium falciparium, a
malarial parasite (21). Finally, the serine protease domain in
IGFBP-rP5 shares a surprisingly high similarity with the bacterial HtrA
protease. Clearly, each C-terminal protein domain in the IGFBP-rPs not
only is modular, but is phylogenetically ancient.
C. Evolutionary models
The structural modularity at both the genomic and protein levels
suggests that the evolution of the IGFBPs and IGFBP-rPs can be examined
in two ways: 1) the evolution of the conserved N-terminal domain; and
2) the evolution of the IGFBP superfamily. The evolutionary
relationship among the N-terminal domains has been discussed above.
More complex is the question of how the N-terminal domain was retained
in the IGFBP superfamily as the remainder of each protein apparently
diversified.
The evolutionary sequence of events that could account for the array of
IGFBPs and IGFBP-rPs are shown in Fig. 9, and include (A) the IGFBPs and related proteins share a common
ancestral gene, which over time, selectively retained the sequences
encoding the N-terminal domain, while the remaining sequences evolved
to encode for the various midsegments and C termini of the IGFBPs and
IGFBP-rPs; (B) the N-terminal domain was originally part of the gene
encoding the IGFBP family and, through gene fusion, was subsequently
acquired by the genes encoding the IGFBP-rP; (C) a gene encoding an
IGFBP-rP originally carried the N-terminal domain DNA sequence, which
was later acquired by the genes encoding for the IGFBP family and other
IGFBP-rPs; or (D) the DNA sequence for the N-terminal domain
constitutes a module which, in the course of evolution, has been
disseminated to genes by various DNA recombinational events.
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X. Summary |
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The classification and nomenclature for the IGFBP superfamily, are, of course, arbitrary; what is ultimately relevant is the underlying biology, much of which still remains to be deciphered. The nomenclature for the IGFBP related proteins was derived from a consensus of researchers working in the IGFBP field (52). Obviously, a more general consensus on nomenclature, involving all groups working on each IGFBP-rP, has yet to be reached. Further understanding of the biological functions of each protein should help resolve the nomenclature dilemma. For the present, redesignating these proteins IGFBP-rPs simplifies the multiple names already associated with each IGFBP related protein, and reinforces the concept of a relationship with the IGFBPs.
Beyond the N-terminal domain, there is a lack of structural similarity between the IGFBP-rPs and IGFBPs. The C-terminal domains do share similarities to other internal domains found in numerous other proteins. For example, the similarity of the IGFBP C terminus to the thyroglobulin type-I domain shows that the IGFBPs are also structurally related to numerous other proteins carrying the same domain (87). Interestingly, the functions of the different C-terminal domains in members of the IGFBP superfamily include interactions with the cell surface or ECM, suggesting that, even if they share little sequence similarities, the C-terminal domains may be functionally related.
The evolutionary conservation of the N-terminal domain and functional
studies support the notion that IGFBPs and IGFBP-rPs together form an
IGFBP superfamily. A superfamily delineates between closely related
(classified as a family) and distantly related proteins. The IGFBP
superfamily is therefore composed of distantly related families. The
modular nature of the constituents of the IGFBP superfamily,
particularly their preservation of an highly conserved N-terminal
domain, seems best explained by the process of exon shuffling of an
ancestral gene encoding this domain. Over the course of evolution, some
members evolved into high-affinity IGF binders and others into
low-affinity IGF binders, thereby conferring on the IGFBP superfamily
the ability to influence cell growth by both IGF-dependent and
IGF-independent means (Fig. 10).
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"But classifications are not passive ordering devices in a world objectively divided into obvious categories. Taxonomies are human decisions imposed upon nature—theories about the causes of nature’s order. The chronicle of historical changes in classification provides our finest insight into conceptual revolutions in human thought. Objective nature does exist, but we can converse with her only through the structure of our taxonomic systems.
"We may grant this general point, but still hold that certain fundamental categories present so little ambiguity that basic divisions must be invariant across time and culture. Not so—not for these, or for any subjects. Categories are human impositions upon nature (though nature’s factuality offers hints and suggestions in return)."
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Footnotes |
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1 Supported by NIH Grants CA-58110 and DK-51513 and by US Army Grants
DAMD 17–96-1–2604 and DAMD 17–97-1–7204. 
