The Trap-like Relaxin-binding Site of the Leucine-rich G-protein-coupled Receptor 7

Abstract

The pleated sheet region of the leucine-rich G-protein-coupled receptor 7 supports a relaxin-binding group of amino acids that perfectly matches the binding cassette of relaxin. Arginines B13 and B17 are each chelated by an aspartic acid/glutamic acid pair and by isoleucine B20, which, offset by a one-quarter helix turn from a straight line connecting the arginines, interacts with a cluster of hydrophobic amino acids. The binding cassette of relaxin cuts at an angle of ∼45° across five parallel leucine-rich repeats. The arginine residues 13 and 17, which evolve parallel from the B-chain α-helix of relaxin, neutralize the charge repulsion of the juxta-posed acidic groups on the receptor and thereby trigger closure of a hydrogen bonding network around the guanidinium groups. Thus, relaxin is bound by synchronized chelation of two arginines and stabilized by hydrophobic interaction of isoleucine B20 with tryptophan, isoleucine, and leucine in neighboring leucine-rich repeats of the receptor. Deletion of any one of the three features diminishes interaction to the level of nonspecific binding. This model explains the exquisite sensitivity of relaxin binding avidity to minute changes in the disposition of the guanidinium and the size dependence of the hydrophobic binding residue in position B20. The pleated sheet region of the leucine-rich G-protein-coupled receptor 7 supports a relaxin-binding group of amino acids that perfectly matches the binding cassette of relaxin. Arginines B13 and B17 are each chelated by an aspartic acid/glutamic acid pair and by isoleucine B20, which, offset by a one-quarter helix turn from a straight line connecting the arginines, interacts with a cluster of hydrophobic amino acids. The binding cassette of relaxin cuts at an angle of ∼45° across five parallel leucine-rich repeats. The arginine residues 13 and 17, which evolve parallel from the B-chain α-helix of relaxin, neutralize the charge repulsion of the juxta-posed acidic groups on the receptor and thereby trigger closure of a hydrogen bonding network around the guanidinium groups. Thus, relaxin is bound by synchronized chelation of two arginines and stabilized by hydrophobic interaction of isoleucine B20 with tryptophan, isoleucine, and leucine in neighboring leucine-rich repeats of the receptor. Deletion of any one of the three features diminishes interaction to the level of nonspecific binding. This model explains the exquisite sensitivity of relaxin binding avidity to minute changes in the disposition of the guanidinium and the size dependence of the hydrophobic binding residue in position B20. The peptide hormone relaxin supports vital processes such as heart function, coronary and peripheral blood flow, and physiological adaptation to the stresses of pregnancy (1.Dschietzig T. Stangl K. Cell. Mol. Life Sci. 2003; 60: 688-700Crossref PubMed Scopus (32) Google Scholar, 2.Bathgate R.A.D. Samuel C.S. Burazin T.C.D. Gundlach A.L. Tregear G.W. Trends Endocrinol. Metabol. 2003; 14: 207-213Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). It acts on the capillary bed as a vasodilator, which, in part, explains its anti-fibrotic action. The positive effect of this hormone on peripheral and cardiac blood flow makes relaxin, along with its receptor, a prime target for research on chronic debilitating diseases. The discovery that the orphan receptor LGR7 1The abbreviations used are: LGR7, leucine-rich G-protein-coupled receptor 7; LRR, leucine-rich repeat; 293T/17, human embryonic kidney 293T clone 17. binds relaxin specifically (3.Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. Hsueh A.J.W. Science. 2002; 295: 671-674Crossref PubMed Scopus (686) Google Scholar, 4.Hsu S.Y.T. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Bathgate R.A.D. Sherwood O.D. Hsueh A.J.W. Mol. Cell. Endocrinol. 