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Molecular Endocrinology 21 (7): 1699-1712
Copyright © 2007 by The Endocrine Society

Defining the LGR8 Residues Involved in Binding Insulin-Like Peptide 3

Daniel J. Scott, Tracey N. Wilkinson, Suode Zhang, Tania Ferraro, John D. Wade, Geoffrey W. Tregear and Ross A. D. Bathgate

Howard Florey Institute (D.J.S., T.N.W., S.Z., T.F., J.D.W., G.W.T., R.A.D.B.) and Department of Biochemistry and Molecular Biology (D.J.S., T.N.W., J.D.W., G.W.T., R.A.D.B.), The University of Melbourne, Melbourne, Victoria 3031, Australia

Address all correspondence and requests for reprints to: Ross A. D. Bathgate, Howard Florey Institute, The University of Melbourne, Melbourne, Victoria 3001, Australia. E-mail: r.bathgate{at}hfi.unimelb.edu.au.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The peptide hormone insulin-like peptide 3 (INSL3) is essential for testicular descent and has been implicated in the control of adult fertility in both sexes. The human INSL3 receptor leucine-rich repeat-containing G protein-coupled receptor 8 (LGR8) binds INSL3 and relaxin with high affinity, whereas the relaxin receptor LGR7 only binds relaxin. LGR7 and LGR8 bind their ligands within the 10 leucine-rich repeats (LRRs) that comprise the majority of their ectodomains. To define the primary INSL3 binding site in LGR8, its LRRs were first modeled on the crystal structure of the Nogo receptor (NgR) and the most likely binding surface identified. Multiple sequence alignment of this surface revealed the presence of seven of the nine residues implicated in relaxin binding to LGR7. Replacement of these residues with alanine caused reduced [125I]INSL3 binding, and a specific peptide/receptor interaction point was revealed using competition binding assays with mutant INSL3 peptides. This point was used to crudely dock the solution structure of INSL3 onto the LRR model of LGR8, allowing the prediction of the INSL3 Trp-B27 binding site. This prediction was then validated using mutant INSL3 peptide competition binding assays on LGR8 mutants. Our results indicated that LGR8 Asp-227 was crucial for binding INSL3 Arg-B16, whereas LGR8 Phe-131 and Gln-133 were involved in INSL3 Trp-B27 binding. From these two defined interactions, we predicted the complete INSL3/LGR8 primary binding site, including interactions between INSL3 His-B12 and LGR8 Trp-177, INSL3 Val-B19 and LGR8 Ile-179, and INSL3 Arg-B20 with LGR8 Asp-181 and Glu-229.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RELAXIN AND INSULIN-LIKE peptide-3 (INSL3) are closely related peptide hormones with a conserved two-chain (A and B chain) structure linked by disulphide bonds (1). INSL3 is abundantly expressed in the fetal testis and is involved in mediation of fetal gonad translocation to the inguinal canal during development (2, 3). Furthermore, evidence suggests that, in adults, INSL3 is involved in reproductive function, with studies in rats demonstrating that INSL3 can initiate oocyte maturation in the ovary, whereas it suppresses germ cell apoptosis in the testes (4).

The relaxin and INSL3 receptors are leucine-rich repeat-containing G protein-coupled receptor 7 (LGR7) and LGR8, respectively (5) [also named relaxin family peptide receptor RXFP1 and RXFP2, respectively (6)]. Similar to their fellow LGR family members, the glycoprotein hormone receptors LH receptor, FSH and TSH receptors, LGR7, and LGR8 possess large extracellular ectodomains containing 10 leucine-rich repeats (LRRs), within which lies the primary hormone binding site (7, 8, 9, 10, 11, 12, 13, 14). It is specific residues in the B chains of relaxin and INSL3 that bind to the LRRs, and although INSL3 has a very poor affinity for LGR7, relaxins from some species will bind with high affinity to LGR8.

LGR7 and LGR8 ligand-mediated activation is complex, with several crucial steps required to stimulate signaling. Although primary hormone binding to the LRRs is the initial stimulus of receptor signaling, at least two secondary interactions are then required to induce signal transduction. A lower-affinity interaction between the hormone and the transmembrane domains of the receptor is required, whereas the LDLa module of each receptor is essential for receptor activation (13, 14, 15, 16). Importantly, hormone binding to the LRRs of these receptors is not affected by the conformational state of the other parts of the receptor. Primary ligand binding to LGR7 and LGR8 had been convincingly demonstrated to be independent of the transmembrane domains, their LDLa modules, and the G protein coupling state of each receptor (13, 16, 17). Thus, we were confident that the primary binding of INSL3 to the LRRs of LGR8 could be investigated independently of receptor signaling.

LRRs are a common structural repeat found in proteins with a wide range of functions. Each LRR unit consists of a ß-strand and an antiparallel linear extended structure connected by short loops (18). These units stack together so that the ß-strands of consecutive LRRs lie parallel to each other, forming an arced solenoid-like structure with a concave ß-sheet lining the inside surface (19, 20). The inner face of each LRR generally adheres to a Lx1x2Lx3Lx4x5N consensus, with L representing hydrophobic residues, forming the strong hydrophobic interior of the structure, and x representing any amino acid, each exposed on the concave surface of the LRR superstructure (18). In the glycoprotein hormone receptors, it is various x residues from multiple LRRs that form the high affinity hormone binding sites (21, 22, 23, 24, 25, 26). Recently, x residues from LRRs IV–VIII of LGR7 were found to be important for high-affinity relaxin binding. Three residues projecting from the {alpha}-helix of the B-chain of relaxin are involved in receptor binding (Arg-B13, Arg-B17, and Ile-B20) and have been proposed to interact with two acidic pockets and one hydrophobic cluster in the LRRs of LGR7 (27). However, the residues in the LGR8 LRRs involved in INSL3 binding are unknown.