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References |
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5ß1 integrin receptor by means of
its Arg-Gly-Asp sequence. Proc Natl Acad Sci USA 90:10553–10557,25-dihydroxyvitamin D3 responsive immediate early
gene in human fetal osteoblasts: regulation by cytokines, growth
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H. Sato, T. Yazawa, T. Suzuki, H. Shimoyamada, K. Okudela, M. Ikeda, K. Hamada, H. Yamada-Okabe, M. Yao, Y. Kubota, et al. Growth Regulation via Insulin-Like Growth Factor Binding Protein-4 and -2 in Association with Mutant K-ras in Lung Epithelia Am. J. Pathol., November 1, 2006; 169(5): 1550 - 1566. [Abstract] [Full Text] [PDF]
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 Y. Mitsui, S. Mochizuki, T. Kodama, M. Shimoda, T. Ohtsuka, T. Shiomi, M. Chijiiwa, T. Ikeda, M. Kitajima, and Y. Okada ADAM28 Is Overexpressed in Human Breast Carcinomas: Implications for Carcinoma Cell Proliferation through Cleavage of Insulin-like Growth Factor Binding Protein-3. Cancer Res., October 15, 2006; 66(20): 9913 - 9920. [Abstract] [Full Text] [PDF]
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 J.-C. Gabillard, B. B. Kamangar, and N. Montserrat Coordinated regulation of the GH/IGF system genes during refeeding in rainbow trout (Oncorhynchus mykiss). J. Endocrinol., October 1, 2006; 191(1): 15 - 24. [Abstract] [Full Text] [PDF]
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L. Peng, P. J. Malloy, J. Wang, and D. Feldman Growth Inhibitory Concentrations of Androgens Up-Regulate Insulin-Like Growth Factor Binding Protein-3 Expression via an Androgen Response Element in LNCaP Human Prostate Cancer Cells Endocrinology, October 1, 2006; 147(10): 4599 - 4607. [Abstract] [Full Text] [PDF]
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 A. Lopez-Bermejo, J. Khosravi, J. M. Fernandez-Real, V. Hwa, K. L. Pratt, R. Casamitjana, M. M. Garcia-Gil, R. G. Rosenfeld, and W. Ricart Insulin Resistance Is Associated With Increased Serum Concentration of IGF-Binding Protein-Related Protein 1 (IGFBP-rP1/MAC25). Diabetes, August 1, 2006; 55(8): 2333 - 2339. [Abstract] [Full Text] [PDF]
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 A. Kunnari, O. Ukkola, M. Paivansalo, and Y. A. Kesaniemi High Plasma Resistin Level Is Associated with Enhanced Highly Sensitive C-Reactive Protein and Leukocytes J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2755 - 2760. [Abstract] [Full Text] [PDF]
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 W. Boers, S. Aarrass, C. Linthorst, M. Pinzani, R. O. Elferink, and P. Bosma Transcriptional Profiling Reveals Novel Markers of Liver Fibrogenesis: GREMLIN AND INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS J. Biol. Chem., June 16, 2006; 281(24): 16289 - 16295. [Abstract] [Full Text] [PDF]
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I. P. Michael, G. Pampalakis, S. D. Mikolajczyk, J. Malm, G. Sotiropoulou, and E. P. Diamandis Human Tissue Kallikrein 5 Is a Member of a Proteolytic Cascade Pathway Involved in Seminal Clot Liquefaction and Potentially in Prostate Cancer Progression J. Biol. Chem., May 5, 2006; 281(18): 12743 - 12750. [Abstract] [Full Text] [PDF]
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B. B. Kamangar, J.-C. Gabillard, and J. Bobe Insulin-Like Growth Factor-Binding Protein (IGFBP)-1, -2, -3, -4, -5, and -6 and IGFBP-Related Protein 1 during Rainbow Trout Postvitellogenesis and Oocyte Maturation: Molecular Characterization, Expression Profiles, and Hormonal Regulation Endocrinology, May 1, 2006; 147(5): 2399 - 2410. [Abstract] [Full Text] [PDF]
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 T. Munzer, C. J. Rosen, S.M. Harman, K. M. Pabst, C. St. Clair, J. D. Sorkin, and M. R. Blackman Effects of GH and/or sex steroids on circulating IGF-I and IGFBPs in healthy, aged women and men Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E1006 - E1013. [Abstract] [Full Text] [PDF]
