title: Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function.
abstract: The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Wacker
authors.forename: David B
authors.initials: DB
authors.lastname: Feller
authors.forename: Xiao-Bo
authors.initials: XB
authors.lastname: Tang
authors.forename: Mia C
authors.initials: MC
authors.lastname: Defino
authors.forename: Yuree
authors.initials: Y
authors.lastname: Namkung
authors.forename: John S
authors.initials: JS
authors.lastname: Lyssand
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Mhyre
authors.forename: Xu
authors.initials: X
authors.lastname: Tan
authors.forename: Jill B
authors.initials: JB
authors.lastname: Jensen
authors.forename: Chris
authors.initials: C
authors.lastname: Hague
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D002460:Cell Line; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism; D030342:Genetic Diseases, Inborn / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007006:Hypogonadism / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D000074502:Receptors, Kisspeptin-1
chemical_list: D044385:GTP-Binding Protein alpha Subunits; C118688:KISS1R protein, human; D043562:Receptors, G-Protein-Coupled; D000074502:Receptors, Kisspeptin-1; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate
references.citation: Science. 1978 Nov 10;202(4368):631-3
references.pmid: 100883
references.citation: Annu Rev Pharmacol Toxicol. 2004;44:559-609
references.pmid: 14744258
references.citation: J Biol Chem. 1995 Sep 29;270(39):22820-6
references.pmid: 7559413
references.citation: J Biol Chem. 1993 Oct 25;268(30):22273-6
references.pmid: 8226735
references.citation: Biochem Biophys Res Commun. 1999 Oct 14;264(1):171-5
references.pmid: 10527859
references.citation: Endocrinology. 2004 Sep;145(9):4073-7
references.pmid: 15217982
references.citation: Science. 1997 Dec 12;278(5345):1907-16
references.pmid: 9417641
references.citation: Nat Clin Pract Endocrinol Metab. 2006 Jun;2(6):328-34
references.pmid: 16932310
references.citation: Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):10972-6
references.pmid: 12944565
references.citation: Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):4877-82
references.pmid: 11320237
references.citation: Pharmacol Rev. 2007 Sep;59(3):225-50
references.pmid: 17878512
references.citation: N Engl J Med. 2003 Oct 23;349(17):1614-27
references.pmid: 14573733
references.citation: Anal Biochem. 2005 Mar 1;338(1):136-42
references.pmid: 15707944
references.citation: Mol Pharmacol. 2007 Aug;72(2):219-30
references.pmid: 17430994
references.citation: Biochem Biophys Res Commun. 2003 Dec 26;312(4):1357-63
references.pmid: 14652023
references.citation: Biochemistry. 1987 May 19;26(10):2657-64
references.pmid: 3038163
references.citation: Eur J Endocrinol. 2004 Nov;151 Suppl 3:U83-8
references.pmid: 15554891
references.citation: Semin Reprod Med. 2007 Jul;25(4):272-86
references.pmid: 17594608
references.citation: J Clin Endocrinol Metab. 2001 Jun;86(6):2680-6
references.pmid: 11397871
references.citation: Nature. 1998 Jul 2;394(6688):35-8
references.pmid: 9665125
references.citation: PLoS Comput Biol. 2006 Feb;2(2):e13
references.pmid: 16485037
references.citation: Neuroendocrinology. 2004;80(4):264-72
references.pmid: 15665556
references.citation: Q Rev Biophys. 2006 May;39(2):117-66
references.pmid: 16923326
references.citation: Pharmacol Ther. 2006 Sep;111(3):949-73
references.pmid: 16616374
references.citation: Science. 2007 Nov 23;318(5854):1258-65
references.pmid: 17962520
references.citation: J Biol Chem. 1995 Jul 28;270(30):17741-8
references.pmid: 7629074
references.citation: Nat Rev Mol Cell Biol. 2008 Jan;9(1):60-71
references.pmid: 18043707
references.citation: Cell. 1997 Apr 18;89(2):251-61
references.pmid: 9108480
references.citation: Mol Pharmacol. 2007 Apr;71(4):959-64
references.pmid: 17192495
references.citation: Mol Pharmacol. 2006 Jan;69(1):397-405
references.pmid: 16210429
references.citation: J Clin Endocrinol Metab. 2005 Dec;90(12):6609-15
references.pmid: 16174713
references.citation: Mol Cell Proteomics. 2007 May;6(5):835-44
references.pmid: 17215244
references.citation: Protein Sci. 2006 Jul;15(7):1679-90
references.pmid: 16815918
references.citation: J Biol Chem. 2006 Feb 3;281(5):2932-8
references.pmid: 16319069
references.citation: J Mol Biol. 2003 Aug 1;331(1):281-99
references.pmid: 12875852
references.citation: J Endocrinol. 2006 Jul;190(1):13-6
references.pmid: 16837606
references.citation: Proc Natl Acad Sci U S A. 2007 Feb 6;104(6):2001-6
references.pmid: 17264214
references.citation: Circ Res. 2002 Oct 18;91(8):672-80
references.pmid: 12386143
references.citation: Endocrinology. 2005 Jan;146(1):156-63
references.pmid: 15375028
references.citation: Science. 2005 Dec 9;310(5754):1686-90
references.pmid: 16339447
references.citation: Endocrinology. 2003 Jun;144(6):2220-8
references.pmid: 12746278
references.citation: Biochem Biophys Res Commun. 2005 Apr 8;329(2):684-92
references.pmid: 15737640
references.citation: J Biol Chem. 1998 Sep 18;273(38):24322-7
references.pmid: 9733718
references.citation: J Clin Endocrinol Metab. 2005 Mar;90(3):1849-55
references.pmid: 15598687
references.citation: J Biol Chem. 2008 Jul 4;283(27):18792-800
references.pmid: 18468998