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J. Biol. Chem., Vol. 275, Issue 45, 35276-35280, November 10, 2000
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From the Department of Biochemistry and Molecular Biology, Medical
University of South Carolina, Charleston, South Carolina 29425
Received for publication, June 29, 2000
Relaxin has a unique, clearly identifiable, mixed
function receptor-binding region comprising amino acid residues that
evolve sequentially from the central portion of the B chain Relaxin, a small, two-chain protein, the physiological mediator of
parturition in most mammals (1), has recently been shown to influence
significantly the symptoms of scleroderma (2, 3). Since its discovery
(4) relaxin has provided a share of unusual features including an
insulin-like structure (5-9) and a receptor-binding site composed of
two charged residues, i.e. arginine in position B13 and B17
(10, 11). The binding residues are positioned one turn apart on the
major B chain helix and are projecting parallel into the surrounding
water (12). This observation led to the suggestion that relaxin would
bind to the receptor by a dual prong mechanism involving the
interaction of the guanidinium groups with two negative charges at the
bottom of a binding pocket in the receptor (11). Although both
arginines are indispensable, the fact that arginine-containing peptides would not interfere with binding suggested that other binding site
members might exist. In this paper, we are reporting that the
receptor-binding site of relaxin includes isoleucine in position B20,
which is located three-quarter of one turn further toward the
C-terminal end of the same helix so that the hydrophobic side chain
opposes the two arginines forming a quasi-prehensile unit that points
to a novel binding mechanism. Evidence presented in this paper supports
the conclusion that Ile-B20 is as important for receptor-binding as
either of the critical arginines and that the relaxin/receptor
interaction is trivalent.
Materials
Amino acid derivatives were purchased from either Advanced
ChemTech (Louisville, KY), Bachem Bioscience (Torrance CA), or Nova
Biochem (San Diego, CA). Solvents for peptide synthesis and HPLC1 were Burdick and
Jackson high quality grade. Reagents for peptide synthesis were
purchased from PerkinElmer Life Sciences. Other chemicals and reagents
were of analytical grade.
Methods
Peptide Syntheses
Human relaxin B29 and B33, Gln-B14, Asp-B14, and
GRER-dpp insulin were synthesized as described (13,
14). All other human relaxin derivatives and GRERI-dpp-insulin B chain
were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl)
chemistry using trifluoroacetic acid labile protecting groups (15, 16)
for all side chains except unprotected tryptophan, methionine
sulfoxides, and S-acetamidomethyl cysteine (B11). The
synthesis was performed on an ABI model 433A protein synthesizer
starting with 0.25 mmol of peptide on the resin up to residue B21.
Thereafter the resin was split into three equal portions, each of which
was used to produce one B chain analog using the standard 0.1-mmol
chemistry protocol. The peptidyl resin was deprotected with
trifluoroacetic acid/ethanedithiol/thioanisole/phenol/water (10:0.25:0.5:0.75:0.5 v/v/v/v/v) (17) for 2 h at room temperature, the resin was filtered off, and the peptide was collected by ether precipitation and dried and purified by preparative HPLC (yield, 15-25
mg). The B chain was dissolved in 5 ml of 1 M acetic acid, and 20 mg of 2,2'-dipyridyldisulfide in 2 ml of methanol added, and the
solution was stirred for 30 min at room temperature. The mixture was
separated by gel filtration on Sephadex G25sf in 1 M acetic
acid and lyophilized (yield, 90-100%). The partially protected B
chain carried an acetamidomethyl group in cysteine B11, a
2-pyridinesulfenyl group in cysteine B23, and sulfoxides in the
methionine side chains.