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M. Kawada, H. Inoue, T. Masuda, and D. Ikeda Insulin-like Growth Factor I Secreted from Prostate Stromal Cells Mediates Tumor-Stromal Cell Interactions of Prostate Cancer. Cancer Res., April 15, 2006; 66(8): 4419 - 4425. [Abstract] [Full Text] [PDF]
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J. D. Veldhuis, J. N. Roemmich, E. J. Richmond, and C. Y. Bowers Somatotropic and Gonadotropic Axes Linkages in Infancy, Childhood, and the Puberty-Adult Transition Endocr. Rev., April 1, 2006; 27(2): 101 - 140. [Abstract] [Full Text] [PDF]
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 M. Novinec, D. Kordis, V. Turk, and B. Lenarcic Diversity and Evolution of the Thyroglobulin Type-1 Domain Superfamily Mol. Biol. Evol., April 1, 2006; 23(4): 744 - 755. [Abstract] [Full Text] [PDF]
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K. A Walters, J. P Binnie, B. K Campbell, D. G Armstrong, and E. E Telfer The effects of IGF-I on bovine follicle development and IGFBP-2 expression are dose and stage dependent. Reproduction, March 1, 2006; 131(3): 515 - 523. [Abstract] [Full Text] [PDF]
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 S. H. Lee, M. Takahashi, K. Honke, E. Miyoshi, D. Osumi, H. Sakiyama, A. Ekuni, X. Wang, S. Inoue, J. Gu, et al. Loss of core fucosylation of low-density lipoprotein receptor-related protein-1 impairs its function, leading to the upregulation of serum levels of insulin-like growth factor-binding protein 3 in fut8-/- mice. J. Biochem., March 1, 2006; 139(3): 391 - 398. [Abstract] [Full Text] [PDF]
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K. Izumi, D. Kurosaka, T. Iwata, Y. Oguchi, Y. Tanaka, Y. Mashima, and K. Tsubota Involvement of Insulin-like Growth Factor-I and Insulin-like Growth Factor Binding Protein-3 in Corneal Fibroblasts during Corneal Wound Healing Invest. Ophthalmol. Vis. Sci., February 1, 2006; 47(2): 591 - 598. [Abstract] [Full Text] [PDF]
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 M. Takaoka, C. E. Smith, M. K. Mashiba, T. Okawa, C. D. Andl, W. S. El-Deiry, and H. Nakagawa EGF-mediated regulation of IGFBP-3 determines esophageal epithelial cellular response to IGF-I Am J Physiol Gastrointest Liver Physiol, February 1, 2006; 290(2): G404 - G416. [Abstract] [Full Text] [PDF]
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G. J. Allan, E. Tonner, M. Szymanowska, J. H. Shand, S. M. Kelly, K. Phillips, R. A. Clegg, I. F. Gow, J. Beattie, and D. J. Flint Cumulative Mutagenesis of the Basic Residues in the 201-218 Region of Insulin-Like Growth Factor (IGF)-Binding Protein-5 Results in Progressive Loss of Both IGF-I Binding and Inhibition of IGF-I Biological Action Endocrinology, January 1, 2006; 147(1): 338 - 349. [Abstract] [Full Text] [PDF]
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 M. Gronborg, T. Z. Kristiansen, A. Iwahori, R. Chang, R. Reddy, N. Sato, H. Molina, O. N. Jensen, R. H. Hruban, M. G. Goggins, et al. Biomarker Discovery from Pancreatic Cancer Secretome Using a Differential Proteomic Approach Mol. Cell. Proteomics, January 1, 2006; 5(1): 157 - 171. [Abstract] [Full Text] [PDF]
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A. Kumar, S. Mohan, J. Newton, M. Rehage, K. Tran, D. J. Baylink, and X. Qin Pregnancy-associated Plasma Protein-A Regulates Myoblast Proliferation and Differentiation through an Insulin-like Growth Factor-dependent Mechanism J. Biol. Chem., November 11, 2005; 280(45): 37782 - 37789. [Abstract] [Full Text] [PDF]
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 M. Matilainen, M. Malinen, K. Saavalainen, and C. Carlberg Regulation of multiple insulin-like growth factor binding protein genes by 1{alpha},25-dihydroxyvitamin D3 Nucleic Acids Res., September 26, 2005; 33(17): 5521 - 5532. [Abstract] [Full Text] [PDF]
