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Fishberg Research Center in Neurobiology (S.C.S.), Departments of Neurology (S.C.S.), Physiology and Biophysics (H.W.), and Pharmacology (H. W.), Mount Sinai School of Medicine, New York, New York 10029; and Medical Research Council Regulatory Peptides Unit (R.P.M.), Departments of Chemical Pathology (R.P.M.) and Medicine (R.P.M.), University of Cape Town Medical School, Observatory 7925, South Africa
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Abstract |
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 Top Abstract I. IntroductionII. Amino Acid Sequences...III. Structure-Activity...IV. Structure and Conformation...V. Functional Structure of...VI. ConclusionsReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. Amino Acid Sequences...III. Structure-Activity...IV. Structure and Conformation...V. Functional Structure of...VI. ConclusionsReferences |
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GONADOTROPIN-RELEASING hormone plays a central role in the biology of reproduction (1), and synthetic GnRH analogs have proven valuable in the treatment of a wide variety of endocrinological and nonendocrinological disorders (2, 3, 4, 5, 6, 7, 8, 9, 10). The decapeptide GnRH is generated in neurons of the medial basal hypothalamus through enzymatic processing of a larger precursor. Released in a pulsatile manner into the portal circulation, GnRH interacts with high-affinity receptors on the gonadotropes in the anterior pituitary, leading to the biosynthesis and release of the gonadotropins LH and FSH. The pulse-timing and concentration levels of GnRH are critical for the maintainence of gonadal steroidogenesis and for normal reproductive function.
Chronic, high concentration agonist stimulation of the pituitary GnRH receptors induce regulatory changes that lead to gonadal hypoactivity. This paradoxical suppression of gonadal function in response to pharmacological levels of agonist is the basis for the utility of GnRH analogs in the treatment of gonadal-steroid sensitive tumors, such as prostate cancer.
The GnRH receptor has been an unabatedly intense and productive subject of research for several decades because of its dual significance both for understanding reproductive biology and for developing medical therapies. The landmark elucidation of the primary sequence of GnRH by the laboratories of Schally (11) and Guillemin (12) inaugurated the field. Previous reviews have documented the subsequent evolution of research into GnRH and its receptors over the intervening decades. The complex regulation of the mammalian receptor, which is critical both for normal reproduction and for therapeutic response to analogs, has been studied in many species (reviewed in Refs. 13–15). The various signal transduction pathways used by the receptor have been investigated (reviewed in Refs. 15–20). Several thousand GnRH analogs have been synthesized and characterized (reviewed in Ref.21) and the amino acid and cDNA sequences for GnRHs have been determined from many vertebrates (reviewed in Refs. 22 and 23).
GnRH receptor clones have recently been isolated. These clones provide the tools and impetus for recent progress in studies of the structure-activity of the receptor-ligand complex (24). The availability of the primary amino acid sequences and cDNAs has made possible the study of the molecular mechanism of action of GnRH and its analogs through receptor mutagenesis and computational modeling of the receptor and peptide (e.g. see Refs. 51 and 174). Thus, the structure-activity of GnRH and its analogs can now begin to be placed in the context of the receptor itself. The present review aims to summarize such work on the structure-activity relations and computational modeling of GnRH analogs and of the receptor. Recent developments will be emphasized, with earlier studies presented in illustrative rather than comprehensive fashion.
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II. Amino Acid Sequences of GnRH Receptors |
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TopAbstractI. IntroductionII. Amino Acid Sequences...III. Structure-Activity...IV. Structure and Conformation...V. Functional Structure of...VI. ConclusionsReferences |
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T3–1 cells (25), as a source of RNA.
Efficient heterologous expression of the mammalian GnRH receptor in
oocytes using T3–1 RNA suggested that this cell line would be a
suitable source for cloning the receptor (26). The first clone was
isolated using a PCR-based homology cloning strategy (27). Mouse GnRH
receptor clones were also identified using Xenopus oocyte
(28) and mammalian cell line (29) expression cloning. After the
elucidation of the mouse receptor sequence, the homologous pituitary
cDNAs were identified in five additional mammalian species and one
nonmammalian vertebrate: human (30, 31), rat (29, 32, 33), sheep (34, 35), cow (36), pig (37), and catfish (38). An alignment of the cloned
GnRH receptor sequences is shown in Fig. 1
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predicted amino acid sequence for the GnRH receptors is more than 85%
conserved overall in the six mammalian species reported and is nearly
identical within the putative transmembrane domains.
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or Ref. 39.) The catfish receptor is 370 amino acids
in length and is notable for having a 49-amino acid cytoplasmic carboxy
terminus domain not present in the mammalian receptors (38). GnRH
receptor cDNAs were also isolated from human breast and ovarian tumors
(40) and from rat gonads (41). The sequences obtained from these
extrapituitary sources were identical to the pituitary GnRH receptor
cDNAs of the corresponding species.
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). However, the three classes do not share any
discernible sequence homology. Included within the rhodopsin-like
family of G protein-coupled receptors are the opsins, G protein-coupled
neurotransmitter receptors (adrenergic, serotonergic, dopaminergic,
muscarinic acetylcholine, etc.), glycoprotein hormone receptors (FSH,
LH/CG, and TSH), and a variety of peptide receptors (49, 50).
A given receptor can be identified as belonging to the rhodopsin-like
GPCR family by the presence of certain amino acid motifs conserved
within the transmembrane helix domains (TMD), a pattern of conservation
that has also facilitated the cloning of a large number of these
receptors (see Fig. 2). Variations in the pattern of
conservation in the GnRH receptor in comparison with other
rhodopsin-like GPCRs has proven valuable in elucidating the functional
and structural roles of some of these side chains (51) (see Figs. 2, and 3 and below).
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T3–1 cells have demonstrated that the G protein involved in
coupling to phospholipase C in this cell line is pertussis-toxin
insensitive, being Gq and/or G11 (54, 55).
A representation of the putative topology of the human GnRH receptor
sequence is presented in Fig. 3. The receptor is
composed of a single polypeptide chain. Hydrophobicity analysis of the
receptor’s primary sequence confirms the presence of seven hydrophobic
stretches corresponding to putative transmembrane helical domains, with
an extracellular amino terminus and an intracellular carboxy terminus.
Direct structural information is available for only two heptahelical
membrane proteins. The structure of bacteriorhodopsin, which has little
if any sequence similarity with the mammalian receptor proteins (see
Ref.56), has been elucidated at 3.5 Å resolution by cryo-electron
microscopy of two-dimensional crystals (57). Using the same approach, a
9-Å projection map of bovine rhodopsin, shown in Fig. 4
(58), and a 6-Å projection map of frog rhodopsin (59) have been
obtained. The maps of rhodopsin are consistent with the presence of
seven transmembrane domains, as had been predicted from primary
sequence analysis. The transmembrane domains of all GPCRs are believed
to be -helical and arranged around a hydrophilic core in a manner
similar to the rhodopsin map (39).
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). Mutation of DRS to DRY in the mammalian
receptor was reported to cause only a small increase in agonist
affinity with no discernible change in signal transduction (60). The
other unique features of the mammalian receptors seem to have
functional significance and will be discussed below in light of recent
experimental results.
C. Covalent modifications
1. Glycosylation. Most GPCRs have consensus glycosylation
sites, and several receptors have been found to be glycosylated at
these sites (61, 62, 63). Biochemical studies of the GnRH receptor have
suggested that it is a sialic acid residue-containing glycoprotein (64, 65). The cow, sheep, pig, and human receptor sequences contain two
potential sites for N-linked glycosylation (N-X-S/T), one in the amino
terminus and one in the first extracellular domain (see Figs. 1 and 3).
The rodent species contain an additional potential glycosylation site
in the amino terminus. The glycosylation at these sites of the mouse
GnRH receptor was investigated by site-directed mutagenesis and
photoaffinity labeling (66). Mutation of Asn1.01(4) or
Asn1.15(18) in the N-terminal domain to Gln caused a lower
apparent molecular weight in gel electrophoresis, whereas mutation of
Asn2.65(102) in the putative first extracellular loop did
not affect mobility. Whereas the ligand-binding affinities of the amino
terminus domain mutants were unchanged, these receptors were expressed
in transfected cells at a lower level than the wild type receptor.
These results suggest that only the amino-terminal domain sites are
glycosylated and that the glycosylation contributes to the level of
receptor expression, consistent with earlier studies in which sialidase
and tunicamycin were reported to decrease the level of receptor
expression but not to alter affinity (65). The mutagenesis studies do
not support an earlier suggestion, based on studying the effects of
periodate on binding, that glycosylation contributes to high affinity
binding (66). The receptor levels of glycosylation-deficient mutants
show similar decreases in both membrane preparations and in whole cell
assays (67). Thus, in contrast to the ß-adrenergic receptor, for
which glycosylation is required for proper transport of the receptor to
the cell surface (62), in the case of the GnRH receptor the observed
decrease in receptor number does not appear to represent altered
receptor transport.
The possibility that differential glycosylation contributes to the differing level of expression observed with transfection of the mouse and human receptor has been examined by introducing a second glycosylation site into the human receptor sequence, thereby recreating the pattern of sites found in the mouse. The second site was found to be glycosylated, and its presence increased the level of receptor expression (68). These studies indicate that the glycosylation of the GnRH receptor does not contribute to receptor affinity but does improve the level of receptor expression, possibly by decreasing the rate of receptor degradation.
2. Phosphorylation sites. Many intracellular serine and
threonine residues are within phosphorylation consensus sequences, and
phosphorylation could be involved in modulating receptor responsiveness
or intracellular trafficking. While desensitization of the GnRH-induced
responses in pituitary cells has been observed, it is not clear whether
this occurs at the level of the receptor (for review see Refs. 13–15).
Of note, however, a lack of rapid receptor-mediated desensitization of
the GnRH receptor has been reported in T3–1 cells and in
transfected cells (69, 70, 71).
3. Disulfide bridges. Most GPCRs contain single conserved cysteines in the first and second extracellular loops that may form a disulfide bond to stabilize the structure of the functional protein. Mutation of these conserved cysteine residues disrupts the function of rhodopsin, muscarinic, ß-adrenergic, and TRH receptors, suggesting that this disulfide bond is required for proper receptor folding (72, 73, 74, 75, 76). Experiments using site-directed mutagenesis and photoaffinity cross-linking support the presence of two extracellular disulfide bridges in the GnRH receptor. The presence of a conserved cystine bridge between C3.25(114)-C5.23(196) was demonstrated in the mouse receptor, and evidence for a second disulfide bond between C1.11(14)-C5.27(200) has been obtained for the human receptor (J. Davidson, personal communication).
