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J. Biol. Chem., Vol. 283, Issue 45, 31068-31078, November 7, 2008

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Disease-causing Mutation in GPR54 Reveals the Importance of the Second Intracellular Loop for Class A G-protein-coupled Receptor Function^*

Jennifer L. Wacker, David B. Feller, Xiao-Bo Tang, Mia C. DeFino, Yuree Namkung, John S. Lyssand, Andrew J. Mhyre, Xu Tan, Jill B. Jensen^¶, and Chris Hague¹

From the Department of Pharmacology and ^¶Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195 and the Clinical Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109

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 _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 G subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of G. Combining our data with a predictive Class A GPCR/G model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of G. Such an interaction could promote opening of switch II of G to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

Received for publication, July 10, 2008 , and in revised form, August 26, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant T32 HD07453 (to J. L. W.); NIGMS National Research Service Award T32 GM07270 (to J. S. L.); T32 GM07750 (to M. C. D.); and T32 GM07108, RO1 NS08174 (BH), and U54HD12629 (to J. B. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: 1959 NE Pacific St., Box 357280, Seattle, WA 98195. Fax: 206-685-3822; E-mail: chague{at}u.washington.edu.

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