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Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
Correspondence: Address all correspondence and requests for reprints to: Robert C. Baxter, Ph.D., D.Sc., Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. E-mail: robaxter{at}med.usyd.edu.au
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Abstract |
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 Top Abstract I. Introductory OverviewII. Modulation of IGF...III. IGF- and IGFRI-Independent...IV. Adhesion and MigrationV. Cell-Binding Sites and...VI. Interactions of IGFBP-3...VII. Cell Cycle, Apoptosis,...VIII. Implications for Animal...IX. Implications for Human...X. Concluding CommentReferences |
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5ß1 integrin, influencing cell adhesion and migration. IGFBP-2, -3, -5, and -6 have heparin-binding domains and can bind glycosaminoglycans. IGFBP-3 and -5 have carboxyl-terminal basic motifs incorporating heparin-binding and additional basic residues that interact with the cell surface and matrix, the nuclear transporter importin-ß, and other proteins. Serine/threonine kinase receptors are proposed for IGFBP-3 and -5, but their signaling functions are poorly understood. Other cell surface IGFBP-interacting proteins are uncharacterized as functional receptors. However, IGFBP-3 binds and modulates the retinoid X receptor-, interacts with TGFß signaling through Smad proteins, and influences other signaling pathways. These interactions can modulate cell cycle and apoptosis. Because IGFBPs regulate cell functions by diverse mechanisms, manipulation of IGFBP-regulated pathways is speculated to offer therapeutic opportunities in cancer and other diseases.
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I. Introductory Overview |
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TopAbstractI. Introductory OverviewII. Modulation of IGF...III. IGF- and IGFRI-Independent...IV. Adhesion and MigrationV. Cell-Binding Sites and...VI. Interactions of IGFBP-3...VII. Cell Cycle, Apoptosis,...VIII. Implications for Animal...IX. Implications for Human...X. Concluding CommentReferences |
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The precursor forms of all six IGFBPs have secretory signal peptides of between 20 and 39 amino acids, and the mature proteins are all found extracellularly. They share a highly conserved structure that is generally described as consisting of three domains of approximately equal size. In addition, important subdomains, or functional motifs, within each domain are now recognized as contributing to their diverse actions. Structural aspects of the IGFBPs have been extensively reviewed (3, 5, 6, 7). The mature proteins have between 216 and 289 amino acids, giving core molecular masses of between 22.8 and 31.3 kDa.
The conserved amino-terminal domain contains six disulfide bonds in all but IGFBP-6, which has five. The organization of these disulfides has been determined for several IGFBPs, revealing that, despite differences in the pairing of cysteines among IGFBP-1, -4, and -6 (8, 9), all form disulfide bonds within the domain. As recently reviewed (6), important IGF-binding residues are found in the amino-terminal domain (Table 1
), predicted by nuclear magnetic resonance studies on IGFBP-5 (10) and confirmed for IGFBP-3 and IGFBP-5 by mutagenesis studies (11, 12, 13). Although no other major functional motifs have been identified in the amino-terminal domain, the observation that amino-terminal proteolytic fragments of IGFBP-3 cause IGF-independent inhibition of mitogenesis (14, 15) implies the presence of another active subdomain in this region.
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), demonstrated by the binding activity of natural carboxyl-terminal fragments of IGFBP-2 (17, 18) and recombinant carboxyl-terminal IGFBP-3 fragments (19, 20), and mutagenesis of IGFBP-5 residues (21). The observation that residues involved in IGF binding occur in both amino- and carboxyl-terminal domains implies the existence of an IGF-binding pocket involving both domains. As shown in Fig. 1, other important subdomains have also been identified within the carboxyl-terminal region of various IGFBPs; for example, Arg-Gly-Asp (RGD) integrin-binding motifs are located at residues 221–223 of IGFBP-1 (22) and residues 265–267 of IGFBP-2 (23). Functionally important 18-residue basic motifs with heparin-binding activity have also been identified at residues 215–232 of IGFBP-3 and residues 201–218 of IGFBP-5 and are involved in interaction with the serum glycoprotein ALS (acid-labile subunit) (24, 25, 26) and other ligands such as plasminogen activator inhibitor-1 (27) and transferrin (28), cell and matrix binding (24, 29), and nuclear transport (30), as discussed in detail later.
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).
B. Relationship between serum and tissue IGFBPs
The term "multifunctional" is very aptly applied to the IGFBPs. Originally described as passive circulating transport proteins for IGF-I and IGF-II, IGFBPs are now recognized as playing a variety of roles in the circulation, the extracellular environment, and inside the cell. The regulation and actions of circulating IGFBPs have been addressed in several reviews (39, 40, 41, 42, 43). In brief, the major IGF transport function can be attributed to IGFBP-3, the most abundant circulating IGFBP. It carries 75% or more of serum IGF-I and IGF-II in heterotrimeric complexes that also contain ALS, a leucine-rich glycoprotein of approximately 85 kDa (44). IGFBP-5, present at about 10% of the molar concentration of IGFBP-3, can form similar ternary complexes (45). Approximately 90% of IGFBP-3 and 55% of IGFBP-5 circulate in these complexes in healthy adults (46). All six IGFBPs are also found in the circulation in the free form or in binary complexes with IGFs. Free or binary-complexed IGFBPs are believed to exit the circulation rapidly, whereas ternary complexes appear to be essentially confined to the vascular compartment (47, 48, 49).
IGFBPs exert a complex array of functions at the cellular level. There is little information on the exact relationship between IGFBPs in the circulation and those in the cellular environment, but it appears that the IGFBPs may be differentially targeted to different tissues depending on both their primary structure and their posttranslational modifications. In some situations, endogenous IGFBPs from circulating ternary complexes may be found at low concentration in the tissues, as first implied by comparison of IGFBPs in serum and lymph (50). Using exogenous IGFBPs, Boes et al. (51) demonstrated in an isolated perfused heart model that IGFBP-4, after crossing the capillary endothelium, preferentially localizes to connective tissue rather than cardiac muscle, the exact distribution depending on the glycosylation state. In contrast, IGFBP-1, -2, and -3 are preferentially localized to cardiac muscle. IGFBP-3 injected iv appears initially in the liver (40% of injected dose) and kidney (4%), within 5 min of administration (52). Uptake by muscle was not examined in this study.
IGFBP-4 administered systemically to mice has also been shown to act on bone, stimulating bone alkaline phosphatase activity and serum osteocalcin by a mechanism that appears to involve IGFBP-4 proteolysis and increased IGF-I availability (53). The influence of circulating IGFBPs at the tissue level is further indicated by the observation that IGFBP-1 administered iv in rats inhibits IGF-I-stimulated 2-deoxyglucose uptake in cardiac and skeletal muscle (54). Similarly, exogenous IGFBP-3 administered to rats blocks the hypoglycemic effect of coadministered IGF-I, an effect that depends explicitly on its ability to form complexes with ALS in the circulation (55).