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W. C. Duncan, S. G. Hillier, E. Gay, J. Bell, and H. M. Fraser Connective Tissue Growth Factor Expression in the Human Corpus Luteum: Paracrine Regulation by Human Chorionic Gonadotropin J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5366 - 5376. [Abstract] [Full Text] [PDF]
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L. Xie, G. Tsaprailis, and Q. M. Chen Proteomic Identification of Insulin-like Growth Factor-binding Protein-6 Induced by Sublethal H2O2 Stress from Human Diploid Fibroblasts Mol. Cell. Proteomics, September 1, 2005; 4(9): 1273 - 1283. [Abstract] [Full Text] [PDF]
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M S Pampusch, G Xi, E Kamanga-Sollo, K J Loseth, M R Hathaway, W R Dayton, and M E White Production of recombinant porcine IGF-binding protein-5 and its effect on proliferation of porcine embryonic myoblast cultures in the presence and absence of IGF-I and Long-R3-IGF-I J. Endocrinol., April 1, 2005; 185(1): 197 - 206. [Abstract] [Full Text] [PDF]
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 F. Lefranc, J. Brotchi, and R. Kiss Possible Future Issues in the Treatment of Glioblastomas: Special Emphasis on Cell Migration and the Resistance of Migrating Glioblastoma Cells to Apoptosis J. Clin. Oncol., April 1, 2005; 23(10): 2411 - 2422. [Abstract] [Full Text] [PDF]
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 A. A. Arslan, L. I. Gold, K. Mittal, T.-C. Suen, I. Belitskaya-Levy, M.-S. Tang, and P. Toniolo Gene expression studies provide clues to the pathogenesis of uterine leiomyoma: new evidence and a systematic review Hum. Reprod., April 1, 2005; 20(4): 852 - 863. [Abstract] [Full Text] [PDF]
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K. Hayashi, K. D Carpenter, T. H Welsh Jr, R. C Burghardt, L. J Spicer, and T. E Spencer The IGF system in the neonatal ovine uterus Reproduction, March 1, 2005; 129(3): 337 - 347. [Abstract] [Full Text] [PDF]
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J. M. Pilewski, L. Liu, A. C. Henry, A. V. Knauer, and C. A. Feghali-Bostwick Insulin-Like Growth Factor Binding Proteins 3 and 5 Are Overexpressed in Idiopathic Pulmonary Fibrosis and Contribute to Extracellular Matrix Deposition Am. J. Pathol., February 1, 2005; 166(2): 399 - 407. [Abstract] [Full Text] [PDF]
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S. C.J.P. Gielen, E. E. Hanekamp, L. J. Blok, F. J. Huikeshoven, and C. W. Burger Steroid-Modulated Proliferation of Human Endometrial Carcinoma Cell Lines: Any Role for Insulin-like Growth Factor Signaling? Reproductive Sciences, January 1, 2005; 12(1): 58 - 64. [Abstract] [PDF]
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 Q.-P. Qin, S. Kokkala, J. Lund, N. Tamm, L.-M. Voipio-Pulkki, and K. Pettersson Molecular Distinction of Circulating Pregnancy-Associated Plasma Protein A in Myocardial Infarction and Pregnancy Clin. Chem., January 1, 2005; 51(1): 75 - 83. [Abstract] [Full Text] [PDF]
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J. L. King and C. Guidry Muller Cell Production of Insulin-like Growth Factor-Binding Proteins In Vitro: Modulation with Phenotype and Growth Factor Stimulation Invest. Ophthalmol. Vis. Sci., December 1, 2004; 45(12): 4535 - 4542. [Abstract] [Full Text] [PDF]
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B. F. Schrijvers, A. S. De Vriese, and A. Flyvbjerg From Hyperglycemia to Diabetic Kidney Disease: The Role of Metabolic, Hemodynamic, Intracellular Factors and Growth Factors/Cytokines Endocr. Rev., December 1, 2004; 25(6): 971 - 1010. [Abstract] [Full Text] [PDF]
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M. Takaoka, H. Harada, C. D. Andl, K. Oyama, Y. Naomoto, K. L. Dempsey, A. J. Klein-Szanto, W. S. El-Deiry, A. Grimberg, and H. Nakagawa Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3 Cancer Res., November 1, 2004; 64(21): 7711 - 7723. [Abstract] [Full Text] [PDF]
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 E. Conti, C. Carrozza, E. Capoluongo, M. Volpe, F. Crea, C. Zuppi, and F. Andreotti Insulin-Like Growth Factor-1 as a Vascular Protective Factor Circulation, October 12, 2004; 110(15): 2260 - 2265. [Full Text] [PDF]