We recently demonstrated the importance of INSL3 His-B12, Arg-B16, Val-B19, and Arg-B20 for high-affinity binding to LGR8 (28). Together, these comprise a relaxin-like binding cassette in INSL3. Unlike relaxin, however, these residues are not the only amino acids responsible for high-affinity binding. Trp-B27, at the C-terminal tail of the B-chain of INSL3, is well characterized as a critical residue for INSL3 binding to LGR8 (28, 29, 30). The unique importance of INSL3 Trp-B27 may explain why INSL3 cannot bind to LGR7 and suggests that INSL3 binds LGR8 in a different manner to relaxin on LGR7. In this paper, we used homology modeling, interspecies sequence conservation, receptor mutational analyses, and competition binding assays with mutant INSL3 peptides to define the particular LRR residues involved in the primary INSL3/LGR8 interaction.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Structural Prediction of LRR Domains
BlastP (basic local alignment search tool) alignment of the LRRs of LGR7 (residues 71–349) and LGR8 (residues 68–345), as numbered from the end of the signal peptide, against the National Center for Biotechnology Information protein database revealed that the Nogo receptor (NgR) was the most homologous protein with a known crystal structure (47% amino acid similarity and 31% identity to the LRRs of LGR7 and LGR8) (data not shown). This homology was predominantly attributable to the conserved hydrophobic residues that are staggered through the consensus sequence for typical type LRRs (LxxLxxLxLxxNxLxxLxxxoFxx, in which x represents any residue, o represents any nonpolar residue, and the ß-strand is underlined) (18).

The LRR protein sequences of LGR7 and LGR8 from human (Homo sapiens), rhesus monkey (Macaca mulatta), chimpanzee (Pan troglodytes), dog (Canis familiaris), mouse (Mus musculus), rat (Rattus norvegicus), cow (Bos taurus), opossum (Monodelphis domestica), and human NgR were aligned using ClustalW. An alignment of the residues predicted to be on the inner face of the LRRs of LGR7 and LGR8 (xxLxLxxN in the consensus sequence) is presented in Fig. 1Go.


Figure 1
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  		Fig. 1. Multiple Sequence Alignment of the Inner LRR Faces of LGR7 and LGR8

Human (H. sapiens), rhesus monkey (M. mulatta), chimpanzee (P. troglodytes), dog (C. familiaris), mouse (M. musculus), rat (R. norvegicus), cow (B. taurus), and opossum (M. domestica) LGR7 and LGR8 sequences corresponding to the inner face of their LRRs are presented, along with the homologous sequence from the human NgR. Residues postulated to be involved in relaxin binding to LGR7 that are conserved in LGR8 are indicated (*). Residues implicated by our model of INSL3 binding to LGR8 that were investigated experimentally are labeled (arrow). Black shading indicates complete conservation, whereas gray indicates there are conservative differences at the particular position.

 
Residues corresponding to x positions in the xxLxLxxN consensus sequence, protrude out from the inner surface of the LRR structure. Nine x residues from LRRs IV–VIII of LGR7 were recently implicated in binding relaxin (27). Seven of these residues, Trp-180, Ile-182, Asp-231, Glu-233, Val-253, Glu-277, and Asp-279, are highly conserved in LGR8 (Fig. 1Go). Especially of interest are LGR7 Asp-231, Glu-233, Glu-277, and Asp-279, which are thought to chelate the crucial arginine residues in the B-chain of relaxin (Arg-B13 and Arg-B17). Conservation of their counterparts in LGR8, Asp-227, Glu-229, Glu-273, and Asp-275, is potentially why relaxin can bind to LGR8 with high affinity. The strict conservation of this relaxin binding site in LGR8, specifically Trp-177, Ile-179, Asp-227, Glu-229, Glu-273, and Asp-275 (Fig. 1Go), implied that these residues may be important for INSL3 binding.

To predict how these conserved x residues are arranged in the LRR structure, the protein sequence corresponding to the LRR domain of human LGR8 was submitted to the Swiss-Model server. The 1.5a crystal structure of the NgR [Protein Data Bank (PDB) accession code 1OZN] was chosen as the preferred template structure because of its high sequence homology to LGR8. The resultant models exhibited solenoid-like structures, typical of proteins containing multiple LRRs (Fig. 2Go). The conserved relaxin binding site identified in LGR8 is labeled in Fig. 2Go. The arrangement of these residues on the inner face of the best LGR8 model was similar to the arrangement of the homologous residues in the LGR7 model reported previously (27). The main difference was that the radius of curvature in our LGR8 model was larger than the LGR7 model reported by Bullesbach and Schwabe, which was based on the ribonuclease inhibitor structure. Like the template structure used, the NgR, the overall curvature of the LGR8 models was more banana-like than the horseshoe-shaped ribonuclease inhibitor (Fig. 2Go).


Figure 2
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  		Fig. 2. Homology Model of the LRRs of LGR8

Using Swiss-Model, a molecular model of the LRRs of human LGR8 was generated using the crystal structure of the NgR as the major template structure. Swiss-Model was unable to model the LRR caps of LGR8; thus models of the LRR caps of LGR7, which display a typical LRR cap structure, have been used and are shown in gray. x residues from the inner face of the LRRs of LGR8 are shaded black. Residues that have been proposed to be crucial for relaxin binding to LGR7 and that are highly conserved in LGR8 are labeled.

 
125I-INSL3 Binding to LGR8 Mutant Receptors
Plasmids were generated encoding mutant LGR8 receptors in which Trp-177, Ile-179, Asp-227, Glu-229, Glu-273, and Asp-275 were individually replaced with alanine. An additional mutant was generated in which LGR8 Asp-227 was replaced with asparagine. These plasmids were transfected into HEK-293T cells, which were subsequently used in [125I]INSL3 binding assays and cell surface expression analysis.

All of the above LGR8 mutants exhibited significantly reduced [125I]INSL3 binding compared with LGR8 (Fig. 3AGo). Presented in order from lowest INSL3 binder to highest compared with LGR8 are as follows: LGR8 D275A (5.69 ± 0.97%, P < 0.001), LGR8 I179A (5.81 ± 0.4%, P < 0.001), LGR8 W177A (19.55 ± 0.33%, P < 0.001), LGR8 D227A (50.43 ± 1.81%, P < 0.001), LGR8 E229A (50.58 ± 1.44%, P < 0.001), LGR8 D227N (56.17 ± 1.36%, P > 0.05), and LGR8 E273A (68.06 ± 3.32%, P < 0.001). Of these mutants, only LGR8 I179A (54.4 ± 3.5%, P < 0.001) and LGR8 E229A (54.4 ± 3.5%, P < 0.001) exhibited significantly reduced cell surface expression compared with LGR8 (Fig. 3BGo). To further define the effects of these mutations on INSL3 binding, competition binding assays were performed.