D. Gene structure
The chromosomal locations of the mouse, sheep, and human genes
have been reported. The human gene was assigned to chromosome 4 by PCR
analysis of somatic hybrid cell lines (77, 78) and to the
4q13.1–4q21.1 region using cell hybrid mapping panels (79). Using
chromosomal in situ hybridization, three groups have
reported the gene localization at band 4q13.2–13.3 (78, 80, 81) and
one group at band 4q21.2 (82). Mapping the gene relative to 4q
microsatellite markers in GnRH receptor YAC clones supports the
4q13.2–13.3 assignment (80). The mouse gene has also been mapped by
linkage analysis to within 1.2 ± 1.2 centimorgans of the
chromosome 5 marker Pmv-11 (79), and the sheep gene has been localized
near the FecB locus of chromosome 6 (83).
The structures of the mouse and human GnRH receptor genes have been
investigated. In contrast to the genes of many GPCRs, which are
intronless and are believed to have arisen by retroposition (84), the
GnRH receptor contains introns within the coding region. The mouse gene
is composed of at least three exons spanning more than 22 kb (85). The
open reading frame is distributed among the three exons, which encode
amino acids 1–174, 175–247, and 248–327, respectively (see Fig. 1
for location of exon junctions). Variant transcripts of the mouse
receptor that are generated by alternative splicing and do not encode
functional receptors have been isolated from T3–1 cells (85). The
alternative transcripts found all include exon 1 but lack either exon 2
or 3. The alternative transcripts form a minority of the cDNAs isolated
from an T3–1 cell library, and the biological function of the
proteins encoded by these cDNAs is not known. The human gene is also
distributed over three exons that span 18.9 kb (77, 86). The amino acid
locations of introns 1 and 2 are homologous to their positions in the
mouse receptor gene. Intron I is located between amino acids 174–175
in the putative TMD 4 domain, and intron 2 is located between amino
acids 248–249. Exon 2 is three nucleotides longer in the human gene in
comparison with the mouse, reflecting the presence of an additional
amino acid (Lys4.77(191)) in the second extracellular loop
of the human receptor (see Fig. 1). Southern blot analysis is
consistent with the presence of a single gene in the mouse (85), rat
(85), and human (77) genomes.
Fan et al. (86) have mapped the 5'- and 3'-flanking
regions of the human receptor gene. Multiple initiation sites and
multiple polyadenylation signals are present. Five consensus TATA
sequences distributed over 669 bases are present in the 5'-flanking
region. Primer extension analysis using human brain RNA indicates the
utilization of several initiation sites. The longest extension was
confirmed by PCR and represents a transcript with 1393 bp of
5'-untranslated sequence. A putative cAMP response element is found at
-1490, a putative glucocorticoid/progesterone response element is
located at -92, and consensus binding sites for several transcription
factors are present. At the 3'-end of the gene, five polyadenylation
signals are found, distributed over 800 bp. In the largest possible
transcript, the 3'-untranslated sequence is 3.1 kb in length. Thus the
exons of the human gene identified appear to account for the largest
5-kb transcript identified on Northern blot analysis (31).
The 5'-flanking region of the mouse gene has been investigated by Albarracin and co-workers (87). In contrast to the human gene, the mouse gene appears to have a smaller 5'-untranslated segment. The major initiation site is found 62 bases upstream of the translation initiation site. Also in contrast to the human gene, the 5'-sequence of the mouse gene lacks TATA sequences. Preliminary studies on the regulation of a 1.2-kb fragment of the 5'-flanking region of the mouse gene have been reported (87).
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III. Structure-Activity Relations of GnRH Peptides |
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TopAbstractI. IntroductionII. Amino Acid Sequences...III. Structure-Activity...IV. Structure and Conformation...V. Functional Structure of...VI. ConclusionsReferences |
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Several thousand GnRH analogs have been synthesized to date, and information on their activities potentially provides a very large data base for the purpose of identifying functional residues in GnRH. However, interpretation is complicated by a number of factors:
1. Frequently, multiple substitutions have been incorporated simultaneously in single GnRH analogs, often without a systematic approach due to the large number of combinatorials involved.
2. Even the effects of single-amino acid substitutions may be difficult to interpret. A single substitution may alter affinity and agonist activity via modification of a side chain that interacts with the binding pocket and/or by altering the conformation of the peptide and thus affecting the presentation of other peptide moieties that interact with the receptor. Substitutions that have a conformational effect cannot be differentiated through structure-activity data alone from those that eliminate receptor contact sites. Any substitution may establish new contacts with the receptor and disturb the normal contacts by altering the families of conformations of the peptide. This difficulty in the interpretation of structure-activity data would be obviated by achieving a more complete understanding of the conformational effects of substitutions and by the analysis of ligand-receptor interactions in a structural context. Progress toward predicting the effects of amino acid substitutions on peptide conformation is described in the next section, whereas the development of three-dimensional models of receptor molecules is discussed in Section V.
3. While substitutions of residues that produce antagonists may remove a contact interaction, they most likely establish new compensatory contact sites, presumably with different sites in the receptor, to retain high-affinity binding.
4. For much of the available data, the activities of analogs cannot be rigorously compared because they have been tested in different assay systems (see Ref. 100 for review). The most commonly employed assays have been in vivo bioassays (e.g. inhibition of ovulation) in which activity is a composite of pharmacokinetics of absorption from the injection site, association with lipophilic compartments (e.g. fat and cell membranes), binding to plasma proteins, degradation, metabolic clearance (including renal clearance), receptor binding affinity, and efficacy. Ideally, comparative data on binding affinity and signal transduction (e.g. second messenger generation) are required. However, as these data are available for relatively few analogs, in adjudging the effects of single-amino acid substitutions we have relied extensively on in vivo data, particularly from early studies. It has been necessary, therefore, to consider possible pharmacodynamic contributions to the activity of the analogs when making inferences about receptor binding and receptor activation based on data obtained from in vivo bioassays (see below).
Even the direct measure of analog affinity in receptor-binding assays may yield misleading results. These assays are usually conducted on membrane preparations (which expose all receptors), as opposed to whole cells, and employ conditions (buffers, pH, temperature, etc.) optimized to give maximal binding. These nonphysiological conditions may affect the binding of a substituted analog or mutated receptor differently than physiological conditions. The radiolabeled GnRH analogs used in binding assays mostly rely on incorporating 125I into Tyr5 of GnRH analogs that have a D-amino acid in position 6. This incorporation of the large electron-withdrawing iodine atom considerably alters the properties of the ligand. Since a large, bulky side chain is allowable when a D-amino acid substitutes for Gly6 in the superactive analogs (see below), we have attempted to overcome this problem by substituting the Tyr5 with His (as in the active chicken GnRH II) and incorporating D-Tyr in position 6 (101). This analog has a higher affinity and increased total binding.
A feature of ligand-receptor complexing is that receptor interaction (affinity) and capacity of the bound ligand to activate the receptor (efficacy) are separable phenomena. Thus particular residues of GnRH are more critical for agonist activity (e.g. His2, Trp3), and others are critical for ligand binding (e.g. Pro9). Various models have been proposed to explain the differing contributions of ligand substituents to affinity and efficacy (102). In the "conformational induction" model, agonists bind to an inactive receptor state and induce the receptor to assume an altered active state that leads to coupling with G proteins. In the "conformational selection" model, the receptor spontaneously fluctuates between inactive and active conformers, and agonists have a higher affinity for the active state whereas antagonists (or inverse agonists) have a higher affinity for the inactive state. Consequently, agonist binding causes the concentration of active receptor to increase by mass action, and inverse agonists have the opposite effect. The separate effects of ligand substitutions on affinity and efficacy can be interpreted within either model. In the case of the induction model, some receptor interaction sites are critical for ligand docking, whereas others are critical for inducing a change in the receptor. In the selection model, some receptor contact sites are accessible in both active and inactive states and thus contribute to affinity, whereas other contact sites are accessible or properly positioned for agonist complexing only when the receptor assumes an activated state. In the following sections, the term "activity" will be used to refer to data derived from functional assays, usually LH release. When radioligand binding data are available, the term "affinity" will be employed.
With the preceding caveats in mind, it is nevertheless possible and useful to review the extant data. When evaluated in concert with studies of the receptor-binding pocket and of analog conformation (reviewed in subsequent sections), the data on the structure-activity of analogs provide insight toward elucidating the interactions in the ligand-receptor complex. For this reason, we present here some indications of the roles of the individual constituent amino acids of GnRH in receptor binding and activation.
B. Comparative structures and activities of vertebrate GnRHs
Identification of the GnRHs present in more than 70 species (for
review see Refs. 22, 23, 92–99, and 103–105) has demonstrated that
two or more forms of GnRH are present in most vertebrate species and in
a protochordate, the tunicate (22, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116) (Fig. 5).
One form is represented by mammalian GnRH and its nonmammalian
counterparts, which have a predominant function as hypophysiotropic
peptides regulating the pituitary. The second form of GnRH, first
identified in chicken brain
(His5Trp7Tyr8GnRH), is the most
ubiquitous form in vertebrates, and most species have this form
along with one or two other GnRHs. As
His5Trp7Tyr8GnRH is present in
fish, amphibians, reptiles, birds, and mammals, referring to this
peptide as "chicken GnRH II" is confusing. We propose that it be
designated "GnRH II." The original mammalian GnRH is then "GnRH
I." Specific chemical identification may be accomplished by
designating the variable amino acids 5–8. Thus chicken GnRH I, salmon
GnRH, and catfish GnRH would be YGLQ GnRH, YGWL GnRH, and HGLN GnRH,
respectively.
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All natural GnRH peptides isolated to date are highly conserved with
respect to their length, to the NH2-terminal domain
(residues pGlu-His-Trp-Ser), and to the COOH-terminal domain
(Pro-Gly.NH2), suggesting that these domains are
functionally essential. However, residue conservation does not
invariably imply functional signficance. For example, Ser4
is highly conserved and yet can be substituted with the retention of
high activity at the mammalian receptor (see below). Among
vertebrate GnRHs, position 8 is most variable, and positions 5 and 7
are highly variable (Fig. 5). Position 6 is invariably Gly in the
higher vertebrates but varies considerably in the lamprey and tunicate
GnRHs. No variation in GnRH sequence is found among mammalian GnRHs.
The comparative activities of the GnRH variants in vertebrates provide
insight into structure-activity relations. In mammals, mammalian GnRH
is highly active at low doses while the other vertebrate GnRHs, with
the exception of GnRH II, have poor activity (binding affinity or
EC50 and/or maximal gonadotropin release) (107, 110, 119, 120, 121) (Fig. 6). Since the single residue that
distinguishes mammalian GnRH from all of the other vertebrate GnRH
structural variants is Arg8 (Fig. 5), this residue was
identified as being critical for high-affinity binding to the mammalian
receptor. However, the substantial activity of GnRH II (20–30%)
(120, 121, 122, 123) suggests that the loss of activity when substituting a
neutral amino acid for Arg8 can be overcome by the
simultaneous substitution of His in position 5 and Trp in position 7.