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M. Matsui, B. Sonntag, S. S. Hwang, T. Byerly, A. Hourvitz, E. Y. Adashi, S. Shimasaki, and G. F. Erickson Pregnancy-Associated Plasma Protein-A Production in Rat Granulosa Cells: Stimulation by Follicle-Stimulating Hormone and Inhibition by the Oocyte-Derived Bone Morphogenetic Protein-15 Endocrinology, August 1, 2004; 145(8): 3686 - 3695. [Abstract] [Full Text] [PDF]
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S. M. Donovan, J. L. Hartke, M. H. Monaco, and M. B. Wheeler Insulin-like Growth Factor-I and Piglet Intestinal Development J Dairy Sci, July 1, 2004; 87(13_suppl): E47 - 54. [Abstract] [Full Text] [PDF]
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 L. Peng, P. J. Malloy, and D. Feldman Identification of a Functional Vitamin D Response Element in the Human Insulin-Like Growth Factor Binding Protein-3 Promoter Mol. Endocrinol., May 1, 2004; 18(5): 1109 - 1119. [Abstract] [Full Text] [PDF]
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 L. J. Spicer Proteolytic Degradation of Insulin-Like Growth Factor Binding Proteins by Ovarian Follicles: A Control Mechanism for Selection of Dominant Follicles Biol Reprod, May 1, 2004; 70(5): 1223 - 1230. [Abstract] [Full Text] [PDF]
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O.J. Ginther, E.L. Gastal, M.O. Gastal, and M.A. Beg Critical Role of Insulin-Like Growth Factor System in Follicle Selection and Dominance in Mares Biol Reprod, May 1, 2004; 70(5): 1374 - 1379. [Abstract] [Full Text] [PDF]
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H.-S. Kim, A. R. Ingermann, J. Tsubaki, S. M. Twigg, G. E. Walker, and Y. Oh Insulin-Like Growth Factor-Binding Protein 3 Induces Caspase-Dependent Apoptosis through a Death Receptor-Mediated Pathway in MCF-7 Human Breast Cancer Cells Cancer Res., March 15, 2004; 64(6): 2229 - 2237. [Abstract] [Full Text] [PDF]
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A. Colao, D. Ferone, P. Marzullo, and G. Lombardi Systemic Complications of Acromegaly: Epidemiology, Pathogenesis, and Management Endocr. Rev., February 1, 2004; 25(1): 102 - 152. [Abstract] [Full Text] [PDF]
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S. Lam, R. N. van der Geest, N. A.M. Verhagen, F. A. van Nieuwenhoven, I. E. Blom, J. Aten, R. Goldschmeding, M. R. Daha, and C. van Kooten Connective Tissue Growth Factor and IGF-I Are Produced by Human Renal Fibroblasts and Cooperate in the Induction of Collagen Production by High Glucose Diabetes, December 1, 2003; 52(12): 2975 - 2983. [Abstract] [Full Text] [PDF]
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 S. W. Song, G. N. Fuller, A. Khan, S. Kong, W. Shen, E. Taylor, L. Ramdas, F. F. Lang, and W. Zhang IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion PNAS, November 25, 2003; 100(24): 13970 - 13975. [Abstract] [Full Text] [PDF]
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K. Mutaguchi, H. Yasumoto, K. Mita, A. Matsubara, H. Shiina, M. Igawa, R. Dahiya, and T. Usui Restoration of Insulin-Like Growth Factor Binding Protein-Related Protein 1 Has a Tumor-Suppressive Activity through Induction of Apoptosis in Human Prostate Cancer Cancer Res., November 15, 2003; 63(22): 7717 - 7723. [Abstract] [Full Text] [PDF]
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 A. M. Y. Nomura, G. N. Stemmermann, J. Lee, and M. N. Pollak Serum Insulin-like Growth Factor I and Subsequent Risk of Colorectal Cancer among Japanese-American Men Am. J. Epidemiol., September 1, 2003; 158(5): 424 - 431. [Abstract] [Full Text] [PDF]
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J. van den Boom, M. Wolter, R. Kuick, D. E. Misek, A. S. Youkilis, D. S. Wechsler, C. Sommer, G. Reifenberger, and S. M. Hanash Characterization of Gene Expression Profiles Associated with Glioma Progression Using Oligonucleotide-Based Microarray Analysis and Real-Time Reverse Transcription-Polymerase Chain Reaction Am. J. Pathol., September 1, 2003; 163(3): 1033 - 1043. [Abstract] [Full Text] [PDF]