Figure 3
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  		Fig. 3. [125I]INSL3 Binding to, and Cell Surface Expression of, LGR8 Mutants I

A, [125I]INSL3 binding to LGR7, LGR8, LGR8 W177A, LGR8 I179A, LGR8 D227A, LGR8 D227N, LGR8 E229A, LGR8 E273A, and LGR8 D275A. B, Cell surface expression of LGR7, LGR8, LGR8 W177A, LGR8 I179A, LGR8 D227A, LGR8 D227N, LGR8 E229A, LGR8 E273A, and LGR8 D275A. *, P < 0.001 compared with LGR8. Data are the mean ± SEM of three to four individual experiments performed in triplicate.

 
[125I]INSL3 Competition Binding to Mutant LGR8 Receptors
INSL3 binds to LGR8 through the B-chain residues His-B12, Arg-B16, Val-B19, Arg-B20, and Trp-B27. Rosengren et al. (28) used mutant INSL3 peptides, in which each of these residues was replaced with alanine, to demonstrate their contribution to INSL3/LGR8 binding. In competition binding assays on LGR8, the pIC50 values of INSL3 Ala-B16 and INSL3 Ala-B19 are 10-fold lower than that of INSL3 (Table 1Go and Fig. 4AGo) (28). INSL3 Ala12/16/20 displays a 100-fold decrease in its pIC50 compared with INSL3, whereas the pIC50 of INSL3 B1–26, which is missing INSL3 Trp-B27, is 500-fold lower than that of INSL3 (Table 1Go and Fig. 4AGo) (28). Of note is that peptides missing INSL3 His-B12 or Arg-B20 exhibit the same pIC50 values as INSL3 (28). Their contribution to LGR8 binding is only unmasked when they are removed in combination with other residues, such as in INSL3 Ala12/16/20. Importantly, these mutant INSL3 peptides exhibit no significant change to their nuclear magnetic resonance (NMR) solution structures, indicating that mutations are revealing specific receptor interactions (28).


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  		Table 1. pIC50 Values of INSL3 Peptides Binding to LGR8, LGR8 D227A/N, LGR8 W177A, LGR8 E229A, and LGR8 E273A

 

Figure 4
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  		Fig. 4. [125I]INSL3 Competition Binding to LGR8 and LGR8 D227A with Mutant INSL3 Peptides

A, Competition of [125I]INSL3 binding to LGR8 using INSL3, INSL3 Ala-B16, INSL3 Ala-B19, INSL3 AlaB12/16/20, and INSL3 B1–26 (missing Trp-B27). B, Competition of [125I]INSL3 binding to LGR8 D227A using INSL3, INSL3 Ala-B16, INSL3 Ala-B19, INSL3 AlaB12/16/20, and INSL3 B1–26 (missing Trp-B27). Data are the mean ± SEM of three to four individual experiments performed in triplicate.

 
Here we used these same mutant peptides in competition binding assays with the mutated LGR8 receptors to ascertain the specific INSL3 residues interacting with these mutated sites. We reasoned that, if the binding site for one of these INSL3 residues was disrupted in a particular LGR8 mutant, then the binding contribution of that particular INSL3 residue would be impaired or lost. This loss would result in the INSL3 mutant peptide exhibiting a similar pIC50 value to INSL3 on the same receptor. In contrast, a decreased pIC50 value would indicate that the mutated INSL3 residue was contributing to INSL3 binding at another site on the receptor. Hence, these competition binding studies will discern the specific INSL3 peptide side-chain interactions with the LRR binding sites.

A set of competition binding experiments were undertaken with all of the mutant LGR8 receptors except LGR8 I179A and LGR8 D275A, which both bound [125I]INSL3 at a level that was too low to conduct competition binding. Of the others, LGR8 D227A and LGR8 D227N produced the most interesting results. Competition binding curves for INSL3 and the mutant INSL3 peptides on LGR8 and LGR8 D227A are presented in Fig. 4Go. As expected, the INSL3 pIC50 for LGR8 D227A was significantly lower than LGR8 (8.72 ± 0.08, P < 0.05), highlighting that this site is involved in INSL3 binding. Additionally, the rank order of pIC50 values for the INSL3 mutant peptides on LGR8 D227A was different from that for LGR8 (Table 1Go and Fig. 4Go, A and B). Importantly, no significant difference was observed between the pIC50 of INSL3 and that of INSL3 Ala-B16 (8.30 ± 0.15, P > 0.05) and INSL3 AlaB12/16/20 (8.34 ± 0.07, P > 0.05) for LGR8 D227A in contrast to their lower pIC50 values than INSL3 on the wild-type receptor (Table 1Go and Fig. 4BGo) (28). Furthermore, the pIC50 values of INSL3 Ala-B19 (6.66 ± 0.08, P < 0.001) and INSL3 B1–26 (< 5) were significantly lower than both the pIC50 of INSL3 for LGR8 D227A and the pIC50 of these peptides for the wild-type receptor, indicating that these residues are the major contributors of INSL3 binding to LGR8 D227A (Table 1Go and Fig. 4BGo). Therefore, His-12, Arg-16, and Arg-20 were not contributing to INSL3/LGR8 D227A binding, clearly indicating that they were somehow involved in binding interactions with LGR8 Asp-227.

To further prove that His-12, Arg-16, or Arg-20 were involved with a specific binding interaction with LGR8 Asp-227, an LGR8 D227N mutant was generated. LGR8 D227N bound to the INSL3 peptides in a similar way to LGR8 D227A. The pIC50 values of INSL3 Ala-B16 (8.35 ± 0.04) and INSL3 AlaB12/16/20 (8.40 ± 0.03) were not significantly different from the pIC50 of INSL3 for this receptor (8.77 ± 0.16, both P > 0.05), revealing that INSL3 His-B12, Arg-B16, and Arg-B20 were not contributing to the INSL3/LGR8 D227N binding. Conversely, the pIC50 values of INSL3 Ala-B19 (6.65 ± 0.09, P < 0.05) and INSL3 B1–26 (<5) were significantly reduced from that of INSL3 (Table 1Go), indicating that INSL3 Val-B19 and Trp-B27 were the major contributors to INSL3/LGR8 D227N binding. Thus, LGR8 Asp-227 was involved in specific binding interactions with INSL3 His-12, Arg-16, or Arg-20.