In addition to these effects of IGFBPs derived from the circulation, there are undoubtedly important local actions of IGFBPs, both autocrine and paracrine. As well as modulating activation of the type I IGF receptor (IGFRI) by IGFs (20, 56, 57), IGFBPs are documented to affect cell motility and adhesion (22, 58), apoptosis and survival, and cell cycle (59, 60, 61). They interact with diverse previously characterized signaling pathways (22, 62, 63, 64) and may have unique signaling pathways of their own (65). Their cellular effects are likely to be influenced by posttranslational modifications, e.g., glycosylation affecting cell interaction (31, 66), phosphorylation affecting IGF binding affinity (67) and susceptibility to proteases (68), and proteolysis affecting both IGF-independent and IGF-dependent actions (15, 69). The purpose of this review is to document and provide a critical discussion of current knowledge on these and related topics.
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II. Modulation of IGF Activity by IGFBPs and Their Proteases |
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TopAbstractI. Introductory OverviewII. Modulation of IGF...III. IGF- and IGFRI-Independent...IV. Adhesion and MigrationV. Cell-Binding Sites and...VI. Interactions of IGFBP-3...VII. Cell Cycle, Apoptosis,...VIII. Implications for Animal...IX. Implications for Human...X. Concluding CommentReferences |
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It is well established, from in vitro systems, that the IGFs acting through the IGFRI have acute anabolic effects on metabolism as well as longer term effects on cell replication and differentiation (40). Apart from their mitogenic activity, the IGFs also have potent inhibitory effects on apoptosis (74, 75). However, the bioactivity of IGFs is not only dependent on their interaction with IGFRI but is also influenced by the family of IGFBPs in the local cellular environment, which can potentially either inhibit or enhance IGF actions depending on the complement of IGFBPs present. Because most cells express more than one IGFBP, it is clear that the regulation of each IGFBP plays an important role in regulating the cellular effects of IGFs. Furthermore, a wide range of proteolytic enzymes can catalyze the limited hydrolysis of IGFBPs. Documented cleavage sites in IGFBP-3, -4, and -5 are shown in Table 2. Some of the resulting fragments have been reported to retain biological activity (76, 77). In certain cell types, IGF-I itself regulates the expression of specific IGFBPs (78) or their proteases (79, 80), thus adding further complexity.
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Parker et al. (79) described a calcium-dependent serine protease secreted by smooth muscle cells, whose activity is induced by IGFs, that specifically cleaves IGFBP-4 into fragments with low affinity for IGF-I. As a consequence, the IGFBP-4 fragment is less inhibitory to IGF-I-stimulated thymidine uptake compared with intact IGFBP-4 (88). It was determined biochemically that the major cleavage site on IGFBP-4 was Lys120-His121 (Table 2), resulting in a 16-kDa amino-terminal fragment (88). This was confirmed by a protease-resistant mutant form of IGFBP-4 (Lys120 and His121 substituted by Asn) that could inhibit DNA synthesis, cell migration, and muscle growth in response to IGFs, similar to intact IGFBP-4 (89, 90).
Similar IGF-dependent IGFBP-4 protease activity has been described in a variety of cells, including fibroblasts (91), osteoblasts (92, 93, 94), endometrial stromal cells (95), decidual cells (96), and granulosa cells (97). Recently, the IGFBP-4 protease was purified from human fibroblasts and identified as pregnancy-associated plasma protein-A (PAPP-A) (98), and the cleavage site on IGFBP-4 was determined to be Met135-Lys136 (Ref. 34 and Table 2). Preexposure of human fibroblasts to IGF-II potentiated subsequent IGF-I-induced DNA synthesis, and this was inhibited by protease-resistant IGFBP-4 mutants but not wild-type IGFBP-4 (34). More recently, it was shown in human osteoblasts using non-IGF-binding mutants that direct interaction between IGFBP-4 and IGF-II was required for optimal proteolysis of IGFBP-4 by PAPP-A (99, 100). Taken together, these studies suggest that IGFBP-4 acts as a potent inhibitor of the anabolic effects of IGF-I or -II by regulating IGF bioavailability, with the corollary that factors that regulate protease activity may thus regulate IGF actions. For example, the high PAPP-A level in estrogen-dominant ovarian follicles has been proposed to account for IGFBP-4 proteolysis leading to IGF release and subsequent dominant follicle development (101, 102). IGFBP-4 proteolysis may also have a role in the repair of arterial injury, because PAPP-A has been shown to increase in injured vascular smooth muscle cell cultures and injured arteries in vivo and might act in these situations by releasing IGF-I from IGFBP-4 (103). Interestingly, both PAPP-A and a related metalloproteinase (PAPP-A2) were reported to have IGFBP-5 proteolytic activity, but in contrast to the requirement of IGF for the cleavage of IGFBP-4 by PAPP-A, cleavage of IGFBP-5 by either PAPP-A or PAPP-A2 was IGF independent (104, 105).
The ability of IGFBP-5 to potentiate the response to IGF-I in smooth muscle cells is dependent upon its binding to extracellular matrix (ECM). As described further in Section V, a highly charged region of IGFBP-5 that contains ten basic amino acids in residues 201–218 (Table 3) has been shown to mediate binding of IGFBP-5 to the ECM of porcine smooth muscle cells (106), and a synthetic peptide containing this sequence inhibited IGFBP-5 binding resulting in reduced cellular responses to IGF-I (107). IGFBP-5 interacts specifically with two ECM proteins, thrombospondin-1 and osteopontin, which not only potentiate the IGF-I effect but may modulate the cooperative interaction between the IGFRI and integrin receptor pathways (108). In human fibroblasts, ECM-bound IGFBP-5 has a 7-fold loss of IGF-I affinity, suggesting that the potentiation of the IGF-I effect may be due to the increased bioavailability of the IGF-I to IGFRI after sequestration and concentration by IGFBP-5 to the cell surface.
