title: Regulation of receptor signaling by relaxin A chain motifs: derivation of pan-specific and LGR7-specific human relaxin analogs.
abstract: Relaxin peptides are important hormones for the regulation of reproductive tissue remodeling and the renal cardiovascular system during pregnancy. Recent studies demonstrated that two of the seven human relaxin family peptides, relaxin H2 (RLN2) and INSL3, signal exclusively through leucine-rich repeat-containing G protein-coupled receptors, LGR7 and LGR8. Although it was well characterized that an RXXXRXXI motif at the RLN2 B chain confers receptor activation activity, it is not clear what roles RLN2 A chain plays in receptor interaction. Analyses of relaxin family genes on syntenic regions of model tetrapods showed that the A chain of RLN2 orthologs exhibited a greater sequence divergence as compared with the receptor-binding domain-containing B chain, foreshadowing a potential role in receptor interactions; hence, defining receptor selectivity in this fast evolving peptide hormone. To test our hypothesis that select residues in the human RLN2 A chain play key roles in receptor interaction, we studied mutant peptides with residue substitution(s) in the A chain. Here, we showed that alanine substitution at the A16 and A17 positions enhances LGR8-activation activity of RLN2, whereas mutation at the A22-23 region (RLN2A22-23) ablates LGR8, but not LGR7, activation activity. In addition, we demonstrated that the functional characteristics of the RLN2A22-23 mutant are mainly attributed to modifications at the PheA23 position. Taken together, our studies indicated that ThrA16, LysA17, and PheA23 constitute part of the receptor-binding interface of human RLN2, and that modification of these residues has led to the generation of novel human RLN2 analogs that would allow selective activation of human LGR7, but not LGR8, in vivo.
authors.affiliation: Reproductive Biology and Stem Cell Research Program, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317, USA.
authors.forename: Jae-Il
authors.initials: JI
authors.lastname: Park
authors.forename: Jenia
authors.initials: J
authors.lastname: Semyonov
authors.forename: Wei
authors.initials: W
authors.lastname: Yi
authors.forename: Chia Lin
authors.initials: CL
authors.lastname: Chang
authors.forename: Sheau Yu Teddy
authors.initials: SY
authors.lastname: Hsu
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D040761:Biomimetic Materials / Q000138:chemical synthesis / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012065:Relaxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D012065:Relaxin
references.citation: J Biol Chem. 2006 Sep 8;281(36):26136-43
references.pmid: 16844694
references.citation: Genes Dev. 2000 Dec 15;14(24):3075-86
references.pmid: 11124800
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3381-5
references.pmid: 12824332
references.citation: J Biol Chem. 2003 Dec 12;278(50):50754-64
references.pmid: 14522968
references.citation: Mol Endocrinol. 2003 Dec;17(12):2639-46
references.pmid: 12933905
references.citation: Mol Cell Endocrinol. 2000 Feb 25;160(1-2):11-6
references.pmid: 10715534
references.citation: Ann Intern Med. 2000 Jun 6;132(11):871-9
references.pmid: 10836913
references.citation: Pharmacol Ther. 2006 Oct;112(1):38-56
references.pmid: 16647137
references.citation: J Biol Chem. 1987 Sep 15;262(26):12496-501
references.pmid: 3624270
references.citation: Mol Endocrinol. 2002 Feb;16(2):244-52
references.pmid: 11818498
references.citation: Endocr Rev. 2004 Apr;25(2):205-34
references.pmid: 15082520
references.citation: Proc Natl Acad Sci U S A. 2004 May 11;101(19):7323-8
references.pmid: 15123806
references.citation: Cancer Res. 2007 May 1;67(9):4399-407
references.pmid: 17483354
references.citation: J Pharmacol Exp Ther. 2005 May;313(2):677-87
references.pmid: 15649866