Other than LGR8 D227A and LGR8 D227N, LGR8 E229A was the only receptor tested in which INSL3 bound with a pIC50 unchanged from its pIC50 on LGR8 (9.42 ± 0.28, P > 0.05) (Table 1Go). Thus, the reduced [125I]INSL3 binding LGR8 E229A exhibited (Fig. 3AGo) was most likely attributable to the low cell surface expression of this receptor (Fig. 3BGo). Interestingly, LGR8 E229A bound to all of the INSL3 mutant peptides with LGR8-like pIC50 values except INSL3 Ala-B19, which bound to LGR8 E229A with significantly lower pIC50 compared with LGR8 (7.59 ± 0.15, P < 0.01) (Table 1Go). The other mutant LGR8 receptors, LGR8 W177A and LGR8 E273A, exhibited reduced INSL3 pIC50 binding values compared with LGR8 (Table 1Go). Unlike LGR8 D227A and LGR8 D227N, however, the pIC50 values of the mutant INSL3 peptides for LGR8 W177A and LGR8 E273A were all shifted in a comparative manner to what is seen when they bind LGR8 (Table 1Go) (28), which indicated that no specific INSL3 residue was losing the ability to bind to these mutant receptors. Thus, characterization of LGR8 D227A and LGR8 D227N yielded the only conclusive evidence for a specific side-chain interaction between INSL3 and LGR8, and the homology model of the LRRs of LGR8 was used to further investigate the binding involvement of LGR8 Asp-227.

Predicting the INSL3/LGR8 Interaction
Molsoft (La Jolla, CA) ICM BrowserPro version 3.4 was used to manually dock the NMR solution structure of INSL3 (PDB accession code 2H8B) to the homology model of the LRRs of LGR8 (Fig. 5Go). LGR8 Asp-227 was seen to be located in the center of an acidic groove running across the inner face of the LRRs (Fig. 5Go). With INSL3 Arg-B16 aligned with LGR8 Asp-227, the other two basic residues from the INSL3 B-chain {alpha}-helix, His-B12 and Arg-B20, were aligned along this groove.


Figure 5
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  		Fig. 5. Surface-Rendered Homology Model of the LRRs of LGR8 Colored According to Electrostatic Charge

Red represents negative charge areas, and blue represents positively charged regions. Of note is the predicted INSL3 binding site, which is composed of a negatively charged (red) groove in the concave surface of the LRRs of LGR8 with Asp-227 (bold) at its center. A putative interaction conformation is highlighted using the NMR solution structure of the B-chain of INSL3 (black) with INSL3 Arg-B16 (dark blue) docking to LGR8 Asp-227. The location of INSL3 Trp-B27 (green) in the structure inferred that LGR8 Phe-131 and Gln-133 might be involved in binding to this INSL3 residue.

 
In this crude model, with INSL3 Arg-B16 bound to LGR8 Asp-227, INSL3 His-B12 was in close proximity to LGR8 Trp-177, INSL3 Val-B19 was close to LGR8 Ile-179, and INSL3 Arg-20 was in the vicinity of LGR8 Glu-229 as well as Asp-181. Of considerable interest was the close proximity of INSL3 Trp-B27, a crucial binding residue, to LGR8 Phe-131 and Gln-133 (Fig. 5Go). Thus, the importance of LGR8 Asp-181, Phe-131, and Gln-133 were investigated experimentally.

[125I]INSL3 Binding to LGR8 F131A and Q133A
LGR8 F131A and LGR8 Q133A mutant receptors were produced using mutagenesis. LGR8 F131A bound [125I]INSL3 at a low level compared with LGR8 (21.54 ± 1.73%, P < 0.001) (Fig. 6AGo), and the cell surface expression of this mutant was reduced compared with that of LGR8 (85.2 ± 3.6%, P < 0.001) (Fig. 6CGo). Similarly, LGR8 Q133A bound to [125I]INSL3 at a significantly lower level than LGR8 (9.67 ± 0.52%, P < 0.001) (Fig. 6AGo) and also exhibited significantly reduced cell surface expression compared with LGR8 (33.9 ± 4.6%, P < 0.001) (Fig. 6CGo).


Figure 6
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  		Fig. 6. [125I]INSL3 Binding to, and Cell Surface Expression of, LGR8 Mutants II

A, [125I]INSL3 (100 pM) binding to LGR7, LGR8, LGR8 F131A, LGR8 Q133A, LGR8 Ala-131/133, LGR8 Ala-131/133/227, LGR8 D181N, and LGR8 D181N/E229. B, [125I]INSL3 (500 pM) binding to LGR7, LGR8, LGR8 Ala-131/133, and LGR8 Ala 131/133/227. C, Cell surface expression of LGR7, LGR8, LGR8 F131A, LGR8 Q133A, LGR8 Ala-131/133, LGR8 Ala-131/133/227, LGR8 D181N, and LGR8 D181N/E229. *, P < 0.001 compared with LGR8. Data are the mean ± SEM of three to four individual experiments performed in triplicate.

 
Because both LGR8 Phe-131 and Gln-133 were predicted to be potential INSL3 Trp-B27 interactors, a mutant LGR8 receptor was produced in which both of these residues were mutated to alanine. LGR8 Ala-131/133 was also combined with LGR8 D227A to form the triple mutant LGR8 Ala-131/133/227.

Both LGR8 Ala-131/133 and LGR8 Ala-131/133/227 displayed [125I]INSL3 binding that was not significantly higher than background using both 100 pM [125I]INSL3 (0.42 ± 0.49%, P > 0.05 and –3.52 ± 1.22%, P > 0.05, respectively) and 500 pM [125I]INSL3 (0.7 ± 1.0%, P > 0.05 and 3.6 ± 0.5%, P > 0.05, respectively) (Fig. 6Go, A and B). The cell surface expression of LGR8 Ala-131/133 and LGR8 Ala-131/133/227, however, was only moderately reduced compared with LGR8 (53.1 ± 9.7%, P < 0.001 and 61.3 ± 2.4%, P < 0.001, respectively) (Fig. 6CGo). Such a loss in INSL3 binding would be expected if LGR8 lost its ability to bind to INSL3 Trp-B27.