2003; 202: 165-170Crossref PubMed Scopus (58) Google Scholar, 5.Sudu S. Kumagai J. Nishi S. Layfields S. Ferraro T. Bathgate R.A.D. Hsueh A.J.W. J. Biol. Chem. 2003; 278: 7855-7862Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) makes it possible to investigate ligand-mediated activation of a member of the group of leucine-rich repeat (LRR) receptors. This genre of receptors, including those for thyrotropin, luteotropin, and human chorionic gonadotropin, is known to have leucine-rich repeats in their ectodomains and to be anchored to the cell surface by seven helical transmembrane segments. The binding sites are thought to be large and to cover much of the inner aspects of the concave pleated sheet structures (6.Smith G. Campillo M. Govaerts C. Janssens V. Richter C. Vassart G. Pardo L. Costagliola S. EMBO J. 2003; 22: 2692-2703Crossref PubMed Scopus (179) Google Scholar, 7.Vassart G. Pardo L. Costagliola S. Trends Biochem. Sci. 2004; 29: 119-126Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). In contrast, our earlier work on relaxin has revealed sharply defined binding structures (8.Büllesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) that would require an equally well defined binding region on the receptor surface. LRR proteins, such as the RNase inhibitor for example, can bind proteins with extraordinarily high affinity (9.Kobe B. Deisenhofer J. Nature. 1995; 374: 183-186Crossref PubMed Scopus (578) Google Scholar, 10.Kobe B. Deisenhofer J. J. Mol. Biol. 1996; 264: 1028-1043Crossref PubMed Scopus (177) Google Scholar). Binding constants reach into the femtomolar range when as many as 26 residues make surface contact (11.Papageorgiou A.C. Shapiro R. Acharya K.R. EMBO J. 1997; 16: 5162-5177Crossref PubMed Scopus (172) Google Scholar). In contrast, our structure function studies with synthetic relaxin showed clearly that the contact with LGR7 is essentially limited to three residues located in the center of the B-chain helix. Thus, the binding energy sufficient to yield an affinity constant of ∼10−10m had to be derived from an unusual receptor/ligand interaction. A binding cassette of the general formula RXXXRXX(I/V) was discovered that appears in every species-specific relaxin regardless of major primary structure variations in the molecule (8.Büllesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In addition, steric effects are exerted by glycine A14, which, by its smallness, controls the position of the A-chain loop (12.Büllesbach E.E. Schwabe C. J. Biol. Chem. 1994; 269: 13124-13128Abstract Full Text PDF PubMed Google Scholar). In this paper we are presenting evidence for the identity of a contact residue on LGR7 for each of the members of the relaxin-binding cassette. Strains, Plasmids, and Oligonucleotides—Escherichia coli strain XL1Blue MRF′ was used for plasmid amplification and the human embryo kidney cell strain 293T/17 (American Type Culture Collection number CRL-11286) for protein expression. Plasmid pUC18 served as cloning vector, and plasmid pcDNA3.1.zeo (Invitrogen) was utilized as expression vector. Mutagenic primers used in this study were custom synthesized and purified by polyacrylamide gel electrophoresis (Integrated DNA Technologies). Sequences are available upon request. Cloning and Site-directed Mutagenesis of the LGR7 Receptor—The full-length cDNA of the human relaxin receptor LGR7 in the pUC-18 cloning vector encoded the prolactin signal peptide and the LGR7 mature protein (courtesy Dr. Hsueh, Department of Gynecology and Obstetrics at Stanford University). This cDNA was further modified to contain the coding sequence for the FLAG epitope at the N terminus of the mature LGR7 protein, a unique BamHI site at the 5′-end and a unique EcoRI site at the 3′-end of the coding sequence. Site-directed mutagenesis was carried out using pairs of custom oligonucleotides in combination with a polymerase chain reaction. The reaction was performed starting with 50 ng of the DNA template, 10 pmol of each primer, 250 μm each deoxynucleotide triphosphate, and 1 unit of Vent polymerase (New England Biolabs) in 50 μl of the reaction buffer. The reaction mixture was heated to 94 °C for 2 min followed by 18 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 6 min, with a final extension of 7 min at 72 °C. Wild-type DNA was digested with 10 units of endonuclease DpnI for 1 h at 37 °C, and the PCR reaction mixture was used to transform competent XL1-Blue-MRF′ cells. Individual colonies of the transformed cells were grown, and the plasmid was isolated and subjected to DNA sequencing using the primers LGR7-14r (5′-GGCATACAGCTTGTTCTC-3′), LGR7-24r (5′-GAAATTCCATCAGTGTTTGG-3′), or LGR7-4f (5′-GAATGTTTGGTCGGTTCTG-3′). The cDNA of the mutant was subcloned into the BamHI/EcoRI site of the suitable pcDNA3.1.zeo vector. Individual clones were cultured in Luria-Bertani broth containing 50 μg/ml ampicillin and purified using a plasmid isolation kit (Qiagen). The primers used to sequence the DNA