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), and protease-resistant IGFBP-5 (Arg138 and Arg139 substituted with Asn) inhibited IGF-I-stimulated DNA and protein synthesis and migration of porcine smooth muscle cells (35). This is consistent with the finding that proteolyzed IGFBP-5 in the conditioned medium had no effect on the IGF-I stimulation of growth in cultured fibroblasts (109) and suggests that, in contrast to ECM-bound IGFBP-5, soluble IGFBP-5 acts as an inhibitor of IGF-I-stimulatory effects and that proteolysis of IGFBP-5 may serve as an important regulatory mechanism of this function. Although the IGFBP-5 protease in medium conditioned by smooth muscle cells has not been identified, similar proteolytic activity against IGFBP-5 in fibroblast-conditioned medium has been attributed to the complement components C1s and/or C1r (110). The biological significance of IGFBP-5 cleavage by these enzymes is as yet unknown. Analogous to the situation in fibroblasts and smooth muscle cells, the IGF-dependent actions of IGFBP-5 in bone cells appear to be dependent on its location. IGFBP-5 is thought to act as a depot for IGF-II in bone via its high affinity for ECM proteins and hydroxyapatite, the mineral constituent of bone, and potentiates the proliferative actions of IGF-II on osteoblastic cells (111, 112). In addition, the stimulation of osteoclastic activity by IGFBP-5 can be blocked by IGF-I antibody (113). These results have led to the proposal that the IGFs, which are sequestered and concentrated in bone by IGFBP-5, may be released during osteoclastic resorption, thus leading to stimulation of osteoblastic activity during bone remodeling. In contrast, IGF-I- and IGF-II-stimulated DNA and glycogen synthesis in a human osteoblastic cell line was inhibited by soluble recombinant IGFBP-5 (114). Likewise, the relative insensitivity of U2 osteosarcoma cells to IGF-I compared with the non-IGFBP-binding analog, des(1–3)IGF-I, was attributed to the inhibitory effect of endogenously secreted intact IGFBP-5 (115). However, proteolyzed IGFBP-5 derived from the medium of U2 cells enhanced IGF-I-stimulated osteoblast mitogenesis (116). Intriguingly, the amount of intact IGFBP-5 was increased significantly in the medium of these cells treated with IGF-I without a concomitant increase in mRNA levels or reciprocal decrease in proteolytic fragments (115), suggesting that IGF-I not only has a protective effect on IGFBP-5 proteolysis, but may also affect the compartmentalization of IGFBP-5.
It is also well established that IGFs can stimulate both proliferation and differentiation of skeletal muscle cells and these actions are mediated through IGFRI. However, the actions of IGFs are modulated by the expression of IGFBP-4, -5, and -6. As described for other cell types, IGFBP-4 is mainly inhibitory (117, 118) whereas IGFBP-5 could be either inhibitory or stimulatory to IGF actions (119, 120). By employing IGF-II analogs, Bach et al. (121) demonstrated that the inhibition of IGF-II-induced proliferation and differentiation of L6A1 rat myoblasts by IGFBP-6 was correlated to its affinity for the analogs. It would appear that the IGFBPs, on balance, are generally inhibitory to IGF actions in myoblasts.
There are relatively few studies on the function of IGFBP-6 and generally, IGFBP-6 appears to inhibit the actions of IGF-II with some selectivity because it has 20- to 100-fold higher affinity for IGF-II than IGF-I (122, 123). Cell systems in which IGFBP-6 has been shown to inhibit IGF-II-induced effects such as proliferation, differentiation, cell adhesion, and colony formation include osteoblasts, keratinocytes, myoblasts, and colon cancer cells (121, 124, 125, 126, 127, 128).
B. IGFBP-1, -2, and -3
Several studies have described both the inhibition and potentiation of IGF actions by IGFBP-1 in a variety of cells (40). The positive or negative modulation by IGFBP-1 is thought to be related to its phosphorylation state because dephosphorylation of human IGFBP-1 reduces its affinity for IGF-I by 6-fold (129). Curiously, phosphorylation appears to have no effect on the affinity of rat IGFBP-1 (130), raising the question whether IGFBP-1 regulatory mechanisms described in humans are relevant to other species. Dephosphorylated IGFBP-1 has been shown to enhance IGF-I-induced DNA synthesis (131, 132, 133), whereas phosphorylated IGFBP-1 inhibits IGF-I effects (131, 133, 134). Although high-affinity phospho-IGFBP-1 is assumed to act by blocking IGF access to the IGFRI, no experimental evidence has yet explained how the low-affinity dephosphorylated form could enhance IGF action (rather than simply not inhibiting it). Polymerization of IGFBP-1 by tissue transglutaminase has also recently been shown to ablate its inhibition of IGF-I-stimulated protein synthesis (135), but the contribution of this effect to net IGFBP-1 cellular activity is not clear.
In the extensively studied paracrine interactions at the maternal-fetal interface during pregnancy, it is thought that IGFBP-1 secreted by the maternal decidua inhibits placental trophoblast invasiveness (further discussed in Section IV), but this inhibitory effect is repressed by the down-regulation of IGFBP-1 production by placental trophoblast-derived IGF-II (136, 137). An alternative mechanism was proposed by Gibson et al. (138), who identified both phosphorylated and nonphosphorylated isoforms of IGFBP-1 secreted by decidualized endometrium under basal conditions. In the presence of trophoblast-derived IGF-II, the nonphosphorylated form of IGFBP-1—noted above to have lower IGF affinity than phospho-IGFBP-1 (67)—was predominantly produced by decidual cells. Placental phosphatases could also generate this form by dephosphorylating phospho-IGFBP-1. In addition, a protease was found to act specifically on nonphospho-IGFBP-1, which would be expected to further decrease its IGF affinity. The net result of these posttranslational modifications would be IGFBP-1 forms with reduced affinity for IGF-I, thus increasing IGF-I bioavailability to enhance tissue growth (138).
IGF-I is actively involved in the process of dermal wound healing by stimulating reepithelialization of the wounds, and this action is potentiated by IGFBP-1 (139, 140, 141, 142, 143), although the contribution of its phosphorylation state to this action is not known. The enhancement of IGF-I actions appears to be related to the ability of IGFBP-1 to bind to 5ß1 integrin (discussed further in Sections III and IV) because a non-integrin-binding IGFBP-1 mutant had no effect (144). IGFBP-2, which has the integrin-binding motif RGD, like IGFBP-1, was ineffective in augmenting the IGF-I enhancement of wound repair (144).
In general, IGFBP-2 appears to inhibit IGF actions, in particular those of IGF-II, possibly related to its higher affinity for IGF-II (40), although this affinity difference is in fact only 2-fold (145). Overexpression of IGFBP-2 in human embryonic kidney fibroblasts results in inhibition of cell proliferation, which can be reversed by the addition of exogenous IGFs, thus suggesting that IGFBP-2 has an inhibitory effect on IGF action (146). This is supported by a previous study that showed growth stimulation of intestinal epithelial cells transfected with an antisense IGFBP-2 construct (147). In addition, Höflich et al. (148) recently reported that giant GH transgenic mice, which had 2- to 3-fold increased expression of serum IGF-I levels, had a significant reduction in growth parameters when crossed with IGFBP-2 transgenic mice, suggesting that IGFBP-2 is also inhibitory to IGF-I actions in vivo.
Addition of equimolar concentrations of IGFBP-2 completely inhibited IGF-II-stimulated DNA synthesis in non-small-cell lung carcinoma (NSCLC) cells but had no significant effect on IGF-I-stimulated DNA synthesis (149, 150). This is not easily explained because, as noted above, the relative affinities for IGF-I and IGF-II do not differ greatly. Interestingly, IGFs bind predominantly to IGF receptors in NSCLC cells, which have relatively low levels of membrane-associated IGFBP-2. In contrast, IGFs bind to high levels of membrane-associated IGFBP-2 in small-cell lung carcinoma (SCLC), which do not respond to IGFs even though IGFRI is present (149). This suggests that both soluble and membrane-associated IGFBP-2 may be competing with the IGF receptors for ligand and may therefore be regulating IGF responsiveness in lung carcinoma.