To confirm that INSL3 Trp-B27 is specifically interacting with LGR8 F131 and LGR8 Gln-133, competition binding assays with mutant INSL3 peptides and the individual mutant receptors were performed. Based on the results above, we would expect that each of these mutant receptors would demonstrate a partial disruption in INSL3 Trp-B27 binding. As expected, INSL3 bound to LGR8 F131 with a reduced pIC50 compared with LGR8 (8.75 ± 0.20, P < 0.05) (Fig. 7AGo and Table 2Go). Importantly, INSL3 Ala-B16 (7.00 ± 0.20, P < 0.001), INSL3 Ala-B19 (6.54 ± 0.17, P < 0.001), and INSL3 AlaB12/16/20 (5.98 ± 0.50, P < 0.01) all displayed reduced pIC50 values compared with their binding to LGR8, whereas INSL3 B1–26 (6.68 ± 0.17, P > 0.05) exhibited a similar pIC50 value on both of these receptors (Table 2Go). This indicated that INSL3 His-B12, Arg-B16, Val-B19, and Arg-B20 are the major contributors to INSL3 binding to LGR8 F131A and thus that the lower pIC50 of INSL3 for LGR8 F131A was attributable to a partial disruption in INSL3 Trp-B27 binding.


Figure 7
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  		Fig. 7. [125I]INSL3 Competition Binding to LGR8 F131A and LGR8 Q133A with Mutant INSL3 Peptides

A, Competition of [125I]INSL3 binding to LGR8 F131A using INSL3, INSL3 Ala-B16, INSL3 Ala-B19, INSL3 AlaB12/16/20, and INSL3 B1–26 (missing Trp-B27). B, Competition of [125I]INSL3 binding to LGR8 Q133A using INSL3, INSL3 Ala-B16, INSL3 Ala-B19, INSL3 AlaB12/16/20, and INSL3 B1–26 (missing Trp-B27). Data are the mean ± SEM of three to four individual experiments performed in triplicate.

 

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  		Table 2. pIC50 Values of INSL3 Peptides Binding to LGR8, LGR8 F131A, LGR8 Q133a, and LGR8 D181N

 
LGR8 Q133A also bound INSL3 with a significantly lower pIC50 than its pIC50 for LGR8 (8.69 ± 0.18, P < 0.05) (Fig. 7BGo and Table 2Go). Importantly, LGR8 Q133A bound to all of the INSL3 mutant peptides in a similar way to LGR8 F131A. The pIC50 values of INSL3 Ala-B16 (7.22 ± 0.01, P < 0.001), INSL3 Ala-B19 (7.47 ± 0.20, P < 0.01), and INSL3 AlaB12/16/20 for LGR8 Q133A were all significantly lower than their pIC50 values for LGR8 (Fig. 7BGo and Table 2Go). However, as with LGR8 F131A, INSL3 B1–26 exhibited a similar pIC50 for LGR8 Q133A as it does for LGR8 (pIC50 of 6.80 ± 0.13, P > 0.05) (Fig. 7BGo and Table 2Go). The very similar peptide pIC50 profiles of LGR8 Q133A and LGR8 F131A suggested that LGR8 Phe-131 and Gln-133 are both involved in binding INSL3 Trp-B27. Thus, the loss of INSL3 binding exhibited by LGR8 Ala-131/133 was attributable to the complete loss of INSL3 Trp-B27 binding.

[125I]INSL3 Binding to LGR8 D181N
INSL3 Arg-B20 was predicted to be in close proximity to both LGR8 Asp-181 and Glu-229 (Fig. 5Go). An LGR8 D181N mutant was produced and was also combined with LGR8 D229A to form the double mutant LGR8 D181N/E229A. LGR8 D181N bound to [125I]INSL3 at a reduced level compared with LGR8 (35.7 ± 0.6%, P < 0.001) (Fig. 6AGo) and significantly disrupted cell surface expression (67.8 ± 3.0%, P < 0.001) (Fig. 6CGo). Although LGR8 D181N/E229A was expressed on the cell surface at a similar level to LGR8 D181N (59.9 ± 1.4%, P < 0.001 compared with LGR8) (Fig. 6CGo), this double mutant exhibited very low [125I]INSL3 binding (5.21 ± 0.50%, P < 0.001) compared with LGR8 (Fig. 6AGo).

To determine whether the loss of [125I]INSL3 binding observed above was a result of the loss of a specific INSL3 interaction, competition binding was undertaken. LGR8 D181N bound INSL3 with a similar pIC50 to LGR8 (9.45 ± 0.10, P > 0.05) (Table 2Go). Additionally, LGR8 D181N bound most of the mutant INSL3 peptides with similar pIC50 values to LGR8 (Table 2Go); however, like LGR8 E229A, it exhibited a lower INSL3 Ala-B19 pIC50 compared with LGR8 (7.54 ± 0.09, P < 0.01) (Table 2Go). This sensitivity to the loss of the INSL3 Val-B19 implied that this INSL3 residue may be compensating for the loss of a minor interaction in these two receptor mutants. Importantly, INSL3 Ala-B20 binds to LGR8 with a similar pIC50 to INSL3, and its binding contribution is only revealed when coupled to another INSL3 mutation. Hence, the increased INSL3 Ala-B19 pIC50 shift seen with LGR8 D181N and LGR8 E229A strongly suggested that a minor binding site such as that of INSL3 Arg-B20 was disrupted in both of these mutants.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
LGR7 and LGR8 ligand-mediated signaling involves at least three steps. Primary hormone binding is the initial driver of receptor activation and occurs between the B-chain of relaxin or INSL3 and the LRRs of LGR7 or LGR8, respectively (13, 14, 16). At least two secondary interactions are then required to stimulate each receptor. A lower-affinity interaction between the hormone and the transmembrane domains of the receptor is required, whereas the LDLa module of each receptor is essential for receptor activation (13, 14, 15, 16). Importantly, experiments have shown that primary hormone binding is completely independent of the other steps involved in receptor activation. When the ectodomains of LGR7 and LGR8 are tethered to a single membrane spanning CD8 domain, the resultant proteins can bind their hormone partners with similar affinity to the wild-type receptors (13, 17). Furthermore, LGR7 and LGR8 receptors missing their LDLa module can also bind their ligands with unchanged affinity but cannot signal (16). Thus, we were confident that the results presented here relate to the primary binding of INSL3 to the LRRs of LGR8 and that the signaling characteristics of the various mutant receptors were irrelevant in this context.