The A chain with the intact intra-chain disulfide bond, an
acetamidomethyl group in position A11, and a sulfhydryl group in position A24 was prepared according to the literature (13, 14). The A
chain was dissolved in 0.1 M acetic acid, pH 4.5, in 8 M guanidinium chloride (5 mg/ml) and added to the dry B
chain (1:1 molar ratio), which dissolved instantaneously. The reaction
was stirred for 24 h at 37 °C, and the products were separated
on Sephadex G50sf in 1 M acetic acid, followed by HPLC on
Synchropak RP-P C18 (10 × 250 mm) (yield, 50% based on the
chains). To remove the acetamidomethyl groups partially protected
relaxin (9 mg) was dissolved in 0.9 ml of 0.1 M HCl,
diluted with 6.1 ml of glacial acetic acid and 2 ml of 50 mM iodine in glacial acetic acid were added. After 15 min
at room temperature excess iodine was reduced by pouring the reaction
mixture slowly into a stirred solution of 40 ml of 0.1 M
ascorbic acid. The relaxin was desalted on Sephadex G25sf in 1 M acetic acid, lyophilized, and further purified by reversed phase HPLC (yield, 1.55 mg; 17.2% for relaxin and 39.4% for
GRERI-dpp insulin). The methionine sulfoxides were reduced with
ammonium iodide in 90% trifluoroacetic acid (13), and the relaxin
analog was HPLC purified (yield, 60-80%).
High Performance Liquid Chromatography
Semipreparative HPLC was performed on Synchropak RP-P
(C18, 10 × 250 mm). The solvent system consisted of 0.1%
trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic
acid in 83% acetonitrile (solvent B). These solvents were used unless
stated otherwise. The flow rate was 3 ml/min, and a 30-min linear
gradient from 30 to 50% B was employed for all separations. Peptides
were detected by UV absorbance at 226 nm.
Chemical Analyses of the Relaxin Analogs
Analytical HPLC--
Two different HPLC systems were used.
System 1: a Bakerbond widebore C18 column (4.1 × 250 mm) was used in combination with a Waters HPLC system. About
10-20 µg of the peptide was injected and separated using a 30-min
linear gradient from 20 to 60% B at a flow rate of 1 ml/min. The
effluent was monitored by UV absorbance at 220 nm.
System 2 consisted of an ABI model 130A chromatograph equipped with an
Aquapore 300 (2.1 mm x 30 mm) C8 column. About 1-2 µg of the
corresponding relaxin was applied via an automatic sample injector.
Separation was achieved at a flow of 100 µl/min, and the eluate was
detected by UV absorbance at 230 nm. Intact relaxins were separated
using a 60-min linear gradient from 25-45% B.
For reduction 2 µg of the protein was dissolved in 30 µl water and
30 µl of 50 mM DTT in 0.2 M Tris/HCl at pH
8.6 in 6 M guanidinium chloride was added. After 60 min at
37 °C the solution was acidified with 10 µl of glacial acetic acid
and the product separated by reversed phase HPLC (system 2) using a
60-min linear gradient from 25-60% B.
For tryptic digestion and peptide mapping 2 µg of the relaxin was
dissolved in 20 µl of 25 mM Tris/HCl at pH 7.5. Tosylphenylalanyl chloromethyl ketone-treated trypsin (EC
3.4.21.4) (100 ng in 2 µl of 50 mM
NH4HCO3, E:S 1:20) was added, and the digest
was maintained at 37 °C for 1 h. Hydrolysis was stopped by the
addition of 38 µl of 0.1% trifluoroacetic acid, and 50 µl were
used for analysis in HPLC (system 2). Tryptic fragments were separated using a 40-min linear gradient from 0 to 40% B.
Amino Acid Composition--
Peptides were hydrolyzed in vapor
phase 6 M HCl containing 0.1% phenol for 1 h at
150 °C. The amino acids were modified with phenylisothiocyanate and
separated by HPLC (Pico·Tag system, Waters).
Sequence Analysis--
Phe-B20 or Ala-B20 relaxin was sequenced
using a Procise protein sequencer (PerkinElmer Life Sciences) connected
to an inline phenylthiohydantoin analyzer.
Protein Determination--
Protein concentrations were measured
by UV spectroscopy using an Olis Cary-15 spectrophotometer conversion
(On-Line Instrument Systems, Inc.). Relaxin analogs (0.2-0.5 mg/ml)
were dissolved in water. The specific absorption coefficient was
calculated with 1.95 cm CD Spectroscopy--
CD spectra were measured on a Jasco J710
spectrapolarimeter at a resolution of 0.2 nm, with a bandwidth of 2 nm.