were T7 (vector), BGHR, LGR7-4f (the coding cDNA), LGR7-4r (5′-CAGAACCGACCAAACATTC-3′), LGR7-14r, LGR7-14f (5′-GAGAACAAGCTGTATGCC-3′), LGR7-24r, and LGR7-24f (5′-CCAAACACTGATGGAATTTC-3′). Cell Culture—Cells (293T/17) were cultured in Dulbecco's modified Eagle's medium (Invitrogen, catalog number 11995-065) in 5% CO2 at 37 °C in a water-saturated atmosphere, detached with trypsin/EDTA (Invitrogen), and transferred to 6-well plates (106 cells/well). The 293T/17 cells were grown for 24 h before transfection with either the pcDNA3.1.zeo vector alone (negative control), the vector bearing the LGR7 coding sequence (positive control), or the vector containing the mutant sequences. For transfections, the Polyfect™ reagent (Qiagen) and protocols provided by the manufacturer were used, and the transfected 293T/17 cells were assayed after a 48-h culture period. Relaxin and Antibody Binding Assays—The transfected 293T/17 cells were dislodged by the addition of 1 ml of 0.5 m EDTA (pH 7.5) per 10 ml of conditioned medium for 10 min at 37 °C. The cells were collected by centrifugation at 2,000 × g for 10 min, suspended two times in 1 ml of ice cold binding buffer (20 mm Hepes, pH 7.5, 1% bovine serum albumin, 0.1 mg/ml lysine, 1.5 mm CaCl2, 50 mm NaCl, and 0.01% NaN3), and recentrifuged at 4 °C for 10 min at 2,000 × g. The pellet, collected and reconstituted to 25 × 106 cells per ml, was transferred to ice-cold binding buffer. For binding assays, 0.25 pmol of 125I-Tyr(B30),Phe(A3) human relaxin-II (based on 80% counting efficiency, the molar activity is 3900 cpm/fmol) (8.Büllesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) in 20 μl of binding buffer, 40 μl of binding buffer with or without 0.16 nmol of human relaxin II, and 40 μl (equal to 106 cells) of the cell suspension was incubated at room temperature for 60 min. Thereafter, the cells were diluted with 1 ml of ice-cold binding buffer and collected by centrifugation (10 min at 2,000 × g at 4 °C), and the pellet washed with 1 ml of the same buffer and centrifuged for 10 min at 5,000 × g at 4 °C. The tips of the vials containing the pellets were placed in tubes and transferred to a γ-counter. Determination of the FLAG Antigen— A radioactive, anti-FLAG antibody (Sigma) was prepared by the chloramine T method (13.Hunter W.M. Greenwood F.C. Nature. 1962; 194: 495-496Crossref PubMed Scopus (5861) Google Scholar). Free 125I was removed from the antibody by size separation on Sephadex G25 in 50 mm phosphate buffer saline at pH 7.4 (ratio of labeled per unlabeled antibody was ∼1:10). Antibody binding assays were performed in parallel with receptor binding assays. About 106 cells were suspended in a total assay volume of 100 μl containing the labeled antibody (100,000 cpm/assay) in the presence or absence of 0.2 μg (0.2 nmol) of the synthetic FLAG peptide and incubated for 1 h at room temperature. The cells were recovered, washed as described and the pellet was in a γ-counter. relaxin binding of the was as × is total is nonspecific is is vector, is and is the to for in protein expression as by the binding of anti-FLAG antibody in the presence and absence of the A is was in are as the of model the leucine-rich repeat of LGR7 on the protein sequence of the the described by Vassart G. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) was used in combination with the The repeats of LGR7 were with the repeats on the structure of the inhibitor The residues of LGR7 were and residues located in the helical region on the surface were Thereafter, chain and were and the molecule was using for at The molecule was for the and were Thereafter, the LGR7 model was The leucine-rich repeat of LGR7 was on the of the inhibitor using the of Vassart G. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The in of the from the acid repeat of inhibitor to the repeat of LGR7, was by the residues in the helical structure of each The of the repeats are in The molecule the parallel pleated and at the of a The on the of the was of the from the as of the were and of the of the structure are the of the structure showed a and located in the repeat 4 and and a angle of the peptide and The of this structure was by the which revealed an of J. J. Mol. Biol. PubMed Scopus Google Scholar, R. 1997; PubMed Scopus Google Scholar). The showed changes to for the pleated sheet and positive for the helical the a the primary structure and the the of a structure is for the surface for the this