A key finding in this study was that seven of the nine residues identified to be crucial for LGR7 to bind relaxin (15) were highly conserved in LGR8 (Fig. 1Go), potentially explaining why relaxin binds LGR8 with high affinity. Recent work, including the solved solution structure of human INSL3 (28), has established that the determinants of INSL3 binding lie in the B-chain, specifically His-B12, Arg-B16, Val-B19, Arg-B20, and Trp-B27 (28, 29, 31). Of particular interest is the "relaxin-like" receptor binding cassette (His-B12, Arg-B16, and Val-B19) on the B-chain {alpha}-helix of INSL3, which offered a possible explanation for the highly conserved relaxin binding site in the LRRs of LGR8.

The NMR solution structure of INSL3 reveals that the basic residues along the B-chain {alpha}-helix, His-B12, Arg-B16, and Arg-B20, form a positively charged cluster that is most probably the initial driver of INSL3 binding to LGR8 (28). In the solution structure of INSL3, the C terminus of the B-chain, containing Trp-B27, loops back to be close to Val-B19. This region was reported to be flexible, and it was postulated that binding of the B-chain basic residues to LGR8 would stabilize Trp-B27, enabling it to interact with its binding partner(s) (28). The low LGR8 binding affinity of INSL3 B1–26, which lacks Trp-B27, indicates how important this interaction is for INSL3 binding to LGR8 (28). Although it was tempting to postulate that relaxin binding to LGR8 is attributable to basic residues along its B-chain (Arg-B13 and Arg-B17), the low affinity exhibited by rhesus monkey relaxin and rat relaxin for LGR8 (13), which both lack a C-terminal tryptophan in their B-chains, indicated that the tryptophan interaction is a unique and crucial characteristic of ligand binding to LGR8.

The conservation of relaxin binding residues in LGR8, Trp-177, Ile-179, Asp-227, Glu-229, Glu-273, and Asp-275, suggested that these residues were important for INSL3 binding to LGR8, and thus they were individually mutated to alanine. All of the mutant receptors exhibited disrupted [125I]INSL3 binding. However, this loss of binding did not give an indication into the cause of the observed loss. Rosengren et al. (28) used competition binding assays with INSL3 mutant peptides to demonstrate which INSL3 residues were contributing to wild-type LGR8 binding. Here we used these same mutant peptides in competition binding assays with the mutated LGR8 receptors to ascertain the specific INSL3 residues interacting with these mutated sites. We reasoned that, if the binding site for one of these INSL3 residues was disrupted in a particular LGR8 mutant, then the binding contribution of that particular INSL3 residue would be impaired or lost. This loss would result in the INSL3 mutant peptide exhibiting a similar pIC50 value to INSL3 on the same receptor. In contrast, a decreased pIC50 value would indicate that the mutated INSL3 residue is contributing to INSL3 binding at another site on the receptor. Hence, these competition binding studies would discern the specific INSL3 peptide side-chain interactions with the LRR binding sites.

We used this paradigm to determine the interactions (if any) that were occurring at the conserved relaxin binding site residues Trp-177 (LGR8 W177A), Asp-227 (LGR8 D227A), Glu-229 (LGR8 E229A), and Glu-273 (LGR8 E273A) of LGR8. These experiments could not be conducted for LGR8 Ile-179 (LGR8 I179A) and Asp-275 (LGR8 D275A) because their maximum INSL3 binding ability was too low to accurately conduct competition binding assays.

Of the initial group of receptor mutants, only LGR8 D227A and LGR8 D227N mutations provided evidence of a specific side-chain interaction with INSL3. The similar pIC50 values of INSL3, INSL3 Ala-B16, and INSL3 AlaB12/16/20 indicated that the binding contributions of INSL3 His-B12, Arg-B16, and Arg-B20 were lost in INSL3 binding to LGR8 D227A and LGR8 D227N because the interaction site for INSL3 His-B12, Arg-B16, and Arg-B20 was disrupted. Although LGR8 Asp-227 may interact with more than one of these INSL3 residues, it is likely that only Arg-B16 interacts with this residue, and the removal of Asp-227 causes a disruption of charge that perturbs the conformation of the surrounding residues as well. Such a chain reaction would explain why the binding of INSL3 His-12 and Arg-20 was also perturbed in LGR8 D227A and LGR8 D227N. The latter possibility became more plausible when the homology model of the LRRs of LGR8 was revisited, especially when this model was visualized in parallel with the solution structure of INSL3. LGR8 Asp-227 was located in the middle of a negatively charged groove (Fig. 5Go), and, when the structure of INSL3 was crudely docked to our model of LGR8, the {alpha}-helix of the B-chain of INSL3 was able to fit along this groove with its basic side chains protruding into the acidic pockets. We predicted that INSL3 Arg-B16, the largest contributor of these three residues to INSL3 binding, was the most likely residue that interacts with LGR8 Asp-227 (Fig. 7Go). This prediction was based on the central position of Arg-B16 in the {alpha}-helix of the B-chain of INSL3 and the competition binding results above that highlight the interaction of INSL3 Ala-B16 with LGR8 Asp-227.

This binding conformation was purely hypothetical and did not take into account the flexible nature of the peptide and receptor. However, it allowed us to predict some more INSL3 binding residues in LGR8. The location of INSL3 Trp-B27, close to LGR8 Phe-131 and Gln-133 (Fig. 5Go), was enough evidence to investigate these residues experimentally. LGR8 F131A and Q133A mutant receptors were produced, and the same experimental paradigms were applied to them. As expected, both of these receptors displayed reduced [125I]INSL3 binding compared with LGR8, and competition binding assays ascertained that the loss of binding exhibited by these receptors was attributable a partial, but not complete, disruption in INSL3 Trp-B27 binding. Hence, it was postulated that both of these residues were contributing to the Trp-B27 interaction. This was confirmed by removing both LGR8 Phe-131 and Gln-133, which resulted in complete loss of [125I]INSL3 binding at both 100 and 500 pM ligand concentration, indicative of the complete loss of INSL3 Trp-B27 binding (Fig. 6Go, A and B), confirming that INSL3 Trp-B27 binds LGR8 Phe-131 and Gln-133.