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Differences in the pharmacology of the GnRH receptors in vertebrate species are illustrated in studies with certain mammalian GnRH antagonist analogs that are pure antagonists in mammals but exhibit agonist activity in chicken (126) and goldfish (127) gonadotropes. Intriguingly, some antagonists stimulate gonadotropin secretion while others release GH in the goldfish (127). It is apparent, therefore, that the structural requirements of GnRH receptors for activation by ligands are variable among vertebrates. Differences, albeit more subtle, between mammalian GnRH receptors in agonist and antagonist binding have also been noted (30, 31, 35, 51, 128).
The studies on GnRH chimeras have also revealed effects of amino acid
substitutions on agonist efficacy at the mammalian GnRH receptor by
comparing relative potencies of the chimeric analogs in stimulating LH
release from sheep pituitary cells and in binding to sheep pituitary
GnRH receptors (Fig. 7) (121). These data showed that
Arg8 substitution by neutral amino acids in mammalian GnRH
resulted in a low binding potency but relatively higher LH-releasing
potency (ratios of 10–150). In contrast, Tyr5 substitution
by His enhanced binding potency but reduced LH-releasing potency
(ratios of 0.14–0.2). Thus, once bound, analogs with a neutral amino
acid in position 8 are more efficient at activating the receptor. On
the other hand, His5 enhances binding, but reduces
efficacy. The role of specific amino acids in affinity and activation
of the mammalian receptor will be addressed in the subsequent section.
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The conservation of the NH2- and COOH-terminal sequences of GnRH through vertebrate evolution and the conclusion that these domains are critically important for receptor binding and activation are substantiated by extensive structure-activity data. Indeed, cognizance of the evolutionary constraints on acceptable structures could have obviated much of the endeavor to produce agonist and antagonist analogs through empirical approaches. It is now clear that both the NH2- and COOH-terminal domains are involved in receptor binding while the NH2-terminal domain plays the major role in receptor activation (see reviews in Refs. 10 and 21). Although the lack of conservation of amino acids 5–8 suggests that these residues are not critical for ligand activity, this is not entirely so, as Arg8 is important for high-affinity binding to the mammalian receptor (see below). This central domain is thus a determinant of receptor selectivity. The role of individual amino acids will be considered within these designated NH2-terminal, central, and COOH-terminal domains.
1. The NH2-terminal domain (pGlu-His-Trp-Ser).
pGlu1. The essential requirement of pGlu was first
noted with the loss of activity of native purified GnRH when treated
with pyroglutamyl aminopeptidase and confirmed by a series of
substitutions in this position. While Leu1,
Gly1, Pro1, Gln1 and
(O=)Thr1-substituted analogs were essentially inactive in
in vivo bioassays (129, 130, 131, 132), several acylated
Gly1 analogs (formyl, acetyl, and propionyl) had low but
significant activity (1%), indicating that the -CO-NHCHCO- group is
the minimal structure required for activity (131). The cyclic (O=)
Ser1 structure (130), which resembles the pGlu structure,
and D-pGlu (133) both had about 5% activity, suggesting that a change
in conformation in the NH2 terminus is not completely
detrimental (134). Although a specific role for pGlu in binding and/or
activation of the receptor has not been revealed by these early
studies, the subsequent universal substitution of pGlu in GnRH
antagonists (see reviews in Refs. 10, 21, and 88–91) identifies this
residue as important in receptor activation.
His2.
His2 of GnRH is a good candidate
for interaction with the receptor (134). The imidazole ring of
histidine has a number of features appropriate for participation in
enzyme actions and also in hormone interactions with receptors. These
include its aromatic character, hydrogen-bonding capacity, and
acid-base properties. The very low activities of Ser-, Ile-, Leu-,
Gln-, Gly-, Thr-, Ala-, Lys-, and Arg-substituted analogs (129, 130, 135) and substantial activity of Phe2 GnRH (1–7%) (130, 135, 136), 3-Me His2 (1%) (129), and Trp2
(40%) (137) demonstrate the need for aromaticity and possibly the
imidazole moiety. The lack of activity of Gln2 GnRH
indicates that the role of His2 does not involve
-position hydrogen-bonding because, were that the case, Gln would be
a suitable substitute. The high activity of Trp2 GnRH has
been confirmed by examining binding to the human GnRH receptor
expressed in COS-1 cells (138). On the other hand, the acid-base and
hydrogen-donor and hydrogen-acceptor capability of His can be modified
with reasonable retention of activity, as demonstrated by
I-Nim-His2 (6%) (139) and
ß-pyrazolyl-3-alanine (19%) (140), although the latter is a much
weaker base. A major breakthrough in understanding the function of
His2 evolved from the observations that the
Gly2 was a partial agonist and the des-His2
analog was an antagonist (141). Bulky hydrophobics (e.g.
D-4-Cl-Phe) have subsequently become the hallmark of substitutions of
His2 in GnRH antagonists (for review see Refs. 10, 21, and
88–91). It is therefore likely that His2 plays a role in
GnRH interaction with the receptor, which leads to signal propagation
and G protein activation. The nature of this interaction appears to
demand an element of aromaticity and may be enhanced by
basicity/H-bonding capability. Mutagenesis studies identify
Lys3.32(121) in the receptor as a possible site of
interaction with His2 (142) (see Section V).
Trp3.
Trp3 is clearly a critical
residue in GnRH. Trp2 His3 GnRH and
Des-Trp3 GnRH were inactive (143). Substitution with
nonaromatic amino acids (e.g. Gly, Leu, and Ala) gives
rise to very low activity (129, 143, 144, 145), whereas some activity is
present with Tyr (0.1%), as well as Phe (0.5%) substitution, and this
is increased substantially in pentamethyl-Phe GnRH (30–70%) (129, 143). Notably, the latter residue resembles Trp in its ability to form
- complexes with aromatic molecules (134). The introduction of an
electron-withdrawing fluorine atom of similar atomic radius in position
5 of Trp3 leads to a marked reduction in activity (6%),
presumably due to reorienting the dipole and forming hydrogen bonds
itself so that an aromatic interaction does not occur (145). The role
of an aromatic side chain in this position is further emphasized by the
natural substitution of Tyr for Trp3 in lamprey GnRH I
(Fig. 5). Even 2-napthyl-Ala substitution results in 50% activity
(129). Since D-Trp3 GnRH has poor gonadotropin-releasing
activity (133) but has been commonly incorporated in antagonists, it is
possible that Trp3 plays a role in receptor activation. The
altered stereochemistry evidently has a critical effect on agonistic
activity. The role of Trp3 in receptor activation is
further suggested by an early study demonstrating antagonist activity
of Leu3 GnRH (145) and recent work showing that
incorporation of NMe in the amino acid in position 3 converts the
peptide to an antagonist (146).
Ser4.
The last of the conserved residues in the
NH2-terminal domain, Ser4, can be substituted
with a number of amino acids (Ala, Thr, Gln, NMeSer) with reasonable
retention of activity (10–20%) (129, 130, 132, 143), yet this is the
most conserved residue in the empirically generated analogs. Because
substitution with larger amino acids such as Ser (But) and Leu is very
detrimental (129), it appears that spatial constraints are paramount.
Recent work has shown that constraint of the peptide bond with NMe does
not decrease activity (146, 147) unlike most other positions in GnRH.
The conclusion from early studies that large side chains are not
tolerated is supported by the recent observation that biotinylated
Ser4 GnRH is inactive (148).
2. The COOH-terminal domain (Pro-Gly·NH2).
Pro9. The conservation of Pro9
in the natural GnRHs and the expected conformational limitations
imposed by Pro on the peptide chain suggest that substitution would not
be readily tolerated. Sarcosine9 GnRH and Ala9
GnRH had low activity (<1%) while N-Me-Gly9 had 10%
activity (129, 132). The exchange of amino acids 8 and 9
(Pro8 Arg9 GnRH) also results in very low
activity (129). The discovery that Pro9 may be hydroxylated
in fetal brain and decreases activity to 10% (149) underlines the
importance of this conserved residue and suggests that this may be a
regulatory mechanism.
Gly-amide10.
Removal of the amide to yield the
free acid of GnRH results in very low activity (150). This has recently
been confirmed for the human GnRH receptor expressed in COS-1 cells
(67). Replacement of the Gly-NH2 moiety with Ala resulted
in a mild reduction in activity (10%) (130), and a similar reduction
was observed with Gly-NMe2 (14%) (132). On the other hand,
substitution of Gly-NH2 with alkylamides maintained
(methylamide and ethanolamide) or increased activity up to 600%
(propylamide and ethylamide) (150, 151) whereas substitution with
larger amides (pyrolidineamide and morpholineamide) (150) or D-amino
acids (129) decreased activity. The incorporation of
electron-withdrawing fluorine atoms into the ethylamide
(2,2,2-trifluoroethylamide) further enhanced activity to about 900%
(151). These findings suggest that the terminal Gly-NH2 is
not essential for activity and that small, uncharged moieties are
acceptable at the COOH terminus. Larger groups are inhibitory, possibly
by sterically hindering ligand access to the binding site. The findings
also suggested that the total chain length might have an important
role in the binding of GnRH to its receptor (150, 151). Recent
mutagenesis of a receptor site (N2.65(102)
A) has
demonstrated a much greater decrease in binding affinity of
Gly-NH2 ligands than N-ethylamide ligands (152)
(see Section V).
3. The central nonconserved domain (Tyr-Gly-Leu-Arg).
Try5. In accordance with the lack of conservation
of Tyr5 in vertebrate GnRHs, substitution in this position
is well tolerated. The 44–64% activity of Phe5 (143, 153)
demonstrated that the hydroxyl group is not required. Interestingly
this substitution has yet to be found in naturally occurring GnRHs,
although it would require only one base change. Substitution of the
hydroxyl group of tyrosine resulted in activities of 37% (amino), 24%
(methoxy), and 5% (nitro) (154). NMe Tyr substitution, which has been
proposed to constrain the peptide backbone and to eliminate one of two
postulated H bonds with Arg8 in a ß-II turn conformation,
led to a reduction in binding affinity to 10–20% (155) Interestingly,
mono-iodo-Tyr-GnRH (129) and mono-chloro-Tyr-GnRH (130) had activities
of 30–80% and 10%, respectively, while di-iodo-Tyr5 GnRH
and di-chloro-Tyr GnRH were devoid of LH-releasing activity (129, 130).
His5 GnRH has very high binding affinity for mammalian GnRH
receptors (121). These findings demonstrate that the hydroxyl group of
Tyr5 is not required, and that simply an aromatic side
chain (Trp, Phe, or His) is adequate for high LH-releasing activity.
The findings suggest that Tyr5 contributes only to receptor
binding and does not play a role in the process of receptor activation.
However, substitution of Tyr5 with His, as in GnRH II,
results in an analog with high receptor potency (aromaticity
maintained) but reduced LH release (partial agonism; (121). Partial
agonism is also observed with His5 D-Trp6 GnRH
and in His5 D-Tyr6 GnRH and Arg5
D-Tyr6 GnRH (R. P. Millar, unpublished). However, when
Arg8 is substituted by Tyr in analogs with His or Arg in
position 5, efficacy is restored. Thus the motif
His5/Arg5-Xxx-Xxx-Arg8 produces
compounds with high binding but diminished receptor activation,
indicating that the Tyr5 does play a role, albeit possibly
indirect, in receptor activation in the mammalian ligand-receptor
complex.