inner surface of the receptor we residues using In parallel 293T/17 cells were transfected with the for the mutant receptor, the LGR7 for positive and the vector for Binding assays were with labeled human relaxin (8.Büllesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) in the presence and absence of unlabeled human relaxin. The was sufficient to the receptor Binding of at to LGR7 in to the number of per cell surface. on the molar activity of the relaxin at 3900 the expression 2,000 and 5,000 The of mutant on the cell surface was using an and for receptor binding were and of the acidic residues and by their the affinity of the mutant receptor for the relaxin of by the same In addition, either or by a in binding. affinity for relaxin was for the LGR7 and the of by the of by receptor binding and of antibody to transfected 293T cells The was in parallel to the receptor binding assays in were performed in a total volume of 100 μl using cells per of antibody in the absence total and presence nonspecific of 2 μg/ml FLAG antibody binding was for 293T cells transfected with The was used to for in protein expression. in a binding of to mutant expression were in 293T/17 cells using of two or three the (positive control), and vector (negative binding was by of the binding of to the receptor or receptor mutant to the number of on the cell surface by the binding of anti-FLAG of of relaxin binding to LGR7 and LGR7 mutant a residue that is of the were in and five of LGR7 and three of were are The arginines B13 and B17 in relaxin, located one helix turn are parallel to each into the a helix turn the terminus the binding into the binding site (8.Büllesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). studies showed that arginines B13 and B17 in relaxin be C. Relaxin and the of Google Scholar) by without a in receptor binding avidity E.E. S. Schwabe C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). B20 in relaxin be for hydrophobic amino acids or binding as a function of (8.Büllesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). investigate which features would to binding we and LGR7 in 293T cells. for was derived from the of the inhibitor (9.Kobe B. Deisenhofer J. Nature. 1995; 374: 183-186Crossref PubMed Scopus (578) Google Scholar, 10.Kobe B. Deisenhofer J. J. Mol. Biol. 1996; 264: 1028-1043Crossref PubMed Scopus (177) Google Scholar, Vassart G. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The surface by the LRR in proteins is at the by that to the of the pleated of the leucine-rich repeats by the provided a starting position for that would the of the binding cassette of relaxin. The the arginine residues was by one helix turn to that any of the into this be In addition, a hydrophobic surface had to on one of a line connecting the upon the of the three binding residues in relaxin, of sites on the receptor was such a be to in a molecule the LGR7 The of our model on the pleated acid and aspartic acid on leucine-rich repeat number and aspartic acid and acid on repeat number 6 would the of arginines in the binding cassette of relaxin 1 and that each pair of residues is on the same are by primary sequence to each This was that the starting for our acid was for by a aspartic acid which the of the pleated was to in a parallel was by of to the FLAG epitope at the N terminus of the LGR7 and The for expression was in any with the total changes in were such as to the receptor that was used in the of to at position a in the binding of relaxin to LGR7, the of the to showed effect and This that an to one of the relaxin arginines, the from a pleated would be to in of for example, as the of our receptor binding studies with relaxin (8.Büllesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). acid a in the guanidinium was by This interaction A interaction with the guanidinium two acidic would the of the primary of in of a guanidinium work on the binding cassette of relaxin, in combination with on the RNase has provided evidence for a interaction either arginine B13 or B17 in relaxin and and on receptor binding of the of a transfection of 293T cells with DNA vector DNA and mutant were performed in a total volume of 100 μl using cells per 106 of 125I-Tyr(B30),Phe(A3) human relaxin in the absence total and presence nonspecific of 10 μg/ml human relaxin for are in a our model is to this the binding residues for relaxin be a of from a on and acidic residues were that this aspartic acid