references.citation: Biochemistry. 2006 Jan 24;45(3):1043-53
references.pmid: 16411781
references.citation: Endocrinology. 2004 Oct;145(10):4712-20
references.pmid: 15256493
references.citation: Ann N Y Acad Sci. 2005 May;1041:82-90
references.pmid: 15956690
references.citation: Mol Endocrinol. 1999 Dec;13(12):2163-74
references.pmid: 10598589
references.citation: J Biol Chem. 2003 Mar 7;278(10):7855-62
references.pmid: 12506116
references.citation: J Biol Chem. 1992 Nov 15;267(32):22957-60
references.pmid: 1331071
references.citation: Mol Cell Biol. 2004 Jan;24(2):687-96
references.pmid: 14701741
references.citation: J Biol Chem. 2008 Jun 20;283(25):17287-97
references.pmid: 18434306
references.citation: Rev Endocr Metab Disord. 2005 Dec;6(4):291-6
references.pmid: 16311947
references.citation: BMC Evol Biol. 2005 Feb 12;5:14
references.pmid: 15707501
references.citation: Oxf Rev Reprod Biol. 1993;15:143-89
references.pmid: 8336976
references.citation: J Biol Chem. 2002 Aug 30;277(35):31283-6
references.pmid: 12114498
references.citation: J Natl Cancer Inst. 2006 Oct 18;98(20):1482-93
references.pmid: 17047197
references.citation: Biol Reprod. 2002 Sep;67(3):699-705
references.pmid: 12193374
references.citation: Biol Reprod. 2000 Sep;63(3):697-703
references.pmid: 10952910
references.citation: J Pept Res. 2004 Feb;63(2):91-8
references.pmid: 15009530
references.citation: Nature. 2000 Nov 23;408(6811):483-8
references.pmid: 11100731
references.citation: Biochemistry. 1999 Mar 9;38(10):3073-8
references.pmid: 10074360
references.citation: J Biol Chem. 2005 Apr 8;280(14):14051-6
references.pmid: 15695505
references.citation: Front Horm Res. 2001;27:105-12
references.pmid: 11450422
references.citation: J Mol Biol. 1991 Sep 5;221(1):15-21
references.pmid: 1656049
references.citation: Curr Opin Rheumatol. 1998 Mar;10(2):123-8
references.pmid: 9567207
references.citation: J Biol Chem. 1999 Aug 6;274(32):22354-8
references.pmid: 10428805
references.citation: Mol Reprod Dev. 2001 Mar;58(3):281-6
references.pmid: 11170269
references.citation: J Biol Chem. 2006 Aug 25;281(34):24889-99
references.pmid: 16728398
references.citation: J Biol Chem. 2002 Jan 11;277(2):1148-57
references.pmid: 11689565
references.citation: Pharm Res. 1993 Jun;10(6):834-8
references.pmid: 8257492
references.citation: Genomics. 1995 Sep 20;29(2):465-70
references.pmid: 8666396
references.citation: J Biol Chem. 1994 May 6;269(18):13124-8
references.pmid: 8175738
references.citation: Genome Res. 2008 Jun;18(6):974-85
references.pmid: 18463305
references.citation: J Biol Chem. 2003 Apr 25;278(17):14798-805
references.pmid: 12590147
references.citation: J Biol Chem. 2003 Dec 12;278(50):50765-70
references.pmid: 14522967
references.citation: Mol Cell Endocrinol. 2003 Aug 29;206(1-2):159-66
references.pmid: 12943998
references.citation: J Biol Chem. 2006 Sep 22;281(38):28287-95
references.pmid: 16867980
references.citation: Trends Biochem Sci. 2002 Feb;27(2):107-9
references.pmid: 11852249
references.citation: Endocrinology. 1999 Jan;140(1):445-53
references.pmid: 9886856
references.citation: Science. 2002 Jan 25;295(5555):671-4
references.pmid: 11809971
references.citation: Br J Pharmacol. 1999 May;127(1):91-8
references.pmid: 10369460
references.citation: J Pept Res. 1999 May;53(5):542-7
references.pmid: 10424349
references.citation: Biol Reprod. 2000 Jun;62(6):1593-9
references.pmid: 10819760
references.citation: Mol Endocrinol. 1992 Sep;6(9):1441-50
references.pmid: 1435788
references.citation: Regul Pept. 2001 Mar 2;97(2-3):139-46
references.pmid: 11164949
references.citation: Biochemistry. 1986 Oct 7;25(20):5998-6004
references.pmid: 3790502
references.citation: Genomics. 1999 Aug 15;60(1):50-6
references.pmid: 10458910
references.citation: J Biol Chem. 1991 Jun 15;266(17):10754-61
references.pmid: 2040595