Gly6.
Gly6 is conserved in all
vertebrates except the ancient jawless lamprey and is also absent in
the tunicate GnRHs (Fig. 5). The presence of this small residue in this
position allows for flexibility and the assumption of the postulated
ß-II-type bend and the preferred conformation for receptor binding
(see below and Section IV). This bend would be energetically
unfavorable in analogs with larger L-amino acid substitutions for
Gly6, and Ile, Val, and Ala analogs were found to have low
activity (132, 137). However, the folded conformation is favored by the
stereochemistry of D-amino acid substitutions (10, 21, 88, 89, 90, 91, 137).
The proposal of a ß-II-type bend for the active conformation of GnRH
was first proposed by Monahan et al. (137) after
demonstrating that D-Ala6 substitution increased activity
to about 400%. This seminal work led to the exploration of numerous
substitutions with D-amino acids in this position (see reviews in Refs.
10, 21, and 88–91). In general, substitution with D-amino acids having
bulky hydrophobic side chains, particularly aromatics, was most
effective (10, 21), and this has been confirmed in numerous binding
studies using pituitary membranes and, more recently, with receptors
expressed in COS-1 cells. A correlation between hydrophobicity (HPLC
retention time) of the D-amino acid and potency has been noted (100).
It appears that there is a large "allowable space" facing away from
the NH2- and COOH-domains which interacts with the
receptor, and this will accommodate the D-amino acids with large side
groups (147). In addition to further favoring the ß-II-type
conformation, the large side chains of the D-amino acids may interact
with nearby residues in the receptor, thereby enhancing the binding
affinity. These potential alternative interactions probably account for
species differences and are likely to be prevalent in GnRH antagonists
with numerous unusual side chains, often aromatic. These features must
be taken into account when analyzing the effects of mutagenesis of
receptor residues on the binding of these analogs.
Leu7.
The comparative studies of activities
of vertebrate GnRHs indicate that substitutions of Leu7
with uncharged L-amino acids with varying size side chains are
generally well tolerated. This supposition is confirmed by the
demonstration that Val, Ile, Nle, Ser, ethoxycarbonyl-Lys,
butoxycarbonal-Lys, and Boc-Lys all had high activities (16–45%)
(129, 130) while Ala and Gly had lower (3–6%) activities (129, 130).
Potential disruption of conformation by D-amino acid D-Leu (133) or Pro
(129) substitution resulted in very low activities as did substitution
with basic residues (Lys, Arg) (see Ref.129). Tolerance of the large,
bulky substitution of Trp for Leu7 was recently
demonstrated by the high LH-releasing activity (110% in sheep
pituitary cells) and receptor binding (37% for sheep and 230% for
rat) for this analog (121, 122). The original proposal of a type-II ß
turn conformation of GnRH also envisaged a hydrogen bond between the
C=0 of Ser and NH of Leu. However, substitution of Leu7
with N-Me-Leu, which would eliminate this H bond, did not reduce
activity (155a).
Arg8.
Comparative activities of vertebrate GnRHs
had indicated that Arg8 is required for high-affinity
binding to mammalian receptors (91, 105, 119, 120, 121). A number of early
studies had shown that D-Arg, Gln, Leu, Orn, His, diaminobutyryl, and
Cit substitution for Arg8 results in a substantial decline
in activity (1–6%) while homoArg, Narg, and Lys retained good
activity (10–20%) (92, 119, 129, 130, 133, 156, 157, 158, 159, 160, 161). A systematic
study on the LH-releasing activities from sheep pituitary cells of
Gln-, Ser-, Tyr-, Phe-, Glu-, His-, Leu-, Lys-, Ile-, and
Trp-substituted analogs confirms the requirement of a basic amino acid
in position 8 for high activity (92, 93). Since receptor-binding is
correlated with LH-releasing activity in all position 8-substituted
analogs studied, it appears that the role of Arg8 may be in
receptor binding. However, as noted above, analogs with neutral amino
acid substitutions display improved efficacy. Two hypotheses may be
invoked to explain the basis of the higher affinities of
Arg8-containing GnRHs. An ionic interaction of
Arg8 with one or more negatively charged residues, either
an amino acid side-chain (162) or a polysaccharide sialic acid residue
(163) in the receptor, were proposed. An alternative or additional
possibility was that the side chain of Arg8 affects the
structure of the ligand, stabilizing the active conformation of GnRH by
hydrogen bonding with the side chains of His2 and
Tyr5 (159, 160). Low pK values were measured for
His2 and Tyr5 in GnRH, and it was suggested
that the more acidic nature of these amino acid side chains was due to
their proximity to the cationic side chain of Arg8 (160).
GnRH analogs with neutral substitutions, Gln and 
-nitro-Arg (159),
in position 8, exhibited normal pK values for His2 and
Tyr5 and extended titration ranges. These results were
interpreted as indicating a decreased interaction of the
His2 and Tyr5 side chains with the neutral
substituents in position 8 (161). The decreased side-chain interaction
was proposed to decrease stabilization of the bioactive conformation
and thus cause the lower bioactivity in the neutral GnRH analogs. Based
on these findings, a folded conformation of GnRH was proposed (159, 160) similar to models of GnRH that were based on energy minimization
and database sequence comparison (137, 164, 165, 166, 167).
The role of Arg8 in determining the preferred conformation of GnRH and in receptor interaction is explored in detail in the following sections. In recent mutagenesis studies, an acidic amino acid residue in extracellular loop III was shown to convey specificity for Arg8 such that mutation to an isosteric amide resulted in the loss of the preferential binding of Arg8 GnRH compared with GnRH with a neutral amino acid in this position (see Section V). The requirement for Arg in position 8 and the acidic residue in the receptor is obviated if the GnRH structure is constrained by incorporation of a D-amino acid in position 6 (see Section V). A recent reexploration of the role of Arg8 in antagonists concluded that this residue may be significant for receptor binding (168) while the demonstration in another study that (Orn(2,4-NAPS0)8 GnRH cross-linked with the receptor (169) was interpreted to support the proposal that Arg8 interacts with receptor moieties.
D. Conclusions from peptide structure-activity data
We have attempted to identify individual residues in GnRH that are
involved in receptor binding and receptor activation, as this
information is critical in undertaking receptor mutagenesis studies
directed at defining ligand contact sites. Although thousands of GnRH
analogs have been synthesized and biologically characterized, the
complexity of most analogs and the predominance of in vivo
testing have complicated the task of clearly identifying the roles of
individual amino acids in ligand conformation and in receptor binding
and activation. Although a more controlled and systematic examination
of the functions of GnRH residues using expressed cloned receptors has
begun (see Section V), relatively few analogs have been
studied to date. Nevertheless it is possible to generate working
hypotheses about ligand requirements of the mammalian pituitary GnRH
receptor for binding and activation (Fig. 8). The
following generalizations may be proffered:
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2. The NH2- and COOH-terminal domains are the most important in receptor binding and activation.
3. Although both domains are involved in receptor binding, residues in the NH2-terminal domain are predominantly responsible for receptor activation.
4. The only residues for which good evidence exists for a role in receptor activation are His2 and Trp3, but pGlu1 may also be involved.
5. Substitution of residues outside of the NH2-terminal domain can affect receptor activation, possibly through effects on the conformation that change the presentation of activating residues, or through restrictions in dynamic ligand conformation changes that occur on binding to the receptor.
6. Nonconserved residues of the central domain are less critical, but Arg8 is required for high-affinity binding to the mammalian receptor. However, the requirement for Arg8 may be obviated in conformationally constrained analogs with D-amino acids in position 6, and also when His5 is present as in chicken GnRH II.
7. The achiral Gly or D-amino acids are required in position 6, presumably to allow assumption of the folded active conformation.
8. Nonmammalian GnRH receptors have different requirements for the nonconserved residues in the central domain. Examples include the lack of requirement for a basic residue in position 8 and the nonacceptance of His5 substitution when Arg8 is present. Nonmammalian GnRH receptors also tend to be less dependent on conformational constraint, and D-amino acid substitution may not enhance activity to the same degree as in mammalian receptors.
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IV. Structure and Conformation of GnRH and Its Analogs |
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TopAbstractI. IntroductionII. Amino Acid Sequences...III. Structure-Activity...IV. Structure and Conformation...V. Functional Structure of...VI. ConclusionsReferences |
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The inherent flexibility of the hormone decapeptide makes it likely that interactions with various sites in the receptor-binding pocket will affect the conformation of any GnRH analog and reduce the ability to define a single "biologically relevant" conformation for the isolated peptide. Moreover, the pharmacophoric patterns of different peptides depend on the specific residues available for interaction with the various receptor sites, even if their conformations are the same. Consequently, the structural determinants for action on the GnRH receptor will have to be sought from a comprehensive characterization of all the conformations accessible to the peptides under given conditions of temperature and environment, i.e. the conformational space of these peptides, as well as of the three-dimensional pattern of the pharmaco-phoric elements that their amino acids present to the receptor. If the ability to adopt certain conformations determines the receptor activity of peptides with similar pharmacophoric elements, then differences in their conformational spaces can reveal the conformations required for receptor interactions. The conformational properties of the most active peptides can thus serve to define the spatial and dynamic requirements for optimal interaction with the receptor. A useful ranking of structure-activity characteristics can be constructed on this basis, provided that peptides with pharmacologically distinct activities such as agonism and antagonism are differentiated. For these reasons, it is not realistic to expect a full understanding to emerge entirely from structure-activity data obtained from probing the activities of various synthetic analogs without specific analysis of their conformational properties. Rather, the mechanistic insights are more likely to emerge when the powerful approaches offered by current experimental and computational methods for conformational analysis (for reviews see Refs. 171 and 172; also Refs. 166, 173, and 174) are applied to the exploration of peptide structure and design.
Pioneering efforts were undertaken to achieve such a comprehensive
exploration of the conformation of GnRH and its active analogs by
computational methods (164, 165, 175). The impetus for such studies
continues to be the assumption that if the peptide conformation
recognized by the receptor (i.e. the bioactive conformation)
corresponds to the most abundant form of the peptide in solution, then
the peptide will have high affinity for the receptor. Because the most
abundant conformers in solution are those corresponding to the lowest
free energy, the computational approaches concentrated on the
calculation of the conformational energies of the peptides. The early
studies (164, 165, 175) identified low-energy conformations of GnRH
that were considered to occur also in solution, although it was not
possible at the time to account for the effects of aqueous solvation.
In spite of the significant limitations of the methods for energy-based
evaluation of peptide conformations (for a review of methods see Ref.176) that were available at the time for studies of peptide molecules
of the size of GnRH, the early studies identified the central
characteristic of the bioactive conformation of GnRH, the ß-bend
involving the Tyr-Gly-Leu-Arg in positions 5–8 (see Fig. 9).