Ten spectra were averaged for each derivative. For far UV spectroscopy
(250-190 nm), the relaxin analogs were dissolved in 25 mM
Tris/HCl, pH 7.5, at a concentration of 0.0833 mg/ml using a cell of
0.1 cm pathlength. Mean residue ellipticity was calculated
according to the literature (18). Protein concentrations were derived from UV spectroscopy and confirmed by amino acid analysis after total
acid hydrolysis.
Matrix-assisted Laser Desorption/Ionization Mass
Spectrometry--
Relaxin analogs (1 µg/µl) were dissolved in
0.1% trifluoroacetic acid and mixed with 50 mM
Biochemical Characterization
Tracer Preparation--
Phe-A3,Tyr-B30 human relaxin protected
at the tryptophan (formyl) and methionine side chains (sulfoxides) was
synthesized as described for the human relaxin synthesis (13).
Radioactive labeling of Tyr-B30 with 125I Receptor-binding Assays--
were performed on crude membrane
preparations of mouse brain (20). Two freshly dissected mouse brains
were dropped into 15 ml of chilled homogenizing buffer (25 mM Hepes, 0.14 M NaCl, 5.7 mM KCl,
8 mg/liter soybean trypsin inhibitor, supplemented with 0.25 M sucrose and 0.4 mM phenylmethylsulfonyl
fluoride, pH 7.5) and homogenized using a Polytron homogenizer at
position 7 for 10 s. After centrifugation for 10 min at 4 °C
and 700 × g, the supernatant was collected. The pellet
was again homogenized in 10 ml, the process was repeated, and the
supernatants were pooled. The crude membranes were collected by
centrifugation at 20,000 × g for 60 min at 4 °C,
the supernatant was discarded, and the pellet was suspended in 25 ml of
25 mM Hepes buffer without sucrose. After a second
centrifugation at 20,000 × g for 60 min at 4 °C,
the supernatant was discarded, and the pellet of each vial was
suspended in 1.5 ml of ice-cold binding buffer (25 mM Hepes, 0.14 M NaCl, 5.7 mM KCl, 2.8 mM glucose, 1.6 mM CaCl2, 25 µM MgCl2, and 1.5 mM
MnCl2) supplemented with 1% bovine serum albumin and 0.2 mM phenylmethylsulfonyl fluoride). The pellets of six
brains were pooled into a 6 ml polypropylene vial, chilled on ice, and
homogenized with a hand-held Polytron for 20-30 s at maximum speed.
Thereafter aliquots of 1.4 ml of the suspension were distributed into
1.5-ml Eppendorf vials and kept on ice. One vial was used for one
dose-response curve. In general, six mouse brains were sufficient to
generate four dose-response curves, each consisting of nine duplicate points.
Assays were performed in 1.5-ml Eppendorf vials. 40 µl of various
concentrations of relaxin or analogs, 20 µl of tracer (60,000-80,000 cpm; final concentration, 125-165 pM), and 60 µl of
crude membranes were added, gently mixed and incubated for 1 h at
room temperature. Thereafter 1 ml of ice-cold wash buffer (25 mM Hepes, 0.14 M NaCl, 5.7 mM KCl,
and 0.2% bovine serum albumin) was added, and the membranes were
collected by centrifugation at 14,000 rpm for 10 min in an Eppendorf
centrifuge. The supernatant was discarded, and the tip of the vial was
cut and counted in a Mouse Symphysis Pubis Assay--
Mouse interpubic ligament
assays were carried out as described by Steinetz et al.
(22), using virgin female mice. Mice were primed with 5 µg of
estrogen cypionate in 100 µl of sesame oil and 5 days later were
injected subcutaneously with relaxin or relaxin analogs in 100 µl of
1% benzopurpurin 4B or with 1% benzopurpurin 4B alone as control.
After 16 h the mice were killed in an atmosphere of
CO2, the symphyses pubis were dissected free of adhering
tissue, and the distance between the interpubic bones was measured with a dissecting microscope fitted with transilluminating fiber optics.