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 D. Nagakubo, T. Murai, T. Tanaka, T. Usui, M. Matsumoto, K. Sekiguchi, and M. Miyasaka A High Endothelial Venule Secretory Protein, Mac25/Angiomodulin, Interacts with Multiple High Endothelial Venule-Associated Molecules Including Chemokines J. Immunol., July 15, 2003; 171(2): 553 - 561. [Abstract] [Full Text] [PDF]
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A. Lopez-Bermejo, J. Khosravi, C. L. Corless, R. G. Krishna, A. Diamandi, U. Bodani, E. M. Kofoed, D. L. Graham, V. Hwa, and R. G. Rosenfeld Generation of Anti-Insulin-Like Growth Factor-Binding Protein-Related Protein 1 (IGFBP-rP1/MAC25) Monoclonal Antibodies and Immunoassay: Quantification of IGFBP-rP1 in Human Serum and Distribution in Human Fluids and Tissues J. Clin. Endocrinol. Metab., July 1, 2003; 88(7): 3401 - 3408. [Abstract] [Full Text] [PDF]
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J. H. Shand, J. Beattie, H. Song, K. Phillips, S. M. Kelly, D. J. Flint, and G. J. Allan Specific Amino Acid Substitutions Determine the Differential Contribution of the N- and C-terminal Domains of Insulin-like Growth Factor (IGF)-binding Protein-5 in Binding IGF-I J. Biol. Chem., May 9, 2003; 278(20): 17859 - 17866. [Abstract] [Full Text] [PDF]
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M. Suzuki, H. Kobayashi, Y. Tanaka, Y. Hirashima, N. Kanayama, Y. Takei, Y. Saga, M. Suzuki, H. Itoh, and T. Terao Bikunin Target Genes in Ovarian Cancer Cells Identified by Microarray Analysis J. Biol. Chem., April 18, 2003; 278(17): 14640 - 14646. [Abstract] [Full Text] [PDF]
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E. Canalis, A. N. Economides, and E. Gazzerro Bone Morphogenetic Proteins, Their Antagonists, and the Skeleton Endocr. Rev., April 1, 2003; 24(2): 218 - 235. [Abstract] [Full Text] [PDF]
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S. L. Franklin, R. J. Ferry Jr., and P. Cohen Rapid Insulin-Like Growth Factor (IGF)-Independent Effects of IGF Binding Protein-3 on Endothelial Cell Survival J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 900 - 907. [Abstract] [Full Text] [PDF]
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E. E. Spangenburg, T. Abraha, T. E. Childs, J. S. Pattison, and F. W. Booth Skeletal muscle IGF-binding protein-3 and -5 expressions are age, muscle, and load dependent Am J Physiol Endocrinol Metab, February 1, 2003; 284(2): E340 - E350. [Abstract] [Full Text] [PDF]
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R. J. Levitt and M. Pollak Insulin-like Growth Factor-I Antagonizes the Antiproliferative Effects of Cyclooxygenase-2 Inhibitors on BxPC-3 Pancreatic Cancer Cells Cancer Res., December 15, 2002; 62(24): 7372 - 7376. [Abstract] [Full Text] [PDF]
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S. M. Firth and R. C. Baxter Cellular Actions of the Insulin-Like Growth Factor Binding Proteins Endocr. Rev., December 1, 2002; 23(6): 824 - 854. [Abstract] [Full Text] [PDF]
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 Y. S. Chang, K. Gong, S. Sun, D. Liu, A. K. El-Naggar, F. R. Khuri, W. K. Hong, and H.-Y. Lee Clinical Significance of Insulin-like Growth Factor-binding Protein-3 Expression in Stage I Non-Small Cell Lung Cancer Clin. Cancer Res., December 1, 2002; 8(12): 3796 - 3802. [Abstract] [Full Text] [PDF]
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 C. McCaig, C. M. Perks, and J. M. P. Holly Intrinsic actions of IGFBP-3 and IGFBP-5 on Hs578T breast cancer epithelial cells: inhibition or accentuation of attachment and survival is dependent upon the presence of fibronectin J. Cell Sci., November 15, 2002; 115(22): 4293 - 4303. [Abstract] [Full Text] [PDF]