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). However, the computational simulations of
molecular conformation and dynamics indicated the large variability in
the structural options of the uncyclized 1–3 fragment, as well as the
fact that several different conformations with equivalent energies and
geometrical features compatible with the NMR data, can be adopted even
by the constrained dicyclic compound (177).
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Early attempts to decompose the structural determinants for activity of cyclized GnRH analogs into contributions from the length of the bridge and the orientation of certain functional groups (e.g. amide bonds considered to be involved in direct interactions with receptor sites) were not successful (170). A main reason is the residual flexibility of the cyclized GnRH analogs, which lose only a portion of their conformational freedom. This insight (e.g. see Refs. 170 and 178) led to the suggestion that a complete structural characterization, rather than the mere identification of minimal energy conformations, will be necessary for both linear and cyclic analogs (170).
C. Exploration of the entire conformation space of GnRH analogs
The results from experimental and computational studies of the
structure and conformation of GnRH analogs emphasize the importance of
a complete exploration of the conformational space of the peptides
(176). Whether the analogs are conformationally constrained or linear,
the complete structural characterization is necessary before a reliable
consensus on the structural features most important for receptor
interaction can be reached (174). Current methods (174, 181) make
possible such extensive explorations of both the conformational and
dynamic properties of the peptides, offering the ability to explore the
entire conformational space of decapeptides such as GnRH and its
analogs (174). Specifically, the application of a recently developed
technique of Conformational Memories (182) to the study of GnRH
conformational properties illustrates the first complete exploration of
the entire conformational space of the peptide using a method of
simulation that includes a satisfactory model for the aqueous solvent.
The novel method overcomes some of the shortcomings of modern molecular dynamics approaches to the study of the peptide hormone: although the molecular dynamics techniques are useful for their ability to describe the molecular motions of the peptide in short time scales, they are unable to explore all the conformational states of the peptide in solution, and hence are not able to characterize their relative abundance. In contrast, the Conformational Memories method utilizes a two-stage process of computation to map, and then characterize, the conformational space of a flexible molecule (174). In the first, exploratory stage, repeated runs of the Monte Carlo method (183) combined with simulated annealing (184) are carried out to map the entire conformational space of a flexible molecule by heating it to very high temperatures and cooling it slowly to body temperature. Once the Conformational Memories are established, the method proceeds to a new Monte Carlo search of the conformations of the peptide, performed at 310K and sampling only from the populated regions. Because only about 50% of the torsional space of the 35 bonds of GnRH is populated at 310K, the two-tiered approach reduces by many orders of magnitude the conformational space that must be explored in this second phase (174). The configurations sampled from the Conformational Memory can be any part of the populated space of dihedral angles defining the conformations of the peptide. Consequently, the notion of a barrier restricting access to any part of the conformational space is eliminated in this procedure without approximations.
1. Conformational families of GnRH. In the application of the
conformational memories approach to GnRH, the second step of the
procedure involved 500,000 steps (174). Structures of the peptide
obtained from the run were clustered in conformational families,
resulting in the five basic structures depicted in Fig. 11. Notably, families of conformations having a
ß-turn between residues 5–8 occur in GnRH with a frequency of
approximately 70%. A distribution showing a superimposition of 70 of
these structures is illustrated in Fig. 12; the
ß-type turn common to all the structures in this family is clearly
evident. In contrast, families that have an extended backbone occur
with a frequency of about 5%. The distribution of side-chain
orientations of Arg8 in all conformational families was
found to be wider than that of any other residues in GnRH.
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. The family of conformations with an
extended backbone has an abundance of more than 70%, while the
ß-type turn conformation of Lys8-GnRH, which is virtually
identical to the major conformational family of the GnRH, has a
probability of only about 3%. A distribution of the members of the
predominant Lys8-GnRH family superimposed upon each other
is shown in Fig. 13, with the entire molecule shown in
red, except for Lys8, which is colored
green. Because Lys8-GnRH is a low affinity
agonist for the GnRH receptor, adoption of a large population of
ß-type turn conformation appears to be a key requirement for
hormone-receptor recognition. This inference agrees with earlier
proposals in the literature (e.g. see Ref.173) and is
supported by results from additional Conformational Memories
simulations on the structural characterization of eight other GnRH
analogs that exhibit different distributions between the ß-turn like
structures and the fully extended conformations of the backbone (F.
Guarnieri, S. C. Sealfon, and H. Weinstein, unpublished).
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Thus, the results of the first conformational study capable of overcoming energy barriers efficiently and achieving a complete sampling of the conformational space of GnRH support a relation between the ß-turn structure identified as the major conformational family of GnRH in solution and high affinity for the GnRH receptor. These inferences support the results from the earlier investigations of conformationally restricted GnRH analogs (164, 173, 178) and provide unbiased support for this mechanistic hypothesis based on a complete exploration of the conformational space of the peptide hormone itself and its unconstrained congeners. Because the method seems to have produced the lowest energy conformers reported for GnRH (174) from a full exploration that is economical and practical, its continued application to the study of structure-function relations of GnRH analogs should produce important mechanistic insights and powerful guides for ligand design.
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V. Functional Structure of the Receptor and Ligand-Receptor Complex |
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TopAbstractI. IntroductionII. Amino Acid Sequences...III. Structure-Activity...IV. Structure and Conformation...V. Functional Structure of...VI. ConclusionsReferences |
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), studies of the
binding pocket have relied on indirect approaches, particularly the
study of the functional effects of site-directed mutagenesis and the
construction of computational three-dimensional molecular models of the
ligand-receptor complexes. Through these approaches, a view of the
receptor and its mode of ligand binding is emerging. Side chains
required for high-affinity binding of peptide ligands have been
identified in both the extracellular and transmembrane domains, and
specific helix-helix proximities within the receptor have been
determined.
As described above, the basic Arg8 of GnRH is critical for
high-affinity agonist activity at the mammalian receptor. Substitutions
at Arg8 [as in Chicken I GnRH (Gln8GnRH)]
cause a marked reduction in the affinity of binding to the mammalian
receptor (119, 121). Based on cation competition experiments, Hazum
(162) originally suggested that the Arg8 of GnRH may
interact with carboxylic groups on the receptor (162). The possibility
that Arg8 of GnRH forms an ionic interaction with an acidic
residue of the GnRH receptor was investigated by site-directed
mutagenesis (186). The chicken GnRH receptor shows little
discrimination between Arg8-GnRH (mammalian GnRH) and
Gln8-GnRH (chicken I GnRH). All acidic residues on the
receptor were mutated to their isosteric amine, and a mutant that
failed to discriminate between Arg8-GnRH and
Gln8-GnRH was sought. One mutant receptor was identified,
Glu7.32(301)-Gln, which had decreased affinity for
mammalian GnRH in comparison with the wild type receptor. The affinity
of this mutant for Gln8- GnRH and for other GnRH analogs
with an uncharged residue in the eight position, however, was
relatively unchanged or improved (see Fig. 14). Most
significant is the marked increase in activity of Glu8-GnRH
in the mutant. These results support a role for the
Glu7.32(301) residue, located in the third extracellular
domain, in conferring the preference of the mammalian receptor for
Arg8 GnRH. A GnRH analog that is conformationally
restricted by having a D-amino acid in position 6
(D-Trp6,Pro9-NHEt)GnRH and its Gln8
congener
(D-Trp6,Gln8,Pro9-NHEt)GnRH have
more similar affinities for both the wild type and
Gln7.32(301) mutant receptors. Thus conformationally
restricted analogs do not seem to require Arg in position 8, suggesting
that the role of Glu7.32(301) in the receptor is to help
induce or select the optimum conformation of the ligand for
high-affinity interaction.
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B. Helix domains
The receptors for the biogenic amine neurotransmitters all contain
an Asp at a homologous location in TMD 3. Mutagenesis studies on
several of these receptors suggest that the Asp anionic side chain
serves as a counterion required for high-affinity interaction with the
cationic head group of the ligand (187, 188, 189, 190, 191, 192, 193). All cloned GnRH receptors
have a lysine at the corresponding 3.32 position (Fig. 1). The role of
Lys3.32(121) in ligand binding and activation of the human
receptor was studied by introducing a series of mutations at this
position (142). Substitution of Arg at this position preserved high
affinity agonist binding, whereas Gln at this position reduced agonist
binding to below the limit of detection. Leu and Asp at this locus
abolished both binding and detectable signal transduction. The
EC50 of concentration-response curves for coupling to
phosphatidyl inositol hydrolysis obtained with the
Gln3.32(121) receptor was more than 3 orders of magnitude
higher than that obtained for the wild-type receptor (see Fig. 15). Receptor inactivation studies confirmed that this
increase in EC50 represented a large decrease in agonist
affinity. In contrast, an antagonist had comparable high affinities for
the wild type, Arg121, and Gln121 mutants. This
study indicated that a charge-strengthened hydrogen-bond donor is
required at this locus for high-affinity agonist binding, but not for
high-affinity antagonist binding. Based on the available
structure-activity data of GnRH analogs, His2 in the ligand
is a potential candidate for interaction with this locus (see
Section III). Although the Lys3.32(121) in the
GnRH receptor appears to serve a function analogous to
Asp3.32 in the neurotransmitter receptors, this
correspondence does not extend to all peptide GPCRs. The corresponding
position of the mouse TRH receptor, Gln3.32(105), appears
to make only a modest contribution to ligand affinity (194). In
contrast, two other TMD 3 side chains in that receptor,
Tyr3.33(106) and Asn3.37(110), have been
identified as major determinants of agonist affinity (194). Thus, while
the side chain at the 3.32 locus is not a critical determinant of
affinity in all receptors, the local properties of this region of TMD 3
seem generally important for ligand-receptor interaction (195).
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As noted above (see Section II.B), an unusual feature of the
GnRH receptor, observed in all mammalian species, is the presence of
Asn2.50(87) in the second putative transmembrane helix at
the location of a highly conserved Asp in the GPCR family, and of
Asp7.49(318) in the putative seventh transmembrane helix
where nearly all other GPCRs have Asn (see Fig. 2). This apparent
interchange of conserved residues in the native receptor raised the
possibility that these residues interact. This possibility was
investigated by expressing mutant receptors in which each residue was
replaced by the other or both residues were exchanged (51). The results
showed that the Asp2.50(87) mutant had no detectable
binding. The double mutant Asp2.50(87)
Asn7.49(318), which recreates the arrangement found in
other GPCRs, regained high-affinity agonist and antagonist binding (see
Fig. 16). The restoration of binding by a second
reciprocal mutation indicates that these two specific residues in TMD 2
and TMD 7 are adjacent in space (Fig. 17) and provides
an empirical basis for refining the structural parameters in the model
of the receptor’s transmembrane helix bundle (see below). Cook and
co-workers (196) reported that the interchange mutation in the rat GnRH
receptor did not show ligand binding. However, these investigators have
subsequently confirmed that this construct is functional (K. Eidne,
personal communication), and two other groups have confirmed the
results with the mouse receptor (K. Catt, personal communication and
Ref.197).