The evidence that two arginine residues (B13 and B17) on the
surface of the B chain helix are in the relaxin-receptor interaction site (11, 13) pointed to a novel binding mechanism. The x-ray structure
of human relaxin shows that these arginines are located on the edge of
the dimerization surface (12), which suggests that relaxin acts as a
monomer and that the two side chains would project away from the
molecular surface. Although it is clear that these two B chain
arginines are indispensable, both in terms of charge and geometry, they
are not sufficient for binding. For example, helical peptides with two
arginines in positions i and i+4 do not compete
for the relaxin receptor-binding site regardless of concentration.
Conversely, relaxins from different species that show more than 50%
sequence differences still bind the test (mouse) receptor quite well.
The display of the Connolly surface (23) derived from the x-ray
structure gives the impression that the two arginines form a contiguous
surface feature in the binding region of relaxin together with Glu-B14,
Val-B16, and Ile-B20 (Fig. 1). Ile-B20
was not an absolutely constant feature, but replacement was rare and
only with large hydrophobic residues such as Leu in hamster (24) and
Val in porcine relaxin (6). Positions B14 and B16 would show a glycine
and alanine, respectively, in a few natural relaxins so that these
positions seemed less critical (25).
To test our ideas we have synthesized the several human relaxin analogs
(Fig. 2) replacing Glu-B14 with either
Ala, Gln, or Asp, replacing valine in position B16 with Ala, and
replacing Ile in position B20 with Ala. The results of the receptor
binding assays (Fig. 3) on crude membrane
preparations of mouse brain suggested that positions B14 and B16 could
be excluded as active site residues. In contrast, substitution of Ala
for Ile-B20 reduced receptor binding by three orders of magnitude
indicating that Ile-B20 is as important for receptor interaction as the
arginines B13 and B17. This conjecture found impressive confirmation
when we redesigned and synthesized our insulin-relaxin Zwitterhormon (GRER-dpp) (14) with Ile instead of Tyr in the position corresponding to B20 and found significant receptor-binding in the mouse brain receptor assay. In fact the binding curve for (GRERI-dpp) runs parallel
to that of relaxin (Fig. 4), whereas the
Zwitterhormon without Ile-B20 recognized only the rat relaxin receptor
as previously reported (14).
The Relaxin Receptor-binding Site Geometry Suggests a Novel
Gripping Mode of Interaction*
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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-helix. Two arginine residues in positions B13 and B17 that project like forefinger and middle finger from the helix provide the electrostatic element opposed by the hydrophobic (thumb) element isoleucine (B20),
offset from the arginines by about 40°. The binding intensity of
relaxin to its receptor decreases by 3 orders of magnitude if alanine
is substituted for the newly discovered binding component isoleucine in
position B20. The arginine residues cannot be replaced by other
positive charges, nor can the guanidinium group be presented on a
longer or shorter hydrocarbon chain. In contrast, the hydrophobic interaction is incremental in nature, and the contribution to the total
binding energy is roughly proportional to the number of hydrocarbon
units in the side chain. It appears that a hydrophobic surface exists
on the receptor that offers optimal van der Waals' interaction with
-branched hydrophobic amino acids. The binding energy increases
roughly 10-fold with each methylene group whereby -branching is more
effective per surface unit than chain elongation. Aromatic side chains
appear to demarcate the extent of the binding region in so far as
residues larger than phenylalanine decrease receptor binding. The
exceptional clarity of binding site geometry in relaxin makes for an
excellent opportunity to design peptido-mimetics.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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2 mg1 for B33
relaxin and relaxin analogs and 2.19 cm2
mg1 for B29 relaxin analogs.
-cyano-4-hydroxycinnamic acid in 80% acetonitrile (1:3 v/v). 1 µl
was placed on a sample probe and air dried. Mass spectra were acquired
with a Voyager-DE Biospectrometry Workstation (Perseptive Biosystems).
Analyses were performed at the MUSC Mass Spectrometry Facility.
was
performed by the chloramine T method, followed by the removal of the
indole protecting groups (19). Phe-A3-125I-Tyr-B30 relaxin
di-sulfoxide was isolated by HPLC on an Aquapore 300 column using a
60-min linear gradient from 25 to 40% B. The eluate was collected into
100 µl of a 1% bovine serum albumin solution in water. For
receptor-binding assays this tracer was remade every 2 weeks.