Proteolyzed IGFBP-2 has been detected in serum (151), milk (18), and cerebrospinal fluid (152) and has decreased affinity for IGFs compared with intact IGFBP-2. Serum withdrawal from porcine aortic smooth muscle cells induces the secretion of a calcium-dependent serine protease for IGFBP-2, the activity of which is relatively more enhanced by IGF-II than IGF-I (153, 154). Menouny et al. (155) reported that the interaction between the plasmin system and IGFBP-2 can modulate the bioavailability of IGF-II, which mediates autocrine proliferation in neuroblastoma cells. However, in the majority of cell studies in which IGFBP-2 is detected by immunoblot, it appears in culture medium at its intact size of approximately 34 kDa. Thus, IGFBP-2 proteolysis may not be a very widespread regulatory mechanism.
Potentiation and inhibition of IGF actions by IGFBP-3 have been demonstrated in many cell culture systems (6, 40). It is thought that cotreatment of cells with IGFBP-3 and IGF-I causes IGFBP-3 to inhibit IGF-I-mediated effects via high-affinity sequestration of the ligand (15, 156, 157, 158), presumably leading to prevention of IGF-I-induced IGFRI autophosphorylation and signaling (20). In contrast, preincubation of cells with IGFBP-3 before IGF-I treatment leads to the accumulation of cell-bound forms of IGFBP-3 with lowered affinity for IGF, which may enhance the presentation of IGF to IGFRI (156, 159, 160). However, this mechanism has never been proven explicitly, and Karas et al. (56) found that cell-bound IGFBP-3 could still attenuate IGF-I-mediated IGFRI signaling. It has also been reported, based on competitive ligand-binding studies, that IGFBP-3 can interact with IGFRI, causing inhibition of IGF-I binding to its receptor (161), although a direct physical interaction between IGFRI and IGFBP-3 has not been demonstrated, for example, by coprecipitation. It is therefore not clear that cell association of IGFBP-3 is the key factor in determining its IGF-stimulatory effects. In addition, it has been suggested that the enhanced IGF-I stimulation of DNA synthesis in MCF-7 cells transfected with IGFBP-3 might result from IGFBP-3 protecting the cells from IGF-I-mediated down-regulation of IGFRI (162) as initially proposed in a previous study in bovine fibroblasts (163). More recently, it has been suggested that the potentiation of IGF action by IGFBP-3 may be mediated through the phosphatidylinositol 3 (PI3)-kinase pathway (62) (Section VI).
As with the other IGFBPs, specific proteases for IGFBP-3 in a variety of cell culture systems have been described (164), including serine proteases, cathepsins, and matrix metalloproteinases. Proteolysis results in IGFBP-3 fragments with decreased affinity for IGFs and is therefore assumed to enhance the availability of IGFs to the cell (77, 165, 166). However, several studies have described the inhibition of IGF actions by IGFBP-3 fragments with low (sometimes undetectable) affinity for IGFs (14, 15, 77, 167). It is unclear whether this inhibitory action of IGFBP-3 may, in some cases, be mediated via its sequestration of IGFs (despite its low affinity) or via an IGF-independent mechanism such as the proposed interaction with the IGFRI directly to prevent IGF-IGFRI interactions as described above.
Figure 2 summarizes proposed IGFBP actions that depend on binding of IGFs and modulation of IGFRI activation. Although studies describing the potentiation or inhibition of IGF activity by IGFBPs have been reported for at least two decades, there is currently no unifying mechanism that would explain these opposite actions. What is evident is that the complex interaction between IGFs and IGFBPs is further complicated by the fact that 1) the production of IGFBPs and IGFBP-specific proteases are often regulated by IGFs, and 2) IGFs can regulate the activity of these proteases.
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III. IGF- and IGFRI-Independent Effects of IGFBPs |
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TopAbstractI. Introductory OverviewII. Modulation of IGF...III. IGF- and IGFRI-Independent...IV. Adhesion and MigrationV. Cell-Binding Sites and...VI. Interactions of IGFBP-3...VII. Cell Cycle, Apoptosis,...VIII. Implications for Animal...IX. Implications for Human...X. Concluding CommentReferences |
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One of the first reports of IGF-independent actions of IGFBPs was the effect of IGFBP-1 on cell motility and adhesion. There is good evidence that this effect is mediated by the RGD integrin-binding motif present in the carboxyl-terminal domain of IGFBP-1 (Table 3), interacting with 5ß1 integrin (22), as further discussed in Section IV. Although a homologous RGD motif is also present in IGFBP-2, the evidence supporting IGF-independent actions of IGFBP-2 mediated by integrin binding is still preliminary (Section IV).
IGFBP-2 has been shown to be mitogenic for uterine endometrial epithelial cells and osteosarcoma cells in the absence of IGFs (168, 169). Overexpression of IGFBP-2 in Y-1 adrenocortical tumor cells resulted in enhanced proliferation and increased cloning efficiency of IGFBP-2-secreting cells (170). In a subsequent preliminary report, several proliferation-associated genes including glutathione S-transferase were shown to be up-regulated in these IGFBP-2-transfected cells, leading to the suggestion that the induced glutathione S-transferase levels may confer resistance to oxidative stress (171). In these studies it was proposed that the proliferative effect of IGFBP-2 overexpression was IGF independent because IGFRI was down-regulated and an IGF-I analog with decreased IGFBP interaction had the same mitogenic potency as IGF-I (170). Although a mechanism of growth stimulation by IGFBP-2 is yet to be established, it may tentatively be concluded that, whereas inhibitory IGFBP-2 effects are likely to involve the sequestration of IGFs (Section II), some of the positive growth effects of IGFBP-2 appear to occur independently of IGF action. This may have implications for the treatment of IGFBP-2-expressing tumors, which include those of colon (172) and prostate (173).
IGF-independent effects of IGFBP-4 and IGFBP-6 are not well established and, as discussed in the previous section, it is generally accepted that IGFBP-4 and IGFBP-6 act primarily through inhibition of IGF actions. Indeed, the majority of reports on IGF-independent effects of IGFBPs have focused on IGFBP-3. The earliest of these is attributable to Harel and her colleagues (174), who determined that murine IGFBP-3 (initially referred to as "inhibitory diffusible factor 45"), in addition to acting through IGF binding, could inhibit DNA synthesis in chick embryo fibroblasts stimulated by serum, fibroblast growth factor, or TGFß, but not insulin or platelet-derived growth factor (175, 176). Subsequently, mouse fibroblasts transfected with IGFBP-3 were shown to have a markedly reduced growth rate and were arrested at a lower cell density than vector-transfected cells. This growth-inhibitory effect, which was demonstrated in the presence of serum-containing media, was not reversible by insulin, leading to the proposal that the IGFBP-3 effect was IGF independent (177).