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I. Y. C. Sun, M. T. Overgaard, C. Oxvig, and L. C. Giudice Pregnancy-Associated Plasma Protein A Proteolytic Activity Is Associated with the Human Placental Trophoblast Cell Membrane J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5235 - 5240. [Abstract] [Full Text] [PDF]
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R. D. Devlin, Z. Du, V. Buccilli, V. Jorgetti, and E. Canalis Transgenic Mice Overexpressing Insulin-Like Growth Factor Binding Protein-5 Display Transiently Decreased Osteoblastic Function and Osteopenia Endocrinology, October 1, 2002; 143(10): 3955 - 3962. [Abstract] [Full Text] [PDF]
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A. Bachelot, P. Monget, P. Imbert-Bollore, K. Coshigano, J. J. Kopchick, P. A. Kelly, and N. Binart Growth Hormone Is Required for Ovarian Follicular Growth Endocrinology, October 1, 2002; 143(10): 4104 - 4112. [Abstract] [Full Text] [PDF]
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E. J. Kim, P. E. Holthuizen, H. S. Park, Y. L. Ha, K. C. Jung, and J. H. Y. Park Trans-10,cis-12-conjugated linoleic acid inhibits Caco-2 colon cancer cell growth Am J Physiol Gastrointest Liver Physiol, August 1, 2002; 283(2): G357 - G367. [Abstract] [Full Text] [PDF]
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T. J. Maures and C. Duan Structure, Developmental Expression, and Physiological Regulation of Zebrafish IGF Binding Protein-1 Endocrinology, July 1, 2002; 143(7): 2722 - 2731. [Abstract] [Full Text] [PDF]
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 H.-M. P. Wilson, R. S. Birnbaum, M. Poot, L. S. Quinn, and K. Swisshelm Insulin-like Growth Factor Binding Protein-related Protein 1 Inhibits Proliferation of MCF-7 Breast Cancer Cells via a Senescence-like Mechanism Cell Growth Differ., May 1, 2002; 13(5): 205 - 213. [Abstract] [Full Text] [PDF]
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A. Hourvitz, A. Kuwahara, J. D. Hennebold, A. B. Tavares, H. Negishi, T. H. Lee, G. F. Erickson, and E. Y. Adashi The Regulated Expression of the Pregnancy-Associated Plasma Protein-A in the Rodent Ovary: A Proposed Role in the Development of Dominant Follicles and of Corpora Lutea Endocrinology, May 1, 2002; 143(5): 1833 - 1844. [Abstract] [Full Text] [PDF]
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 J. Liu, V.-M. Kosma, T. Vanttinen, C. Hyden-Granskog, and R. Voutilainen Gonadotrophins inhibit the expression of insulin-like growth factor binding protein-related protein-2 mRNA in cultured human granulosa-luteal cells Mol. Hum. Reprod., February 1, 2002; 8(2): 136 - 141. [Abstract] [Full Text] [PDF]
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 P Vorwerk, H Wex, B Hohmann, K Mohnike, U Schmidt, and U Mittler Expression of components of the IGF signalling system in childhood acute lymphoblastic leukaemia Mol. Pathol., February 1, 2002; 55(1): 40 - 45. [Abstract] [Full Text] [PDF]
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C. H. Lang, G. J. Nystrom, and R. A. Frost Burn-induced changes in IGF-I and IGF-binding proteins are partially glucocorticoid dependent Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2002; 282(1): R207 - R215. [Abstract] [Full Text] [PDF]
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J. Nakae, Y. Kido, and D. Accili Distinct and Overlapping Functions of Insulin and IGF-I Receptors Endocr. Rev., December 1, 2001; 22(6): 818 - 835. [Abstract] [Full Text] [PDF]
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A. Hoeflich, R. Reisinger, H. Lahm, W. Kiess, W. F. Blum, H. J. Kolb, M. M. Weber, and E. Wolf Insulin-like Growth Factor-binding Protein 2 in Tumorigenesis: Protector or Promoter? Cancer Res., December 1, 2001; 61(24): 8601 - 8610. [Full Text] [PDF]
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C. G. Goodyer, R. M. O. Figueiredo, S. Krackovitch, L. De Souza Li, J. A. Manalo, and G. Zogopoulos Characterization of the growth hormone receptor in human dermal fibroblasts and liver during development Am J Physiol Endocrinol Metab, December 1, 2001; 281(6): E1213 - E1220. [Abstract] [Full Text] [PDF]