-counter. Nonspecific binding was determined in
the presence of 2600 nM B33 human relaxin. In a typical
experiment total binding was 7-10% of the total radioactivity added,
and specific binding was 35-50% of the total binding. Each point was
measured in duplicate, and each analog was determined in at least three
independent experiments. As a control each set of experiments contained
a dose-response curve of human relaxin. Data were averaged and fitted
as described by De Lean et al. (21).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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View larger version (96K):
[in a new window]
Fig. 1.
Three-dimensional structure of human relaxin
with view on the B chain helix emphasizing Arg-B13, Arg-B17, and
Ile-B20 in blue. The arrow
marks the purported path of the binding ridge on the receptor.
View larger version (12K):
[in a new window]
Fig. 2.
Sequence of human relaxin II, human insulin
and a insulin-relaxin hybrid (GRERI-dpp insulin) in which relaxin
residues are substituted for the corresponding insulin residues.
Circles indicate relaxin residues that were investigated
during this study. Z, pyroglutamine).
View larger version (30K):
[in a new window]
Fig. 3.
Receptor binding assays of B14, B16, and B20
modified relaxins using crude membrane preparations of mouse brain and
125I-Phe-A3-Tyr-B30 human relaxin for tracer. Three
independent dose-response curves of each analog were averaged.
View larger version (22K):
[in a new window]
Fig. 4.
Receptor binding assays of GRERI-dpp insulin
on crude membrane preparations of mouse brain using
125I-Phe-A3-Tyr-B30 human relaxin for tracer. The
effect was compared with human relaxin and GRER-dpp insulin, which were
run in parallel. Three independent dose-response curves were acquired.
GRER-dpp insulin lacks the critical Ile-B20.
Probing for fine structural binding requirements as concerns position
B20, we extended alanine by one methylene group (-aminobutyric acid)
and by three methylene groups (norleucine) (Fig.
5). Remarkably, each CH2
group gives a 10-fold increase in binding energy. Reducing the chain
length from norleucine and adding a -branch (valine) resulted in a
further 2.5-fold increase in binding intensity, suggesting that bulk at
the base of the side chain is favorable. In line with this argument we
noted that threonine, which is isosteric with valine, produced a
significant improvement over alanine despite its polar character at the
-carbon. On the other hand, the larger surface of phenylalanine does
not compensate for the missing -branch (Fig. 5). Plotting the log of
the binding affinity (nM) against the surface area shows a
nearly linear relationship from alanine to isoleucine with norleucine
and phenylalanine lying outside possibly because of size limitations
(Fig. 6). We have synthesized a B20
p-benzoylphenylalanine relaxin derivative that, despite its
hydrophobic character, does not bind, possibly because it exceeds the
dimensions of the surface on the receptor and thus prohibits the proper
alignment of Arg-B13 and Arg-B17 with the binding site.
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These considerations invoke the rather unique but certainly plausible
mode of prehensile binding action at the molecular level. Viewed normal
to the axis of the B chain -helix, comparison with portions of a
human hand suggests itself, with arginine fingers on one side opposed
by a thumb that, if diminished in surface, will cause reduced holding
power of the ligand to its receptor. The idea also invites the proposal
that relaxin interacts with a ridge on the receptor as opposed to the
more commonly found pocket. One is hard pressed to explain the drastic
destabilization of the di-arginine-mediated relaxin/receptor
interaction by removal of one member of the binding triad (Ile) if
relaxin slides into a deep binding pocket on the receptor surface.
To confirm that our studies are based upon relaxin derivatives with the proper structure, the homogeneity of each analog was verified in two reversed phase HPLC systems. Upon reduction each analog yielded two chains; the A chain showed identical retention times for all analogs, whereas the retention times of B chains differed. Tryptic digest and peptide mapping by HPLC of the B (20) relaxin analogs showed the expected difference for the C-terminal cystinyl peptide A (23-24)/B (18-30), and all other fragments were identical. Mass spectrometry indicated the correct molecular mass of each analog. Amino acid analysis of the total acid hydrolysate resulted in the expected amino acid composition, and sequence analysis of Ala-B20 and Phe-B20 relaxin confirmed the structure.