Although the possibility remained that IGF signaling somehow contributed to these early observations, IGFBP-3 was subsequently shown unambiguously to have IGFRI-independent growth-inhibitory effects in a mouse fibroblast cell line derived from IGFRI-knockout mice where transfection of IGFBP-3 resulted in cell growth inhibition (178). Whether IGFBP-3 might have acted in these cells by blocking IGF activity through a different receptor [e.g., IGF-II signaling through the type A insulin receptor (179)] has never been explicitly excluded. This illustrates the need to distinguish between IGFRI independence (which was clearly demonstrated in this study) and IGF independence, which is difficult to prove rigorously in cell culture studies.
An interesting illustration of transition from IGF-independent to -dependent IGFBP-3 action is seen in the changing response to exogenous IGFBP-3 in differentiating chondrocytes (180). In this study in a chondrogenic cell line, IGFBP-3 inhibited thymidine incorporation stimulated by insulin, IGF-I, or low-IGFBP-affinity IGF-I analogs, when the cells were in the predifferentiated state. In contrast, when the cells had undergone terminal differentiation, only IGF-I action was inhibited by IGFBP-3, suggesting that it could only inhibit the actions of ligands that it could sequester with high affinity. Although there is as yet no mechanistic explanation for this transition, this cell system appears to offer a valuable model in which to examine the molecular basis of IGF-independent inhibitory signaling by IGFBP-3.
Several studies using human breast cancer cells have correlated the induction of IGFBP-3 mRNA and protein expression with growth-inhibitory effects of various antiproliferative agents including TGFß, retinoic acid (181), antiestrogens (182), vitamin D analogs (183, 184), and TNF (Ref. 185 and Fig. 3). IGFBP-3 expression is also up-regulated by the transcription factor p53 in colon carcinoma cells (186), and by the histone deacetylase inhibitors trichostatin A (187) and butyrate (188). Studies from several independent laboratories using breast or prostate cancer cell lines have shown that decreasing the expression of IGFBP-3 by antisense IGFBP-3 oligodeoxynucleotide treatment, or sequestering IGFBP-3 using antibodies, partially abrogates the antiproliferative effects of some of these factors. This phenomenon, demonstrated for TGFß, retinoic acid, the antiestrogen ICI 182780, and 1,25-dihydroxyvitamin D3, has been cited as evidence that the induction of IGFBP-3 contributes to the growth-inhibitory functions of these agents (181, 182, 184, 189). Interestingly, the induction of IGFBP-3 by TGFß has also been associated with growth-stimulatory effects of TGFß in airway smooth muscle cells and colon cancer cells (190, 191), and this TGFß-induced cell growth can also be blocked by antisense IGFBP-3 oligodeoxynucleotides or neutralizing antibodies. These studies raise interesting questions, representing as they do a powerful accumulation of evidence that IGFBP-3 plays a mediating role in the actions of a diverse range of growth effectors. Balancing this is abundant literature describing alternative mechanisms of action of these effectors, so that it will be important to determine the extent to which IGFBP-3 induction is necessary to mediate their actions.
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Other, independent evidence supports the proposal that IGFBP-3 amino-terminal fragments have intrinsic activity. Using a semipurified mixture of plasmin-digested IGFBP-3, Booth et al. (194) demonstrated the bioactivity of approximately 20-kDa amino-terminal and about 8-kDa carboxyl-terminal fragments in stimulating glucose uptake by microvessel endothelial cells, in contrast to intact IGFBP-3, which was ineffective. It was not clear which fragment was responsible for the activity and whether the approximately 20-kDa fragment was the same as the 16-kDa fragment described above. A similar 16-kDa amino-terminal fragment, derived from proteolysis by an enzyme secreted by MCF-7 breast cancer cells (80), was identified by sequencing and mass spectrometry as IGFBP-3[1–97]. This fragment inhibits basal DNA synthesis in MCF-7 cells in the absence of IGF-I (15). The mechanism by which intact IGFBP-3, and amino-terminal IGFBP-3 fragments, might exert their effects independent of the modulation of IGF-IGFRI interactions remains to be determined. As described in Section V, possible cell-interacting sites have been identified in the central (38) and carboxyl-terminal domains (24) of IGFBP-3 but not in its amino-terminal domain.
Apart from binding its well described ligands, IGF-I and IGF-II, IGFBP-3 has a number of other interacting partners, as summarized in Table 4. In the serum, the binary complex of IGF-IGFBP-3 can bind ALS, to form high molecular mass heterotrimeric complexes (195). These complexes are thought to prevent the efflux of the IGFs from the vasculature and hence play a crucial role in regulating IGF bioavailability to target tissues. In pregnancy serum, IGFBP-3 appears to undergo limited proteolysis but still circulates in the full-sized ternary complex form (196). However, it is hypothesized that IGFs in complexes containing proteolyzed IGFBP-3 are more readily bioavailable, and it was suggested recently that ADAM 12, a disintegrin metalloprotease, may be involved in this process (197). Residues 228–232 of IGFBP-3 play an important role in ALS binding (24); these residues are part of the 18-residue basic domain implicated in cell binding by IGFBP-3 described above (Table 3). It may be speculated that competition between the endothelial cell surface and ALS for the same residues on IGFBP-3 is involved in the dissociation of the ternary complex and the release of IGFs.
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. Binding of heparin and certain other glycosaminoglycans, as well as cell surface proteoglycans, to this heparin-binding motif of IGFBP-3 has been demonstrated (29, 199, 200). Recently, it was found that both IGFBP-3 and IGF-I-IGFBP-3 complexes bind fibrinogen, fibrin (201), and plasminogen (202) with high affinity via the heparin-binding domain (HBD) (Table 3). It was also shown that activation of plasminogen to plasmin, a specific protease for IGFBP-3, is not inhibited by IGFBP-3 binding (202). In addition, fibronectin binding to IGFBP-3 and IGF-I-IGFBP-3 complexes has also been recently demonstrated (203). Because all of these proteins are involved in the process of wound healing, these studies suggest a role for IGFBP-3 in concentrating IGF-I at wound sites, and, conceivably, after proteolysis of IGFBP-3 by plasmin, IGF-I is released to exert its mitogenic effects. IGFBP-3 has also been shown to bind to human serum transferrin, the effect depending on the degree of iron saturation (28). In an independent study, transferrin itself was shown to bind IGFs, although with an affinity 200-fold lower than the IGF-IGFBP-3 affinity (204). Interestingly, the affinity of IGF-II for IGFBP-3 was increased 5-fold in the presence of transferrin. Transferrin, a key component of iron transport and metabolism, is involved in various aspects of cell-cell interactions and cell viability (205) and reported to regulate programmed cell death (206). IGFBP-3 induced cell proliferation in bladder smooth muscle cells, and apoptosis in prostate cancer cells was blocked by transferrin cotreatment (28). However, it is not clear whether these effects were due to the modulation of IGFBP-3 or transferrin (or both) interacting with their respective receptors, or whether IGF binding by either protein was involved. The related iron-binding protein, lactoferrin, has also been described as binding IGFBP-3, competing with IGF-binding, and affecting IGFBP-3 nuclear entry in mammary cells (207). Because IGFs may modulate the cellular interactions between IGFBP-3 and transferrin or lactoferrin, or may even be central to their effects, these studies do not necessarily represent IGF-independent actions of IGFBP-3. They do, however, raise the possibility of cellular entry of IGFBP-3 through transferrin receptors.