and acid in repeat number was by and by and the two were in transfected 293T cells. bind relaxin, that LGR7 bound the two arginines of relaxin each with an aspartic and acid The arginines would neutralize the charge repulsion residues and and a binding the guanidinium group two acidic interaction is with a binding which is of a on the receptor that is on the inner LGR7 surface. the of the inner of LGR7 that to the relaxin one is with an interaction the arginines from an α-helix in relaxin and acidic residues from a pleated sheet of the receptor surface. The inner of the LGR7 receptor is concave without the that a binding in a protein this our would relaxin to bind the receptor in two either with the the cell or the This is when one to for the signal site of relaxin. The hydrophobic interaction region that would have to B20 in relaxin is located to B17 arginine in relaxin, offset from the straight line connecting the arginine This an of that would the on the receptor surface. isoleucine leucine and such a hydrophobic cluster at the to isoleucine B20 in relaxin. This cluster of hydrophobic residues is located on 4 and of LGR7 1 and by the of to by of which is we of on repeat number to binding to of The of isoleucine on the same repeat binding to and of leucine on repeat number by binds relaxin to of the receptor. are by the structure function work that this study (8.Büllesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). and in position B20 (the binding cassette of would the same essentially the same binding isoleucine for either or acid binding it out with the of to a A was when we members of the contact site in LGR7, isoleucine or leucine to the of on repeat number for in binding a for the of the hydrophobic binding upon we that the hydrophobic cluster on repeats 4 and of LGR7 is the interaction site for B20 in relaxin. The discovery of the hydrophobic interaction site the binding for relaxin on the LRR surface such that the of the chain the of the receptor. of be into the that into the final for the of the the relaxin-binding site on the receptor have derived from the of the binding cassette of relaxin. It that the binding residues on the receptor have to be a of the binding cassette. one binding residue would be a the discovery of three binding at the from each as by the of relaxin have by our have that the two arginines, B13 and in relaxin have charge and chain and our that any of is binding be For example, of one of the acid in one of the two either or to the is sufficient to binding. It was equally to that a in by one without the of one of the acids binding as the that aspartic acid to acid binding of a it the groups with to each with a that be by This with the binding sites on relaxin, the binding site the of the LGR7 leucine-rich The two arginines with acidic of two alone the arginine interaction in the of the relaxin B-chain helix be from a interaction which was provided by B20 in relaxin and its in the hydrophobic cluster of the receptor, including and of either of the residues or in LRR number 7 by a of relaxin binding without a of interaction with the The contact residues and in relaxin are located the two binding arginines in relaxin and from the receptor interaction In addition, residues can be by and residues without binding affinity (8.Büllesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). and C. It is that the in LGR7 the acidic residues on repeats number 6 and number to the with the arginines on the chain helix. A of LGR7 without the region bind relaxin. structure function work with relaxin has revealed residues that receptor binding the binding cassette and glycine (8.Büllesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, C. E.E. J. 1994; PubMed Scopus Google Scholar). The that of the region binding that the binding site has by the region the pleated sheet of the receptor. In the LRR surface of proteins a make this structure for the of binding that to when one of interaction is unique in that the is by arginine from an α-helix in relaxin, that are by the acidic of the receptor, and of this is by hydrophobic binding residues that the of a our this is the of such a binding site that well be for protein binding leucine-rich repeat receptors. model explains the of that sequence variations as as the binding cassette is of the cassette in relaxin and the of its members has provided of the model of LGR7, at to the of the relaxin contact and for and and and for