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C. Intracellular loop domains
Mutagenesis, chimera, and deletion studies indicate that the
intracellular domains of GPCRs are involved in mediating G protein
coupling. Particularly important domains are the membrane-proximal
segments of the third intracellular loop, the second intracellular
loop, and the membrane-proximal segment of the carboxy-terminal domain
(for review see Refs. 49, 50, and 200). The second intracellular loop
domains of the mouse and human receptors have been investigated by
site-directed mutagenesis (60, 201). The GnRH receptor has a Ser at
position 3.51(140), a locus where most receptors have the
Tyr of the Asp-Arg-Tyr ("DRY") motif. Mutation of this locus to the
consensus Tyr by Arora and co-workers (60) caused an increase in
agonist affinity and in rates of internalization in comparison to the
wild-type receptor. Mutation of the second intracellular loop
Leu3.58(147) to Ala or Asp was found to impair receptor
coupling and internalization.
A specific conformational structure of this loop domain appears to be required for efficient G protein coupling. Mutation of Arg3.56(145) to Pro has been found to disrupt significantly the efficiency of coupling to signal transduction (201). This mutation introduces a Pro-Pro motif that disrupts most known secondary structure. We have performed computational simulations and protein database searches with the the wild-type and Pro-Pro mutant receptor loop segment sequences. By comparing the structures accessible to the loop segments in the wild-type and mutant receptors, the conformations likely to be preferred for G protein coupling have been identified. We find by incorporating these results into the computational model of the receptor that an association of the second intracellular loop with other loop domains may be required for efficient receptor coupling (F. Guarnieri, L. Chi, V. Rodic, L. Ballesteros, H. Weinstein, and S. C. Sealfon, unpublished data).
One feature of the mammalian GnRH receptor, unique among the several
hundred GPCRs cloned to date, is the complete absence of an
intracellular C-terminal domain. In most rhodopsin-family GPCRs, this
domain contains a cysteine that has been shown to be palmitoylated in
several receptors (202, 203, 204, 205). This domain also contains sites involved
in phosphorylation-mediated regulation and desensitization of several
GPCRs (206, 207). In the TRH receptor, for example, agonist-induced
receptor internalization requires specific domains in the C terminus
(208). Truncation or mutation of specific C-terminal domains has been
reported to diminish agonist-mediated internalization of the
gastrin-releasing peptide receptor (209) and the angiotensin receptor
(210, 211), and to diminish desensitization of the LH receptor (212)
(see, however, Ref.213), the substance P receptor (214), the
neurokinin receptor (215), and the -1B-adrenergic receptor (216).
Nonetheless, it is likely that the functional role of the carboxyl
terminus domain is not identical in different GPCRs. For example,
truncation of the ß-adrenergic receptor was reported not to affect
sequestration (217), and truncation of the FSH receptor was found not
to alter desensitization (218). While the ultimate response elicited
after GnRH receptor stimulation (e.g. LH release) undergoes
desensitization (reviewed in Refs. 13–15), it has not been
demonstrated that this desensization occurs at the level of the
receptor. In view of the role of the missing carboxyl-terminal domain
in desensitization of a number of receptors, it is interesting to note
that minimal rapid desensitization of the phosphoinositol response
mediated by the endogenous T3–1 GnRH receptor or the cloned GnRH
receptor expressed in several cell lines has been observed (69, 70, 71).
D. Computational modeling of three-dimensional receptor structure
Inferences from the probing of the GnRH receptor with biochemical
approaches, mutagenesis, and biophysical considerations, as described
above, have validated its initial structural classification as a member
of the family of rhodopsin-like GPCRs. Although no direct structural
information at atomic resolution is available for any GPCR, strong
inferences about structural characteristics of the transmembrane
portion of the GnRH receptor rest on the projection map of the electron
density of bovine rhodopsin (Fig. 4) and the results of extensive
probing of the other members of the rhodopsin-like GPCR family. The
extension of these inferences to the GnRH receptor is based on the
extensive and pervasive sequence homologies and identities of specific
motifs observed among the various rhodopsin-like receptors that include
the GnRH receptors. Such sequence comparisons are used to identify the
likely determinants for the structural commonalty expressed in the
template of protein families, such as the seven loop-connected
transmembrane helix bundles of the GPCRs (39, 219).
It has been shown that such sequence comparisons are useful in the characterization of structural properties and can also serve to identify the basis for the different functional properties of receptor proteins, e.g. those that determine ligand binding as well as the response of the GPCR to the actions of a large variety of ligands (for a review see Ref.39). Not surprisingly, sequence alignments of the rhodopsin-family GPCRs are often the first steps in the modeling process of probing structure-function relations of these proteins, and in the construction of three-dimensional molecular models of specific receptors (39). The basic assumptions underlying the extraction of structural information about GPCRs from a set of aligned sequences are that they all share a structural framework, and that highly conserved residues can be considered essential for the structural and/or functional integrity of the receptor. Sequence sites observed to have a lower degree of conservation are considered to play a lesser role in determining the structure and/or function of the GPCR. The criteria guiding the construction of a sequence alignment of GPCRs have been reviewed, together with the conceptual and practical limitations of the sole use of sequence alignments for the prediction and construction of three-dimensional molecular models of GPCRs (39).
Using a complex array of interrelated criteria after a set of well
defined methods for the construction and computational probing of such
models (39), a three-dimensional model of the transmembrane helix
bundle of the GnRH receptor has been developed (51) (Fig. 18). Specific
criteria in the construction of this model included the structural
inferences derived from the analysis of sequence conservation patterns
(220, 221), the physico-chemical properties of conserved and partially
conserved residues (219, 222), and specific protein motifs such as
Pro-kinks (223, 224, 225), the projection map of rhodopsin (58, 219), and
experimental results. The predicted helix boundaries take into account
the role of Arg and Lys residues at the membrane-cytoplasm interface,
where these residues belong, as described (39), to the transmembrane
helix acting as an anchor to the membrane through ionic pairs with
phospholipid head-groups (224). The model is consistent with the
overall template of rhodopsin-family GPCRs (39), including the proposed
interactions between helix 2 and 7 (51, 198), the mutual orientation of
TMDs 1 and 7 (226), the counter-clockwise connectivity of the TMD
domains when viewed from the extracellular side (227, 228), as well as
the detailed deployment of sites in the interior of the TMD bundle and
in the extracellular loops that have been suggested by experiments to
contribute to the ligand-binding pocket. As illustrated by model-based
investigations of structure-function relations of other GPCRs, both in
the family of neurotransmitter receptors (195, 229) and in peptide
receptors (194, 230, 231, 232), the discrete molecular models of the GnRH
receptor should provide key insight into mechanisms of receptor
specificity and activation (e.g. see Refs. 51, 142, 195, and
198).
|
). The computational model of GnRH is based on the
predominant conformer observed in conformational family simultations of
the peptide (174), and the ligand positioning reflects the following
assumptions derived from experimentation: 1) Arg8 of GnRH
is in proximity to Asp7.32(302) (186); 2) the C terminus
glycinamide of GnRH may interact with Asn2.65(102) (152);
3) His2 of GnRH may interact with Lys3.31(121)
(142) and/or Asp2.61(980 (195a) of the receptor.
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VI. Conclusions |
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TopAbstractI. IntroductionII. Amino Acid Sequences...III. Structure-Activity...IV. Structure and Conformation...V. Functional Structure of...VI. ConclusionsReferences |
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Acknowledgments |
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Footnotes |
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TopAbstractI. IntroductionII. Amino Acid Sequences...III. Structure-Activity...IV. Structure and Conformation...V. Functional Structure of...VI. ConclusionsReferences |
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1-adrenergic receptors coupled to inositol
phospholipid metabolism in DDT1 MR-2 smooth muscle cells. J Biol
Chem 262:3098–31051-adrenergic receptor subtypes identify critical residues that
modulate active state isomerization. J Biol Chem 271:7956–7964
-opioid
receptor. J Biol Chem 271:7875–7878This article has been cited by other articles:
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J H Li, F Sicard, M A Salam, M Baek, J LePrince, H Vaudry, K Kim, H B Kwon, and J Y Seong Molecular cloning and functional characterization of a type-I neurotensin receptor (NTR) and a novel NTR from the bullfrog brain J. Mol. Endocrinol., June 1, 2005; 34(3): 793 - 807. [Abstract] [Full Text] [PDF]
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J. H. Li, H. Choe, A. F. Wang, K. Maiti, C. Wang, A. Salam, S. Y. Chun, W.-K. Lee, K. Kim, H. B. Kwon, et al. Extracellular Loop 3 (EL3) and EL3-Proximal Transmembrane Helix 7 of the Mammalian Type I and Type II Gonadotropin-Releasing Hormone (GnRH) Receptors Determine Differential Ligand Selectivity to GnRH-I and GnRH-II Mol. Pharmacol., April 1, 2005; 67(4): 1099 - 1110. [Abstract] [Full Text] [PDF]
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S. J. Gregory, C. T. Lacza, A. A. Detz, S. Xu, L. A. Petrillo, and U. B. Kaiser Synergy between Activin A and Gonadotropin-Releasing Hormone in Transcriptional Activation of the Rat Follicle-Stimulating Hormone-{beta} Gene Mol. Endocrinol., January 1, 2005; 19(1): 237 - 254. [Abstract] [Full Text] [PDF]