The first description of IGF-independent effects of IGFBP-5 was reported in 1992 when a 23-kDa IGFBP-5 fragment was shown to stimulate normal osteoblast mitogenesis in the absence of IGF-I (116). Using the recombinant amino-terminal fragment IGFBP-5[1–169], Andress et al. (208) confirmed the initial study and suggested that the IGFBP-5 fragment-stimulated mitogenesis may be mediated by low-affinity binding of the fragment to the osteoblast surface. This is further supported by reports that IGFBP-5 binds to cell surfaces via its basic carboxyl-terminal region (residues 201–218; Refs. 29 and 209), shown in Table 3. In contrast, IGFBP-5[1–169] did not display intrinsic bioactivity in mesangial cells but inhibited IGF-I-stimulated migration (210). It is currently unexplained how IGFBP-5[1–169], shown to have decreased affinity for IGF-I (208), could be a more potent inhibitor of IGF-I than intact IGFBP-5.
Analogous to IGFBP-3, the HBD region of IGFBP-5 has been implicated as the binding region for several different molecules including ALS (25, 26), heparin, and various glycosaminoglycans (211, 212), IGF-I (21, 213), plasminogen activator-1 (27), osteopontin (108), thrombospondin (108), hydroxyapatite (214), and importin ß (30) as well as to the ECM (Refs. 29 and 209 and Table 4). At present, it is not entirely clear if the same specific residues within the 201–218 region of IGFBP-5 (Table 3) are required for interacting with this diverse group of molecules. This issue is addressed in more detail in a recent review of IGFBP mutagenesis studies (7).
As described above, IGFBP-5 binds to ECM (215) and hydroxyapatite (214), indicating that it may accumulate in bone and sequester IGFs to bone. IGFs are important regulators of bone formation because IGF-I knockout mice show severe impairment of bone growth (216). Administration of recombinant human IGFBP-5 to mice increased bone formation parameters and decreased bone resorption parameters. The increase in bone formation was not mediated by increases in circulating levels of IGF-I, providing indirect evidence of an IGF-independent effect (217). This finding was further extended by a similar study in IGF-I knockout mice. Treatment of osteoblast cells, derived from IGF-I knockout mice, with recombinant human IGFBP-5 significantly increased proliferation and alkaline phosphatase activity, a marker of osteoblast differentiation. When injected in vivo into IGF-I knockout mice, recombinant human IGFBP-5 increased local levels of alkaline phosphatase activity and osteocalcin, markers of bone formation, whereas an equimolar administration of IGF-I did not have a significant effect (112). A second study using ovariectomized mice also showed that administration of recombinant human IGFBP-5 stimulated osteoblast activity and bone accretion in the femur and spine (218). Although the mechanism involved in this IGF-independent effect of IGFBP-5 is yet to be established, IGFBP-5 has been shown to stimulate the binding of GH to GH receptors, resulting in the potentiation of GH-stimulated mitogenesis in rat osteoblasts (219), an intriguing result if confirmed.
The concept of IGF-independent effects by IGFBPs on cellular growth has gained wide acceptance in recent years, yet the mechanisms underlying these activities are still poorly understood. Although many questions remain unanswered regarding specific receptors and intracellular signaling (Sections V and VI), the complexity of having several IGFBPs expressed in the same cell system, each possibly existing in different isoforms due to posttranslational modifications, and potentially having opposing effects that may be IGF dependent or independent, adds to the challenge faced by researchers. Recent studies using overexpression systems have shown such complexity of IGFBP regulation and the necessity for caution in interpreting the data. Transfection of IGFBP-2 into an epidermoid carcinoma cell line, which normally does not secrete IGFBP-2, resulted in increased tumor growth. However, concomitant with the expression of IGFBP-2, there was a decrease in IGFBP-1 expression and an increase in IGFBP-3 proteolysis (220). When IGFBP-4 was transfected into prostate carcinoma cells, the delayed onset of tumorigenesis was accompanied by a decrease in IGFBP-2 expression (221). However, when an antisense IGFBP-4 construct was transfected into the same cell line, tumor growth was also decreased but in this instance, this was accompanied by an increase in IGFBP-3 and IGFBP-6 expression (222). Thus, it may well be that changes in cell activity attributed to a single IGFBP are, in reality, the result of alterations in several proteins.
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IV. Adhesion and Migration |
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TopAbstractI. Introductory OverviewII. Modulation of IGF...III. IGF- and IGFRI-Independent...IV. Adhesion and MigrationV. Cell-Binding Sites and...VI. Interactions of IGFBP-3...VII. Cell Cycle, Apoptosis,...VIII. Implications for Animal...IX. Implications for Human...X. Concluding CommentReferences |
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). Integrins function as cell adhesion receptors, transducing extracellular signals both through phosphorylation cascades and through direct connection with cytoskeletal elements (228).
Jones et al. (22) first reported the increased migration of Chinese hamster ovary (CHO) cells transfected to express human IGFBP-1. Cells expressing a mutated form of IGFBP-1 containing WGD in place of the 221RGD motif failed to show increased migration, and the stimulatory effect of wild-type IGFBP-I could be blocked by the addition of a synthetic peptide containing the RGD sequence. The interacting cell surface protein, isolated by affinity chromatography on immobilized IGFBP-1, was identified as 5ß1 integrin, the fibronectin receptor (22). Despite its RGD motif, IGFBP-2 was unable to stimulate smooth muscle cell migration under conditions where IGFBP-1 was stimulatory (229). However, both IGFBP-1 and IGFBP-2 were found to inhibit IGF-stimulated migration of smooth muscle cells, in contrast to the stimulatory effect of IGFBP-1 seen in the absence of IGFs.
IGF-independent actions of IGFBP-1 mediated by integrins have been demonstrated in several systems, including the stimulation of healing in a dermal wound model (144) and the stimulation of cell detachment and apoptosis in breast cancer cells (58). Although focal adhesion kinase (FAK) has been implicated in the IGFBP-1-induced changes in cellular adhesion and migration, the mechanism is unclear, with both dephosphorylation (58) and increased phosphorylation (230) of FAK reported as a consequence of IGFBP-1 action. The most extensively studied system for IGFBP-1 signaling through 5ß1 integrin is that of human trophoblast cell migration. Gleeson et al. (230) reported that IGFBP-1 stimulated the migration of extravillous trophoblast cells, the effect again being dependent on RGD interaction with 5ß1 integrin. In vivo, the IGFBP-1 is assumed to come from the decidua, an abundant source of this protein (231). In contrast, decidua-derived IGFBP-1 has been shown by others to prevent cytotrophoblast attachment to fibronectin and was inhibitory to cytotrophoblast invasiveness (137). As noted earlier, trophoblast-derived IGF-II, by inhibiting decidual IGFBP-1 production, has been proposed to overcome the inhibitory effect, thus allowing the trophoblast to invade (136).