The conclusive interactions, INSL3 Arg-B16 with LGR8 Asp-227 and INSL3 Trp-B27 with LGR8 Phe-131 and Gln-133, were used as constraints to triangulate where the other INSL3 B-chain residues may be binding to LGR8 (Fig. 7Go). The first of these residues, INSL3 His-B12, was predicted to interact with LGR8 Trp-177 (Fig. 7Go). LGR8 W177A exhibited the largest INSL3 affinity shift of all of the mutant receptors (Table 1Go). The parallel pIC50 shifts seen with all of the mutant INSL3 peptides indicated that the binding contribution of no particular INSL3 residue had been lost and, coupled with the low INSL3 pIC50, was indicative of a gross structural change in this receptor. Such a change, in which all of the individual interaction points were disrupted, would remove the sensitivity required to elucidate the loss of the minor His-B12 interaction. A similar story may apply to the adjacent LGR8 residue, Ile-179, which, when replaced with alanine, resulted in a receptor that bound INSL3 at a very low level. Although this was likely the result of a similar structural perturbation, LGR8 Ile-179 was predicted to interact with INSL3 Val-B19, which is a significant contributor to the INSL3 binding site (Fig. 8Go). The large loss of INSL3 binding exhibited by LGR8 I179A (Fig. 3AGo) meant that we were unable to investigate this potential interaction experimentally. Importantly, the location of LGR8 Trp-177 and Ile-179 in our model, both in the center of the predicted INSL3 binding site (Fig. 7Go), may bolster the explanation for their drastically reduced INSL3 binding.


Figure 8
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  		Fig. 8. INSL3 Binding to LGR8

Our model interaction of INSL3 with the LRRs of LGR8 is shown with INSL3 folded back from the predicted binding site. The confirmed interactions, INSL3 Arg-B16 with LGR8 Asp-227 and INSL3 Trp-B27 with LGR8 Phe-131 and Gln-133, are indicated with bold arrows. More inferred pairings are indicated with dashed arrows and include INSL3 His-B12 with LGR8 Trp-177, INSL3 Val-B19 with LGR8 Ile-179, and INSL3 Arg-B20 with LGR8 Asp-181 and Glu-229.

 
The final minor contributor to the INSL3 binding site is INSL3 Arg-B20, which was predicted to be interacting with LGR8 Glu-229 or Asp-181 or both (Fig. 7Go). Direct evidence for this interaction could not be obtained experimentally, mainly because this is one of the minor contributors to INSL3 binding (28). As would be expected if these residues are involved in minor binding contributors, INSL3 bound both LGR8 E229A and LGR8 D181N with pIC50 values not significantly different from that on LGR8 (Tables 1Go and 2Go). This was consistent with the affinity of INSL3 Ala-20 for LGR8, which was unchanged from that of INSL3 (28). Only when coupled to the mutation of another INSL3 residue is the importance of INSL3 Arg-B20 revealed (28). The increased sensitivity of both LGR8 E229A and LGR8 D181N to the loss of INSL3 Val-B19 (Tables 1Go and 2Go) strongly supported our predicted binding model. This result suggested that Val-B19 was compensating for the loss of another minor interactor, such as Arg-B20, when INSL3 bound LGR8 D181N and LGR8 E229A (Fig. 8Go). The predicted role of these residues in INSL3 binding was further justified by the removal of LGR8 Asp-181 and Glu-229 in LGR8 D181N/E229A, which resulted in low [125I]INSL3 binding (Fig. 6AGo). Unfortunately, this low level of INSL3 binding meant that competition binding assays could not be undertaken to further define the reason for this loss. Overall, the characterization of LGR8 D181N, LGR8 E229A, and LGR8 D181N/E229A revealed that LGR8 Asp-181 and Glu-229 were involved in minor contributions to INSL3 binding. This, together with the location of these residues in the acidic groove of the LRRs of LGR8, indicated that the most probable role these residues are playing was the binding of INSL3 Arg-B20.

Interestingly, five of the seven residues that have been implicated by our model as being crucial INSL3 interaction points in LGR8 (Gln-133, Trp-177, Ile-179, Asp-227, and Glu-229) were totally conserved in LGR7 (Fig. 1Go). The residue corresponding to Phe-131 in LGR8 was not well conserved in LGR7, being Tyr-134 in human, rhesus monkey, chimpanzee, and opossum LGR7, but is Cys-134 in dog, mouse, rat, and cow LGR7 (Fig. 1Go). Considering that INSL3 Trp-27 potentially interacts at this site in LGR8, Phe-131 may be a crucial residue in determining the specificity of LGR8 for INSL3. The binding of INSL3 to LGR8 is reliant on five B-chain residues, whereas relaxin interacts with LGR7 through only three residues. We postulated that, although the LRRs of LGR7 contain most of the residues needed for INSL3 binding, these residues are not arranged in the specific surface topology needed to accommodate INSL3. In contrast, relaxin can likely bind to LGR8 through interaction of Arg-B13 and Arg-B17 with the acidic grove, which would then allow the Trp-B27 of relaxin to mimic the role of INSL3 Trp-B27.