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A. Rose, P. Froment, V. Perrot, M. J. Quon, D. LeRoith, and J. Dupont The Luteinizing Hormone-releasing Hormone Inhibits the Anti-apoptotic Activity of Insulin-like Growth Factor-1 in Pituitary {alpha}T3 Cells by Protein Kinase C{alpha}-mediated Negative Regulation of Akt J. Biol. Chem., December 10, 2004; 279(50): 52500 - 52516. [Abstract] [Full Text] [PDF]
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G. J. Reinhart, Q. Xie, X.-J. Liu, Y.-F. Zhu, J. Fan, C. Chen, and R. S. Struthers Species Selectivity of Nonpeptide Antagonists of the Gonadotropinreleasing Hormone Receptor Is Determined by Residues in Extracellular Loops II and III and the Amino Terminus J. Biol. Chem., August 13, 2004; 279(33): 34115 - 34122. [Abstract] [Full Text] [PDF]
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C. J. Caunt, J. N. Hislop, E. Kelly, A.-L. Matharu, L. D. Green, K. R. Sedgley, A. R. Finch, and C. A. McArdle Regulation of Gonadotropin-Releasing Hormone Receptors by Protein Kinase C: Inside Out Signalling and Evidence for Multiple Active Conformations Endocrinology, August 1, 2004; 145(8): 3594 - 3602. [Abstract] [Full Text] [PDF]
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D. Bonfil, D. Chuderland, S. Kraus, D. Shahbazian, I. Friedberg, R. Seger, and Z. Naor Extracellular Signal-Regulated Kinase, Jun N-Terminal Kinase, p38, and c-Src Are Involved in Gonadotropin-Releasing Hormone-Stimulated Activity of the Glycoprotein Hormone Follicle-Stimulating Hormone {beta}-Subunit Promoter Endocrinology, May 1, 2004; 145(5): 2228 - 2244. [Abstract] [Full Text] [PDF]
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P. M. Gault, K. Morgan, A. J. Pawson, R. P. Millar, and G. A. Lincoln Sheep Exhibit Novel Variations in the Organization of the Mammalian Type II Gonadotropin-Releasing Hormone Receptor Gene Endocrinology, May 1, 2004; 145(5): 2362 - 2374. [Abstract] [Full Text] [PDF]
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D. Gonzalez-Martinez, T. Madigou, E. Mananos, J. M. Cerda-Reverter, S. Zanuy, O. Kah, and J. A. Munoz-Cueto Cloning and Expression of Gonadotropin-Releasing Hormone Receptor in the Brain and Pituitary of the European Sea Bass: An In Situ Hybridization Study Biol Reprod, May 1, 2004; 70(5): 1380 - 1391. [Abstract] [Full Text] [PDF]
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R. P. Millar, Z.-L. Lu, A. J. Pawson, C. A. Flanagan, K. Morgan, and S. R. Maudsley Gonadotropin-Releasing Hormone Receptors Endocr. Rev., April 1, 2004; 25(2): 235 - 275. [Abstract] [Full Text] [PDF]
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A. R. Finch, L. Green, J. N. Hislop, E. Kelly, and C. A. McArdle Signaling and Antiproliferative Effects of Type I and II Gonadotropin-Releasing Hormone Receptors in Breast Cancer Cells J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1823 - 1832. [Abstract] [Full Text] [PDF]
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A. Ulloa-Aguirre, J. A. Janovick, A. Leanos-Miranda, and P. M. Conn Misrouted cell surface GnRH receptors as a disease aetiology for congenital isolated hypogonadotrophic hypogonadism Hum. Reprod. Update, March 1, 2004; 10(2): 177 - 192. [Abstract] [Full Text] [PDF]
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R. G. Romanelli, T. Barni, M. Maggi, M. Luconi, P. Failli, A. Pezzatini, E. Pelo, F. Torricelli, C. Crescioli, P. Ferruzzi, et al. Expression and Function of Gonadotropin-releasing Hormone (GnRH) Receptor in Human Olfactory GnRH-secreting Neurons: AN AUTOCRINE GnRH LOOP UNDERLIES NEURONAL MIGRATION J. Biol. Chem., January 2, 2004; 279(1): 117 - 126. [Abstract] [Full Text] [PDF]
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C. Wang, O. Yun, K. Maiti, D. Y. Oh, K. K. Kim, C. H. Chae, C. J. Lee, J. Y. Seong, and H. B. Kwon Position of Pro and Ser near Glu7.32 in the Extracellular Loop 3 of Mammalian and Nonmammalian Gonadotropin-Releasing Hormone (GnRH) Receptors Is a Critical Determinant for Differential Ligand Selectivity for Mammalian GnRH and Chicken GnRH-II Mol. Endocrinol., January 1, 2004; 18(1): 105 - 116. [Abstract] [Full Text] [PDF]
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F. Liu, D. A. Austin, and N. J. G. Webster Gonadotropin-Releasing Hormone-Desensitized L{beta}T2 Gonadotrope Cells Are Refractory to Acute Protein Kinase C, Cyclic AMP, and Calcium-Dependent Signaling Endocrinology, October 1, 2003; 144(10): 4354 - 4365. [Abstract] [Full Text] [PDF]
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Y. Okada, A. Murota-Kawano, S. S. Kakar, and S. J. Winters Evidence that Gonadotropin-Releasing Hormone (GnRH) II Stimulates Luteinizing Hormone and Follicle-Stimulating Hormone Secretion from Monkey Pituitary Cultures by Activating the GnRH I Receptor Biol Reprod, October 1, 2003; 69(4): 1356 - 1361. [Abstract] [Full Text] [PDF]
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A. J. Pawson, S. R. Maudsley, J. Lopes, A. A. Katz, Y.-M. Sun, J. S. Davidson, and R. P. Millar Multiple Determinants for Rapid Agonist-Induced Internalization of a Nonmammalian Gonadotropin-Releasing Hormone Receptor: A Putative Palmitoylation Site and Threonine Doublet within the Carboxyl-Terminal Tail Are Critical Endocrinology, September 1, 2003; 144(9): 3860 - 3871. [Abstract] [Full Text] [PDF]
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A. M. Navratil, S. P. Bliss, K. A. Berghorn, J. M. Haughian, T. A. Farmerie, J. K. Graham, C. M. Clay, and M. S. Roberson Constitutive Localization of the Gonadotropin-releasing Hormone (GnRH) Receptor to Low Density Membrane Microdomains Is Necessary for GnRH Signaling to ERK J. Biol. Chem., August 22, 2003; 278(34): 31593 - 31602. [Abstract] [Full Text] [PDF]
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B. Fromme, P. Eftekhari, M. van Regenmortel, J. Hoebeke, A. Katz, and R. Millar A Novel Retro-Inverso Gonadotropin-Releasing Hormone (GnRH) Immunogen Elicits Antibodies That Neutralize the Activity of Native GnRH Endocrinology, July 1, 2003; 144(7): 3262 - 3269. [Abstract] [Full Text] [PDF]
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B. Karges, W. Karges, M. Mine, L. Ludwig, R. Kuhne, E. Milgrom, and N. de Roux Mutation Ala171Thr Stabilizes the Gonadotropin-Releasing Hormone Receptor in Its Inactive Conformation, Causing Familial Hypogonadotropic Hypogonadism J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1873 - 1879. [Abstract] [Full Text] [PDF]
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V. D. Dixit, R. Sridaran, M. A. Edmonsond, D. Taub, and W. E. Thompson Gonadotropin-Releasing Hormone Attenuates Pregnancy-Associated Thymic Involution and Modulates the Expression of Antiproliferative Gene Product Prohibitin Endocrinology, April 1, 2003; 144(4): 1496 - 1505. [Abstract] [Full Text] [PDF]
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K. Morgan, D. Conklin, A. J. Pawson, R. Sellar, T. R. Ott, and R. P. Millar A Transcriptionally Active Human Type II Gonadotropin-Releasing Hormone Receptor Gene Homolog Overlaps Two Genes in the Antisense Orientation on Chromosome 1q.12 Endocrinology, February 1, 2003; 144(2): 423 - 436. [Abstract] [Full Text] [PDF]
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J. Y. Seong, L. Wang, D. Y. Oh, O. Yun, K. Maiti, J. H. Li, J. M. Soh, H. S. Choi, K. Kim, H. Vaudry, et al. Ala/Thr201 in Extracellular Loop 2 and Leu/Phe290 in Transmembrane Domain 6 of Type 1 Frog Gonadotropin-Releasing Hormone Receptor Confer Differential Ligand Sensitivity and Signal Transduction Endocrinology, February 1, 2003; 144(2): 454 - 466. [Abstract] [Full Text] [PDF]
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D. Harris, D. Chuderland, D. Bonfil, S. Kraus, R. Seger, and Z. Naor Extracellular Signal-Regulated Kinase and c-Src, But Not Jun N-Terminal Kinase, Are Involved in Basal and Gonadotropin-Releasing Hormone-Stimulated Activity of the Glycoprotein Hormone {alpha}-Subunit Promoter Endocrinology, February 1, 2003; 144(2): 612 - 622. [Abstract] [Full Text] [PDF]
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E. Terasawa Gonadotropin-Releasing Hormone II: Is this Neuropeptide Important for Mammalian Reproduction? Endocrinology, January 1, 2003; 144(1): 3 - 4. [Full Text] [PDF]
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C. Prioleau, I. Visiers, B. J. Ebersole, H. Weinstein, and S. C. Sealfon Conserved Helix 7 Tyrosine Acts as a Multistate Conformational Switch in the 5HT2C Receptor. IDENTIFICATION OF A NOVEL "LOCKED-ON" PHENOTYPE AND DOUBLE REVERTANT MUTATIONS J. Biol. Chem., September 20, 2002; 277(39): 36577 - 36584. [Abstract] [Full Text] [PDF]
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K. D. G. Pfleger, J. Bogerd, and R. P. Millar Conformational Constraint of Mammalian, Chicken, and Salmon GnRHs, But Not GnRH II, Enhances Binding at Mammalian and Nonmammalian Receptors: Evidence for Preconfiguration of GnRH II Mol. Endocrinol., September 1, 2002; 16(9): 2155 - 2162. [Abstract] [Full Text] [PDF]
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L. F. G. Silveira, P. M. Stewart, M. Thomas, D. A. Clark, P. M. G. Bouloux, and G. S. MacColl Novel Homozygous Splice Acceptor Site GnRH Receptor (GnRHR) Mutation: Human GnRHR "Knockout" J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2973 - 2977. [Abstract] [Full Text] [PDF]
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G. Maya-Nunez, J. A. Janovick, A. Ulloa-Aguirre, D. Soderlund, P. M. Conn, and J. P. Mendez Molecular Basis of Hypogonadotropic Hypogonadism: Restoration of Mutant (E90K) GnRH Receptor Function by a Deletion at a Distant Site J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2144 - 2149. [Abstract] [Full Text] [PDF]
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T. R. Ott, B. E. Troskie, R. W. Roeske, N. Illing, C. A. Flanagan, and R. P. Millar Two Mutations in Extracellular Loop 2 of the Human GnRH Receptor Convert an Antagonist to an Agonist Mol. Endocrinol., May 1, 2002; 16(5): 1079 - 1088. [Abstract] [Full Text] [PDF]
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D. Grove-Strawser, S. A. Sower, P. M. Ronsheim, J. B. Connolly, C. G. Bourn, and B. S. Rubin Guinea Pig GnRH: Localization and Physiological Activity Reveal That It, Not Mammalian GnRH, Is the Major Neuroendocrine Form in Guinea Pigs Endocrinology, May 1, 2002; 143(5): 1602 - 1612. [Abstract] [Full Text] [PDF]
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C. Castro-Fernandez, J. A. Janovick, S. P. Brothers, R. A. Fisher, T. H. Ji, and P. M. Conn Regulation of RGS3 and RGS10 Palmitoylation by GnRH Endocrinology, April 1, 2002; 143(4): 1310 - 1317. [Abstract] [Full Text] [PDF]