The results of CD spectroscopy shown in Fig.
7 support the idea that the reduced
affinity is not due to a conformational change. In contrast, the
dichroic intensity of Phe-B20 relaxin is reduced (Fig.
8), and a red shift of the maximum to 197 nm is observed as well as a reduced maximum to minimum ratio
(
197 nm/209 nm = 0.93) when compared
with human relaxin (195 nm/209 nm = 1.71). The changes seem, however, mostly quantitative and of such
nature that this analog retained a relatively high potency.
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To assure that binding would be an indicator of bioactivity, a selected
number of analogs were tested in the mouse symphysis pubis assay.
Relaxin derivatives with modification in positions B14 (Gln and Asp),
B16 (Ala), and Phe(B20) were active at a dose of 1 µg/mouse, whereas
Ala-B20 relaxin was inactive at a dose of 5 µg/mouse (Fig.
9). These results are in full agreement
with the receptor binding assays and suggest that binding may be
synonymous with bioactivity.
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We conclude that the relaxin receptor-binding site comprises three
crucial binding residues, Arg-B13, Arg-B17, and Ile-B20, which form a
triangular contact region on the relaxin surface (Fig. 1). Remarkably,
the components of this binding site are strongly hydrophilic on one
side, opposed by a strongly hydrophobic component on the other, and all
held in proper relation to each other by the geometry of the same
-helix.
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ACKNOWLEDGEMENTS |
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We thank Robert Bracey and George Fullbright for technical assistance and Dr. Kevin Schey and the mass spectrometry facility at the Medical University of South Carolina for help. Computergraphics were made possible by the Biomolecular Computing Resource at the Medical University of South Carolina.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 48893.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Medical University of South Carolina, 173 Ashley
Ave., P.O. Box 250509, Charleston, SC 29425. Tel.: 843-792-9929; Fax:
843-792-4322; E-mail: schwabec@musc.edu.
Published, JBC Papers in Press, August 23, 2000, DOI 10.1074/jbc.M005728200
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ABBREVIATIONS |
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The abbreviations used are: HPLC, high pressure liquid chromatography; GRER-dpp, Gly (A10), Arg(B9), Glu(B10), Arg(B13) des-pentapeptide(B26-30)insulin amide.
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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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M. A. Hossain, K. J. Rosengren, L. M. Haugaard-Jonsson, S. Zhang, S. Layfield, T. Ferraro, N. L. Daly, G. W. Tregear, J. D. Wade, and R. A. D. Bathgate The A-chain of Human Relaxin Family Peptides Has Distinct Roles in the Binding and Activation of the Different Relaxin Family Peptide Receptors J. Biol. Chem., June 20, 2008; 283(25): 17287 - 17297. [Abstract] [Full Text] [PDF]
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C. Kuei, S. Sutton, P. Bonaventure, C. Pudiak, J. Shelton, J. Zhu, D. Nepomuceno, J. Wu, J. Chen, F. Kamme, et al. R3(B{Delta}23 27)R/I5 Chimeric Peptide, a Selective Antagonist for GPCR135 and GPCR142 over Relaxin Receptor LGR7: IN VITRO AND IN VIVO CHARACTERIZATION J. Biol. Chem., August 31, 2007; 282(35): 25425 - 25435. [Abstract] [Full Text] [PDF]
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 J. D. Silvertown, J. C. Symes, A. Neschadim, T. Nonaka, J. C. H. Kao, A. J. S. Summerlee, and J. A. Medin Analog of H2 relaxin exhibits antagonistic properties and impairs prostate tumor growth FASEB J, March 1, 2007; 21(3): 754 - 765. [Abstract] [Full Text] [PDF]