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A. Decensi, H. Johansson, R. Miceli, L. Mariani, T. Camerini, E. Cavadini, M. G. Di Mauro, A. Barreca, A. G. Gonzaga, S. Diani, et al. Long-Term Effects of Fenretinide, a Retinoic Acid Derivative, on the Insulin-like Growth Factor System in Women with Early Breast Cancer Cancer Epidemiol. Biomarkers Prev., October 1, 2001; 10(10): 1047 - 1053. [Abstract] [Full Text] [PDF]
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M. W. Elmlinger, I. Mayer, D. Schnabel, B. S. Schuett, D. Diesing, G. Romalo, H. A. Wollmann, W. Weidemann, K.-D. Spindler, M. B. Ranke, et al. Decreased Expression of IGF-II and Its Binding Protein, IGF-Binding Protein-2, in Genital Skin Fibroblasts of Patients with Complete Androgen Insensitivity Syndrome Compared with Normally Virilized Males J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4741 - 4746. [Abstract] [Full Text] [PDF]
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C. K. Buckway, E. M. Wilson, M. Ahlsen, P. Bang, Y. Oh, and R. G. Rosenfeld Mutation of Three Critical Amino Acids of the N-Terminal Domain of IGF-Binding Protein-3 Essential for High Affinity IGF Binding J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4943 - 4950. [Abstract] [Full Text] [PDF]
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Z Laron Insulin-like growth factor 1 (IGF-1): a growth hormone Mol. Pathol., October 1, 2001; 54(5): 311 - 316. [Abstract] [Full Text] [PDF]
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P Vilmos, K Gaudenz, Z Hegedus, and J L Marsh The Twisted gastrulation family of proteins, together with the IGFBP and CCN families, comprise the TIC superfamily of cysteine rich secreted factors Mol. Pathol., October 1, 2001; 54(5): 317 - 323. [Abstract] [Full Text] [PDF]
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E. M. Wilson, Y. Oh, V. Hwa, and R. G. Rosenfeld Interaction of IGF-Binding Protein-Related Protein 1 with a Novel Protein, Neuroendocrine Differentiation Factor, Results in Neuroendocrine Differentiation of Prostate Cancer Cells J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4504 - 4511. [Abstract] [Full Text] [PDF]
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T. C. Voss, M. P. Flynn, and D. L. Hurley IGF-I Causes an Ultrasensitive Reduction in GH mRNA Levels via an Extracellular Mechanism Involving IGF Binding Proteins Mol. Endocrinol., September 1, 2001; 15(9): 1549 - 1558. [Abstract] [Full Text] [PDF]
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C. Martinerie, C. Gicquel, A. Louvel, M. Laurent, P. N. Schofield, and Y. Le Bouc Altered Expression of novH Is Associated with Human Adrenocortical Tumorigenesis J. Clin. Endocrinol. Metab., August 1, 2001; 86(8): 3929 - 3940. [Abstract] [Full Text] [PDF]
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T. Nickerson, F. Chang, D. Lorimer, S. P. Smeekens, C. L. Sawyers, and M. Pollak In Vivo Progression of LAPC-9 and LNCaP Prostate Cancer Models to Androgen Independence Is Associated with Increased Expression of Insulin-like Growth Factor I (IGF-I) and IGF-I Receptor (IGF-IR) Cancer Res., August 1, 2001; 61(16): 6276 - 6280. [Abstract] [Full Text] [PDF]
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K. Prelle, M. Stojkovic, K. Boxhammer, J. Motlik, D. Ewald, G. J. Arnold, and E. Wolf Insulin-Like Growth Factor I (IGF-I) and Long R3IGF-I Differently Affect Development and Messenger Ribonucleic Acid Abundance for IGF-Binding Proteins and Type I IGF Receptors in in Vitro Produced Bovine Embryos Endocrinology, March 1, 2001; 142(3): 1309 - 1316. [Abstract] [Full Text] [PDF]
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R. G. Rosenfeld, V. Hwa, and Y. Oh Nomenclature of the Insulin-Like Growth Factor-Binding Protein Superfamily J. Clin. Endocrinol. Metab., February 1, 2001; 86(2): 946 - 946. [Full Text]
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