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D. Pati and H. R. Habibi Involvement of Protein Kinase C and Arachidonic Acid Pathways in the Gonadotropin-Releasing Hormone Regulation of Oocyte Meiosis and Follicular Steroidogenesis in the Goldfish Ovary Biol Reprod, March 1, 2002; 66(3): 813 - 822. [Abstract] [Full Text] [PDF]
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B. J. Fromme, A. A. Katz, R. W. Roeske, R. P. Millar, and C. A. Flanagan Role of Aspartate7.32(302) of the Human Gonadotropin-Releasing Hormone Receptor in Stabilizing a High-Affinity Ligand Conformation Mol. Pharmacol., December 1, 2001; 60(6): 1280 - 1287. [Abstract] [Full Text] [PDF]
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H. M. Everest, J. N. Hislop, T. Harding, J. B. Uney, A. Flynn, R. P. Millar, and C. A. McArdle Signaling and Antiproliferative Effects Mediated by GnRH Receptors After Expression in Breast Cancer Cells Using Recombinant Adenovirus Endocrinology, November 1, 2001; 142(11): 4663 - 4672. [Abstract] [Full Text] [PDF]
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J. N. Hislop, H. M. Everest, A. Flynn, T. Harding, J. B. Uney, B. E. Troskie, R. P. Millar, and C. A. McArdle Differential Internalization of Mammalian and Non-mammalian Gonadotropin-releasing Hormone Receptors. UNCOUPLING OF DYNAMIN-DEPENDENT INTERNALIZATION FROM MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING J. Biol. Chem., October 19, 2001; 276(43): 39685 - 39694. [Abstract] [Full Text] [PDF]
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L. Wang, D. Y. Oh, J. Bogerd, H. S. Choi, R. S. Ahn, J. Y. Seong, and H. B. Kwon Inhibitory Activity of Alternative Splice Variants of the Bullfrog GnRH Receptor-3 on Wild-Type Receptor Signaling Endocrinology, September 1, 2001; 142(9): 4015 - 4025. [Abstract] [Full Text] [PDF]
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 F. Van Goor, Y.-X. Li, and S. S. Stojilkovic Paradoxical Role of Large-Conductance Calcium-Activated K+ (BK) Channels in Controlling Action Potential-Driven Ca2+ Entry in Anterior Pituitary Cells J. Neurosci., August 15, 2001; 21(16): 5902 - 5915. [Abstract] [Full Text] [PDF]
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R. Millar, S. Lowe, D. Conklin, A. Pawson, S. Maudsley, B. Troskie, T. Ott, M. Millar, G. Lincoln, R. Sellar, et al. A novel mammalian receptor for the evolutionarily conserved type II GnRH PNAS, August 1, 2001; (2001) 141048498. [Abstract] [Full Text] [PDF]
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R. D. Horvat, D. A. Roess, S. E. Nelson, B. G. Barisas, and C. M. Clay Binding of Agonist but Not Antagonist Leads to Fluorescence Resonance Energy Transfer between Intrinsically Fluorescent Gonadotropin-Releasing Hormone Receptors Mol. Endocrinol., May 1, 2001; 15(5): 695 - 703. [Abstract] [Full Text]
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 K. W. Cheon, H.-S. Lee, I. S. Parhar, and I. S. Kang Expression of the second isoform of gonadotrophin-releasing hormone (GnRH-II) in human endometrium throughout the menstrual cycle Mol. Hum. Reprod., May 1, 2001; 7(5): 447 - 452. [Abstract] [Full Text] [PDF]
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L. Wang, J. Bogerd, H. S. Choi, J. Y. Seong, J. M. Soh, S. Y. Chun, M. Blomenröhr, B. E. Troskie, R. P. Millar, W. H. Yu, et al. Three distinct types of GnRH receptor characterized in the bullfrog PNAS, December 14, 2000; (2000) 11508498. [Abstract] [Full Text]
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J. N. Hislop, M. T. Madziva, H. M. Everest, T. Harding, J. B. Uney, G. B. Willars, R. P. Millar, B. E. Troskie, J. S. Davidson, and C. A. McArdle Desensitization and Internalization of Human and XenopusGonadotropin-Releasing Hormone Receptors Expressed in {{alpha}}T4 Pituitary Cells Using Recombinant Adenovirus Endocrinology, December 1, 2000; 141(12): 4564 - 4575. [Abstract] [Full Text] [PDF]
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T. Madigou, E. Mañanos-Sanchez, S. Hulshof, I. Anglade, S. Zanuy, and O. Kah Cloning, Tissue Distribution, and Central Expression of the Gonadotropin-Releasing Hormone Receptor in the Rainbow Trout (Oncorhynchus mykiss) Biol Reprod, December 1, 2000; 63(6): 1857 - 1866. [Abstract] [Full Text]
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R. Sosnowski, P. L. Mellon, and M. A. Lawson Activation of Translation in Pituitary Gonadotrope Cells by Gonadotropin-Releasing Hormone Mol. Endocrinol., November 1, 2000; 14(11): 1811 - 1819. [Abstract] [Full Text]
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S. H. Hoffmann, T. t. Laak, R. Kühne, H. Reiländer, and T. Beckers Residues within Transmembrane Helices 2 and 5 of the Human Gonadotropin-Releasing Hormone Receptor Contribute to Agonist and Antagonist Binding Mol. Endocrinol., July 1, 2000; 14(7): 1099 - 1115. [Abstract] [Full Text]
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F. E. M. Rebers, P. T. Bosma, W. van Dijk, H. J. T. Goos, and R. W. Schulz GnRH stimulates LH release directly via inositol phosphate and indirectly via cAMP in African catfish Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2000; 278(6): R1572 - R1578. [Abstract] [Full Text] [PDF]
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J. Cui, R. G. Smith, G. R. Mount, J.-L. Lo, J. Yu, T. F. Walsh, S. B. Singh, R. J. DeVita, M. T. Goulet, J. M. Schaeffer, et al. Identification of Phe313 of the Gonadotropin-Releasing Hormone (GnRH) Receptor as a Site Critical for the Binding of Nonpeptide GnRH Antagonists Mol. Endocrinol., May 1, 2000; 14(5): 671 - 681. [Abstract] [Full Text]
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B. E. Troskie, J. P. Hapgood, R. P. Millar, and N. Illing Complementary Deoxyribonucleic Acid Cloning, Gene Expression, and Ligand Selectivity of a Novel Gonadotropin-Releasing Hormone Receptor Expressed in the Pituitary and Midbrain of Xenopus laevis Endocrinology, May 1, 2000; 141(5): 1764 - 1771. [Abstract] [Full Text] [PDF]
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J. Carolsfeld, J. F. F. Powell, M. Park, W. H. Fischer, A. G. Craig, J. P. Chang, J. E. Rivier, and N. M. Sherwood Primary Structure and Function of Three Gonadotropin-Releasing Hormones, Including a Novel Form, from an Ancient Teleost, Herring Endocrinology, February 1, 2000; 141(2): 505 - 512. [Abstract] [Full Text] [PDF]
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H.-O. Chung, Q. Yang, K. J. Catt, and K. K. Arora Expression and Function of the Gonadotropin-releasing Hormone Receptor Are Dependent on a Conserved Apolar Amino Acid in the Third Intracellular Loop J. Biol. Chem., December 10, 1999; 274(50): 35756 - 35762. [Abstract] [Full Text] [PDF]
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M. Blomenröhr, A. Heding, R. Sellar, R. Leurs, J. Bogerd, K. A. Eidne, and G. B. Willars Pivotal Role for the Cytoplasmic Carboxyl-Terminal Tail of a Nonmammalian Gonadotropin-Releasing Hormone Receptor in Cell Surface Expression, Ligand Binding, and Receptor Phosphorylation and Internalization Mol. Pharmacol., December 1, 1999; 56(6): 1229 - 1237. [Abstract] [Full Text]
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C. A. Flanagan, W. Zhou, L. Chi, T. Yuen, V. Rodic, D. Robertson, M. Johnson, P. Holland, R. P. Millar, H. Weinstein, et al. The Functional Microdomain in Transmembrane Helices 2 and 7 Regulates Expression, Activation, and Coupling Pathways of the Gonadotropin-releasing Hormone Receptor J. Biol. Chem., October 8, 1999; 274(41): 28880 - 28886. [Abstract] [Full Text] [PDF]
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F. P. Pralong, F. Gomez, E. Castillo, S. Cotecchia, L. Abuin, M. L. Aubert, L. Portmann, and R. C. Gaillard Complete Hypogonadotropic Hypogonadism Associated with a Novel Inactivating Mutation of the Gonadotropin-Releasing Hormone Receptor J. Clin. Endocrinol. Metab., October 1, 1999; 84(10): 3811 - 3816. [Abstract] [Full Text] [PDF]
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K. K. Arora, H.-O. Chung, and K. J. Catt Influence of a Species-Specific Extracellular Amino Acid on Expression and Function of the Human Gonadotropin-Releasing Hormone Receptor Mol. Endocrinol., June 1, 1999; 13(6): 890 - 896. [Abstract] [Full Text]
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J. D. Neill, L. C. Musgrove, L. W. Duck, and J. C. Sellers High Efficiency Method for Gene Transfer in Normal Pituitary Gonadotropes: Adenoviral-Mediated Expression of G Protein-Coupled Receptor Kinase 2 Suppresses Luteinizing Hormone Secretion Endocrinology, June 1, 1999; 140(6): 2562 - 2569. [Abstract] [Full Text]
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N. Illing, B. E. Troskie, C. S. Nahorniak, J. P. Hapgood, R. E. Peter, and R. P. Millar Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus) PNAS, March 2, 1999; 96(5): 2526 - 2531. [Abstract] [Full Text] [PDF]
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S. Nelson, R. D. Horvat, J. Malvey, D. A. Roess, B. G. Barisas, and C. M. Clay Characterization of an Intrinsically Fluorescent Gonadotropin-Releasing Hormone Receptor and Effects of Ligand Binding on Receptor Lateral Diffusion Endocrinology, February 1, 1999; 140(2): 950 - 957. [Abstract] [Full Text]
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X. Lin and P. M. Conn Transcriptional Activation of Gonadotropin-Releasing Hormone (GnRH) Receptor Gene by GnRH: Involvement of Multiple Signal Transduction Pathways Endocrinology, January 1, 1999; 140(1): 358 - 364. [Abstract] [Full Text]
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X. Lin, J. A. Janovick, and P. M. Conn Mutations at the Consensus Phosphorylation Sites in the Third Intracellular Loop of the Rat Gonadotropin-Releasing Hormone Receptor: Effects on Receptor Ligand Binding and Signal Transduction Biol Reprod, December 1, 1998; 59(6): 1470 - 1476. [Abstract] [Full Text]
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K. K. Arora, L. Z. Krsmanovic, N. Mores, H. O'Farrell, and K. J. Catt Mediation of Cyclic AMP Signaling by the First Intracellular Loop of the Gonadotropin-releasing Hormone Receptor J. Biol. Chem., October 2, 1998; 273(40): 25581 - 25586. [Abstract] [Full Text] [PDF]
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, •) and
[Gln8]-GnRH (•,
) to membranes from wild-type
(broken line) or mutant (unbroken line)
receptors. [Reprinted with permission of the publisher from C. A.
Flanagan et al.: J Biol Chem
269:22636–22641, 1994 (186).]
; Leu3.32(121)-receptor 
;
Gln3.32(121)-receptor 
;
Arg3.32(121)-receptor •; wild-type receptor 
. [Reprinted with permission of the publisher from W.
Zhou et al.: Mol Pharmacol 45:165–170,
1994 (51).]