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E. E. Bullesbach and C. Schwabe The Mode of Interaction of the Relaxin-like Factor (RLF) with the Leucine-rich Repeat G Protein-activated Receptor 8 J. Biol. Chem., September 8, 2006; 281(36): 26136 - 26143. [Abstract] [Full Text] [PDF]
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M. P. Del Borgo, R. A. Hughes, R. A. D. Bathgate, F. Lin, K. Kawamura, and J. D. Wade Analogs of Insulin-like Peptide 3 (INSL3) B-chain Are LGR8 Antagonists in Vitro and in Vivo J. Biol. Chem., May 12, 2006; 281(19): 13068 - 13074. [Abstract] [Full Text] [PDF]
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 R. A. Bathgate, R. Ivell, B. M. Sanborn, O. D. Sherwood, and R. J. Summers International Union of Pharmacology LVII: Recommendations for the Nomenclature of Receptors for Relaxin Family Peptides. Pharmacol. Rev., March 1, 2006; 58(1): 7 - 31. [Abstract] [Full Text] [PDF]
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 M. L. Halls, C. P. Bond, S. Sudo, J. Kumagai, T. Ferraro, S. Layfield, R. A. D. Bathgate, and R. J. Summers Multiple Binding Sites Revealed by Interaction of Relaxin Family Peptides with Native and Chimeric Relaxin Family Peptide Receptors 1 and 2 (LGR7 and LGR8) J. Pharmacol. Exp. Ther., May 1, 2005; 313(2): 677 - 687. [Abstract] [Full Text] [PDF]
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E. E. Bullesbach and C. Schwabe The Trap-like Relaxin-binding Site of the Leucine-rich G-protein-coupled Receptor 7 J. Biol. Chem., April 8, 2005; 280(14): 14051 - 14056. [Abstract] [Full Text] [PDF]
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C. Liu, C. Kuei, S. Sutton, J. Chen, P. Bonaventure, J. Wu, D. Nepomuceno, F. Kamme, D.-T. Tran, J. Zhu, et al. INSL5 Is a High Affinity Specific Agonist for GPCR142 (GPR100) J. Biol. Chem., January 7, 2005; 280(1): 292 - 300. [Abstract] [Full Text] [PDF]
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 C. Liu, J. Chen, C. Kuei, S. Sutton, D. Nepomuceno, P. Bonaventure, and T. W. Lovenberg Relaxin-3/Insulin-Like Peptide 5 Chimeric Peptide, a Selective Ligand for G Protein-Coupled Receptor (GPCR)135 and GPCR142 over Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 7 Mol. Pharmacol., January 1, 2005; 67(1): 231 - 240. [Abstract] [Full Text] [PDF]
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 S. Hombach-Klonisch, J. Schon, A. Kehlen, S. Blottner, and T. Klonisch Seasonal Expression of INSL3 and Lgr8/Insl3 Receptor Transcripts Indicates Variable Differentiation of Leydig Cells in the Roe Deer Testis Biol Reprod, October 1, 2004; 71(4): 1079 - 1087. [Abstract] [Full Text] [PDF]
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 O. D. Sherwood Relaxin's Physiological Roles and Other Diverse Actions Endocr. Rev., April 1, 2004; 25(2): 205 - 234. [Abstract] [Full Text] [PDF]
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S. Sudo, J. Kumagai, S. Nishi, S. Layfield, T. Ferraro, R. A. D. Bathgate, and A. J. W. Hsueh H3 Relaxin Is a Specific Ligand for LGR7 and Activates the Receptor by Interacting with Both the Ectodomain and the Exoloop 2 J. Biol. Chem., February 28, 2003; 278(10): 7855 - 7862. [Abstract] [Full Text] [PDF]
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R. A.D. Bathgate, A. L. Siebel, P. Tovote, A. Claasz, M. Macris, G. W. Tregear, and L. J. Parry Purification and Characterization of Relaxin from the Tammar Wallaby (Macropus eugenii): Bioactivity and Expression in the Corpus Luteum Biol Reprod, July 1, 2002; 67(1): 293 - 300. [Abstract] [Full Text] [PDF]
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R. A. D. Bathgate, C. S. Samuel, T. C. D. Burazin, S. Layfield, A. A. Claasz, I. G. T. Reytomas, N. F. Dawson, C. Zhao, C. Bond, R. J. Summers, et al. Human Relaxin Gene 3 (H3) and the Equivalent Mouse Relaxin (M3) Gene. NOVEL MEMBERS OF THE RELAXIN PEPTIDE FAMILY J. Biol. Chem., January 4, 2002; 277(2): 1148 - 1157. [Abstract] [Full Text] [PDF]
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