There is considerably less evidence that the RGD motif in IGFBP-2 can initiate IGF-independent signaling. Indeed, only preliminary data in published abstracts currently support this hypothesis. Mutation to Arg-Gly-Glu (RGE) decreased IGFBP-2 cell association in ovine choroid plexus cells (232); in a more recent report, IGFBP-2 was shown to cell associate through its RGD to 5ß1 integrin (233), similarly to IGFBP-1. In contrast, IGFBP-2 with the RGE mutation was identical with the wild-type protein in cell association and growth inhibition when expressed in a transgenic mouse model (234). In two adhesive tumor cell lines (Ewing sarcoma A673 and Hs578T breast cancer) IGFBP-2 was reported to induce FAK dephosphorylation and affect cell adhesion and migration, suggesting that its cell interaction is functional (233). Overall, the lack of published data on an IGFBP-2-integrin interaction, together with cell migration studies where IGFBP-2 does not mimic IGFBP-1 (229), makes this an area where further investigation is needed.
The effect of IGFBP-5 on the migration of glomerular mesangial cells has been studied by Abrass et al. (210). Although IGFBP-5 was inhibitory to IGF-I-stimulated migration, it was stimulatory when added alone. At high concentration, the 18-residue carboxyl-terminal fragment IGFBP-5[201–218] also showed potent stimulatory activity. This peptide represents the basic motif (Table 3) known to be involved in ALS binding, cell and matrix interaction, and nuclear translocation of IGFBP-3 and IGFBP-5. IGF-I-induced cell migration involves Vß3 integrin and is blocked by the disintegrin kistrin (235); however, migration induced by IGFBP-5[201–218] was not inhibited by kistrin (210), indicating a different mechanism. IGFBP-5 also induced marked morphological changes in mesangial cells, with multiple filopodia developing (236). The small GTPase Cdc42, known to be involved in filopodia formation, was shown to be activated by the IGFBP-5 peptide, and early addition of staurosporine inhibited the IGFBP-5 effect, consistent with signaling through a serine-threonine protein kinase. The possible role of a putative IGFBP-5 receptor in this function is discussed in Section V.
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V. Cell-Binding Sites and Putative Receptors |
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TopAbstractI. Introductory OverviewII. Modulation of IGF...III. IGF- and IGFRI-Independent...IV. Adhesion and MigrationV. Cell-Binding Sites and...VI. Interactions of IGFBP-3...VII. Cell Cycle, Apoptosis,...VIII. Implications for Animal...IX. Implications for Human...X. Concluding CommentReferences |
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5ß1 integrin, the fibronectin receptor, was discussed as a cell surface protein complex of known structure that binds IGFBP-1 through an identified domain, initiating a definable response of cellular events. Despite intensive investigation and the reporting of numerous putative receptor proteins, there are, to date, no other examples in the literature of a fully characterized cell surface protein that would satisfy the usual criteria for a signaling receptor for any IGFBP, namely reversible and saturable ligand binding, and the initiation of a definable intracellular signaling pathway. Although the characterization of cell surface IGFBP receptors has proved elusive, cell binding of IGFBPs has been reported in many systems, resulting in the partial description of several interacting proteins. In some of the early literature, before the identification of the six IGFBPs and the establishment of specific analytical reagents, it was unclear which IGFBP was being studied. Clemmons et al. (239) used affinity labeling to demonstrate the association of a 35- to 40-kDa IGFBP with fibroblast monolayers. Although no cell binding site was identified, the interaction was proposed to modify cell responsiveness to IGFs (237). By using [Q3A4Y15L16]IGF-I, which has greatly reduced affinity for IGFBPs but near-normal affinity for the IGFRI, it was estimated that surface-bound IGFBPs could contribute up to 80% of the total IGF-I binding sites in human glioblastoma cells and fetal fibroblasts (238). Data from the laboratories of Clemmons (239) and Conover (240) further showed, using IGF-I analogs, that fibroblasts exposed to IGF-I released an unidentified IGFBP of approximately 40 kDa from the cell surface, independently of IGFRI interaction. Martin et al. (241) extended these observations using specific immunological detection of IGFBP-3 to show IGF-I-dependent release of IGFBP-3 from the fibroblast cell surface, with reciprocal appearance in the culture medium. Receptor-inactive IGF-I analogs were fully active in this process. It thus appears that, whereas IGFs can bind to cell surface IGFBP-3 and other IGFBPs, the IGF-IGFBP interaction may, paradoxically, also act to release IGFBP-3 from the cell.
A. Glycosaminoglycan-binding domains on IGFBPs
In addition to the integrin system discussed earlier for IGFBP-1 and IGFBP-2, other mechanisms of IGFBP-2 interaction with cells have been reported. In the rat olfactory bulb, IGFBP-2 has been shown to interact with cell surface proteoglycan binding sites. In vitro, IGFBP-2 bound to chondroitin-4 and -6-sulfate, keratan sulfate, and the proteoglycan aggrecan (242). Arai et al. (243) also demonstrated IGFBP-2 interaction with glycosaminoglycans, but only if IGF-I or IGF-II was present. IGFs were similarly required for IGFBP-2 interaction with cell matrix in fibroblast cultures. Residues 180KKLR in the central domain of human IGFBP-2 represent a consensus short HBD of the form B-B-X-B, where B is a basic residue (198), although the role of these residues in IGFBP-2 cell binding has not been demonstrated. The consequence of IGFBP-2 binding for cell function is unknown, but it may serve to concentrate IGFs near type I IGF receptors as it can increase IGF-stimulated proliferation in some cell types (170, 220), although paradoxically it is growth inhibitory in other cells (146), and when overexpressed in vivo (244).
IGFBP-4 has no consensus HBD, whereas a putative long HBD of the form B-B-B-X-X-B (198) is found in human IGFBP-6 residues 173RKRQCR (Table 3). IGFBP-4 is not known to associate with cell surface-binding sites, but a single report describes nonglycosylated IGFBP-6 binding to heparan sulfate, chondroitin sulfate, and other glycosaminoglycans (66), as further discussed below. In contrast, there are numerous reports of the cell association of IGFBP-3 and IGFBP-5, and evidence for growth-regulatory signaling by these proteins. The observation that heparin, like IGF-I, was effective in releasing IGFBP-3 from the fibroblast cell surface into the culture medium led to the suggestion that its binding sites might involve proteoglycans (241). A variety of sulfated glycosaminoglycans in addition to heparin are partially effective in competing with radioiodinated IGFBP-3 or IGFBP-5 for binding to endothelial cell monolayers, as is an 18-residue basic peptide corresponding to IGFBP-3 [215–232] (29). This sequence, like the corresponding motif in IGFBP-5 (and IGFBP-6), contains a consensus long HBD at residues 220KKKQCR (Table 3). However, removal of sulfated proteoglycans by growing various cell types in 5 mM sodium chlorate, or treating them with a mixture of heparinases, was unable to prevent IGFBP-3 or IGFBP-5 binding (29, 245), leading to the conclusion that the inhibitory effect of heparin probably resulted from a direct interaction between heparin and the binding protein. The interaction of heparin with these basic residues presumably also accounts for its ability to block IGFBP-3 binding to ALS (246).