In summary, the results presented here have conclusively defined the LGR8 LRR residues that are involved in binding to the two most important INSL3 residues, Arg-B16 and Trp-B27. Additionally, they allowed the prediction of the LGR8 residues involved in the minor binding residues in INSL3, His-B12, Val-B19, and Arg-B20. Understanding the molecular determinants of primary INSL3 binding to LGR8 is the first step to thoroughly characterize the mechanism of LGR8 signaling. A B-chain only mimetic of INSL3 was recently characterized as an LGR8 antagonist in vitro that was able to block testicular function in vivo in male rats (32). It was able to do this by competing with INSL3 for the primary binding site in LGR8. Knowledge of the residues involved in INSL3 binding to LGR8 will allow additional development of such peptides, which can be made to target the Trp-B27 and Arg-B16 sites in LGR8. Recently, the N terminus of the A-chain of INSL3 and the N-terminal LDLa module of LGR8 were shown to be crucial for LGR8 activation but not primary binding (15, 16). Combining the results presented here with those from future efforts to characterize the roles of the A-chain of INSL3 and the receptor LDLa module will lead to a more complete understanding of how these unique receptors function and thus greatly aid in the development of agonists and antagonists.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Hormones
H2 relaxin was kindly provided by BAS Medical (San Mateo, CA). INSL3 was synthesized as described previously (33) and labeled with 125I by Dr. Pierre Demeyts (Hagedorn Research Institute, Gentofte, Denmark) or Steve Sutton (Johnson and Johnson Pharmaceutical Research and Development, La Jolla, CA). INSL3 Arg->Ala-B16 (INSL3 Ala-B16), INSL3 Val->Ala-B19 (INSL3 Ala-B19), INSL3 His->Ala-B12 + Arg->Ala-B16 + Arg->Ala-B20 (INSL3 AlaB12/16/20), and the truncated INSL3 B1–26 were synthesized as described previously (28).

Secondary Structure Prediction and Molecular Modeling
The protein sequences of the LRR subdomains of LGR7 and LGR8 were submitted into the PROF secondary structure prediction server (34), and the outputs were used to estimate the positions of each ß-strand. The identity of each ß-strand was further clarified by aligning LGR7 and LGR8 protein sequences to that of their closest structural homolog, the NgR, using BlastP and by judging sequence conformity to the typical LRR ß-strand consensus, Lx1x2Lx3Lx4x5N. Molecular models of the LRRs of LGR7 and LGR8 were generated by submitting the relevant protein sequences to Swiss-Model using the first approach mode (35, 36, 37). Swiss-Model outputs were opened with the program DeepView 3.7 (38) for quality assessment. LRR domain models were analyzed using UCSF Chimera (39) and Molsoft BrowserPro version 3.4 molecular visualization software packages. Structural alignments were performed with the MatchMaker function in UCSF Chimera (39). The solution structure of INSL3 (PDB accession no. 2H8B) was manually docked to the best LGR8 LRR model using Molsoft BrowserPro version 3.4.

Sequence Conservation Analysis
The amino acid sequences of LGR7 and LGR8 from the human (H. sapiens) (Q9HBX9 and Q8WXD0), mouse (M. musculus) (AAR97515 and Q91ZZ5), and rat (R. norvegicus) (AAR97516 and AAW84088) were retrieved from the GenBank database at National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). Sequence similarity searches of the available genomes at Ensembl (http://www.ensembl.org) using TBlastN (40) using a representative full-length sequence of each receptor were conducted. Predicted orthologs of LGR7 and LGR8 from the rhesus monkey (M. mulatta), chimpanzee (P. troglodytes), dog (C. familiaris), cow (B. taurus), and opossum (M. domestica) genomes were identified (Wilkinson, T., G. W. Tregear, T. P. Speed, R. A. D. Bathgate, unpublished data). Sequences were aligned using ClustalW (41) with default parameters and shaded using Boxshade. All LGR7 and LGR8 protein sequences are numbered from the predicted signal peptide cleavage sites.

Site-Directed Mutagenesis
LGR8 mutants were generated using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) using a pcDNA3.1/zeo plasmid encoding N-terminal FLAG-tagged human LGR8 as the template for each reaction. Mutagenic primer pairs were designed following the protocol described previously (42). Reactions were undertaken as described by the manufacturer, incubated with 1 µl Dpn1 for 60 min before 1 µl was transformed into XL-1 supercompetent cells (Stratagene), which were then grown overnight on 100 µg/ml ampicillin containing agar plates. Plasmid DNA was extracted from selected clones and sequenced.

Ligand Binding Assays
HEK-293T cells were transfected with plasmids encoding the receptor of interest, and [125I]INSL3 binding assays were conducted as described previously (43). Briefly, 300,000 cells were seeded into each well of poly-L-lysine-coated 48-well plates and left to attach overnight before receptor transfection the following morning using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Twenty-four hours later, the media were aspirated from each well, and the cells washed with PBS, and treated with solutions containing 100 pM [125I]INSL3 together with a selected concentration of the unlabeled peptide of interest. Double LGR8 mutants that displayed very low INSL3 binding using 100 pM [125I]INSL3 were assayed again using 500 pM [125I]INSL3. Data are expressed as mean ± SEM of percentage specific binding of triplicate measurements pooled from at least three independent experiments. Data were analyzed using Prism (GraphPad Software, San Diego, CA), and a nonlinear regression one-site binding model was used to plot curves and calculate pIC50 values. Final pooled pIC50 and total binding data were analyzed using one-way ANOVA coupled to Newman-Keuls multiple comparison test for multiple group comparisons.

Cell Surface Expression Assays
All of the receptors produced in this study contained an N-terminal FLAG epitope (Sigma, St. Louis, MO), which does not affect the pharmacology of LGR7 or LGR8 (5, 13). This tag enabled us to quantitate the cell surface expression of each receptor. Cell surface expression assays were performed as described previously (16). Empty vector transfected cells were used to determine nonspecific background of this cell surface expression assay. All data points were performed in triplicate, and the data are expressed as the mean ± SEM from triplicate measurements pooled from three separate experiments. These values were compared using one-way ANOVA coupled to Newman-Keuls comparison test for multiple group comparisons.


   ACKNOWLEDGMENTS
 
We thank Prof. Pierre De Meyts and Dr. Steve Sutton for 125I-labeling of INSL3, Sharon Layfield for technical assistance, and Assoc. Prof. Paul Gooley for assistance with the manual docking.


   FOOTNOTES
 
This work was supported by Australian National Health and Medical Research Council Project Grants 30012 and 454375 (to R.A.D.B., J.D.W., and G.W.T.) and 350245 (to J.D.W. and R.A.D.B.). D.J.S. is a recipient of an Australian Postgraduate Award.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 1, 2007

Abbreviations: Blast, Basic local alignment search tool; INSL3, insulin-like peptide 3; LGR, leucine-rich repeat-containing G protein-coupled receptor; LRR, leucine-rich repeat; NgR, Nogo receptor; PDB, Protein Data Bank.

Received for publication February 20, 2007. Accepted for publication April 26, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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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.
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