Site-directed mutagenesis of IGFBP-3 residues 228KGRKR to the corresponding IGFBP-1 residues MDGEA (Table 3) substantially abolished binding to Chinese hamster ovary cells (24, 247) and other cell types (our unpublished data). This region is adjacent to, but not overlapping, the carboxyl-terminal consensus HBD, and the mutant protein retained considerable affinity for a heparin-agarose column (24), despite the loss of cell binding. A contribution to the residual heparin binding may come from a secondary short consensus HBD at residues 149KKGH, which has been shown to be functional (199), and provides further evidence against glycosaminoglycans as IGFBP-3 cell surface-binding sites.
Although IGFBP-3[1–184], representing the amino-terminal and central domains, shows no binding to CHO cells, in contrast to the full-length protein (24), a central domain cell-binding site on IGFBP-3 has been proposed on the basis of competition studies between nonglycosylated IGFBP-3 and the central domain peptides IGFBP-3[88–148] and IGFBP-3[88–183] for binding to Hs578T human breast cancer cells (38). This result, contrary to the observation of Firth et al. (24), is possibly explained by cell-specific differences in IGFBP-3 binding sites. It remains unclear whether a central domain cell-binding site for IGFBP-3 would involve the HBD residues 149KKGH, because a peptide containing these residues had extremely low activity in competing for bound IGFBP-3 (38). A central domain heparin binding site, involving consensus short HBD residues in this region, has also been described for IGFBP-5 (37). This appears to be masked by the carboxyl-terminal domain in the intact protein, but is active in a carboxyl-terminally truncated form of IGFBP-5. Whether it has a role in cell association of IGFBP-5 is unknown.
B. The role of posttranslational modifications in cell surface association
Of the four IGFBPs with well documented cell and matrix association, only IGFBP-3 has consensus sites for N-glycosylation (248), whereas IGFBP-5 is reported to be O-glycosylated (249), and the low level of carbohydrate on IGFBP-1 is presumably also O-linked (Ref. 250 and Table 5). In contrast to IGFBP-1 and IGFBP-5, the primary structures of IGFBP-2 and IGFBP-3 predict no O-glycosylation sites (Ref. 31 and Table 5). In IGFBP-3, carbohydrate increases the core protein size of 29 kDa to forms estimated to be 40–43 kDa. Of the three potential glycosylation sites at Asn89, Asn109, and Asn172, the first two are always used, carrying an estimated 4 kDa and 4.5 kDa of carbohydrate, respectively, whereas the third site alternatively contains either undetectable or about 5 kDa of carbohydrate, accounting for the characteristic doublet form of the protein (31). Comparison of Escherichia coli-derived and CHO cell-derived IGFBP-3 indicates that glycosylation has no significant effect on the binding of IGF-I (251) or ALS (31).
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). This suggests that the carbohydrate present in natural IGFBP-3 might mask potential cell-association sites and raises the question whether cell binding studied using E. coli IGFBP-3 reflects the binding of the native protein.
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). Binding to glycosaminoglycans is greatly inhibited by O-glycosylation, and the nonglycosylated protein, which is not known to occur in nature, has been shown to bind to PC12 cell membranes, whereas the natural, O-glycosylated form shows no binding (66). This suggests that, as seen to a smaller degree in IGFBP-3, cell binding sites of IGFBP-6 are permanently masked by carbohydrate.
IGFBP-1, IGFBP-3, and IGFBP-5 are all secreted as phosphoproteins (Ref. 33 and Table 5). IGFBP-1 phosphorylation increases IGF-I affinity 6-fold for the human protein (67) but, as noted in Section II, has no effect on the rat protein (130). Phosphorylation differences in IGFBP-1 appear to account for two distinct species isolated from human amniotic fluid, the more weakly anionic of which was found to enhance IGF-I-stimulated DNA synthesis (131), whereas the more strongly anionic was inhibitory. Only the less anionic form, presumably in a lower phosphorylation state, bound to smooth muscle cells (131), suggesting that phosphorylation may be inhibitory to cell surface interaction of IGFBP-1. Although, as described earlier, IGFBP-1 can interact with cells through 5ß1 integrin, it is not clear how this interaction is modulated by phosphorylation.
There is also evidence that phosphorylation inhibits IGFBP-3 cell binding (Table 6). Human skin fibroblasts secrete IGFBP-3 into the culture medium as a phosphoprotein, but release of surface-bound IGFBP-3 from fibroblasts using an IGFRI-inactive IGF-I analog was found to increase total IGFBP-3 but not phospho-IGFBP-3 in the culture medium, implying that surface-bound IGFBP-3 was nonphosphorylated (253). More recently, phosphorylation of IGFBP-3 in vitro by protein kinase CK2 has been shown by direct binding studies to be inhibitory to cell surface association (68).
The functional implications of the effects of phosphorylation and glycosylation on cell binding, especially in the case of IGFBP-3, are uncertain. Clearly, if cell signaling by IGFBP-3 can be initiated by cell surface binding, this process may be modulated by phosphorylation, possibly in a dynamic way. Modification of signaling by changes in glycosylation is less likely to be biologically relevant, at least as a form of acute regulation. A key limitation in interpreting these studies is the unknown relationship between general cell surface binding of IGFBPs, e.g., the heparin-displaceable binding of IGFBP-3 by fibroblasts (241), and binding to true functional receptors. For example, if phosphorylated IGFBP-3 shows decreased cell surface binding (Table 6), does this imply decreased signaling through a cell surface receptor? In the case of IGFBP-3 and IGFBP-5 it would be premature to conclude that the extensively studied binding involving carboxyl-terminal basic residues, which appears to account for the majority of cell-binding sites (29), represents receptor binding. As discussed elsewhere, IGFBP-3 mutated in these residues, and truncated IGFBP-5 lacking these residues, both elicit biological effects. Therefore it may be surmised that functional, low-abundance receptors do not necessarily make a major contribution to overall cell binding of IGFBPs.
C. Other putative IGFBP receptors
A number of other cell surface proteins have been designated in the literature as IGFBP receptors, with varying degrees of experimental support. Iodinated nonglycosylated IGFBP-3 exhibited cation-stimulated binding to Hs578T breast cancer cells, displaceable by unlabeled ligand with an EC50 of approximately 10 nM (254). Subsequent kinetic studies yielded affinity constants of 8 nM for IGFBP-3 binding to both Hs578T and MCF-7 breast cancer cells (38). Iodo-IGFBP-3 binding to other cell types has revealed a higher apparent affinity, with a Kd estimated as 0.22 n
