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J. Biol. Chem., Vol. 280, Issue 32, 28981-28988, August 12, 2005

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Identification of Ser¹⁵³ in ICL2 of the Gonadotropin-releasing Hormone (GnRH) Receptor as a Phosphorylation-independent Site for Inhibition of G_q Coupling^*

Sharon Shacham, Maya N. Cheifetz, Mati Fridkin¶, Adam J. Pawson||, Robert P. Millar||, and Zvi Naor||**

From the Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel, the ^¶Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel, and the ^||Human Reproduction Sciences Unit, Medical Research Council, the University of Edinburgh Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, United Kingdom

Type I gonadotropin-releasing hormone (GnRH) receptor (GnRHR) is unique among mammalian G-protein-coupled receptors (GPCRs) in lacking a C-terminal tail, which is involved in desensitization in GPCRs. Therefore, we searched for inhibitory sites in the intracellular loops (ICLs) of the GnRHR. Synthetic peptides corresponding to the three ICLs were inserted into permeabilized T3-1 gonadotrope cells, and GnRH-induced inositol phosphate (InsP) formation was determined. GnRH-induced InsP production was potentiated by ICL2 > ICL3 but not by the ICL1 peptides, suggesting they are acting as decoy peptides. We examined the effects of six peptides in which only one of the Ser or Thr residues was substituted with Ala or Glu. Only substitution of Ser¹⁵³ with Ala or Glu ablated the potentiating effect upon GnRH-induced InsP elevation. ERK activation was enhanced, and the rate of GnRH-induced InsP formation was about 6.5-fold higher in the first 10 min in COS-1 cells that were transfected with mutants of the GnRHR in which the ICL2 Ser/Thr residues (Ser¹⁵¹, Ser¹⁵³, and Thr¹⁴²) or only Ser¹⁵³ was mutated to Ala as compared with the wild type GnRHR. The data indicate that ICL2 harbors an inhibitory domain, such that exogenous ICL2 peptide serves as a decoy for the inhibitory site (Ser¹⁵³) of the GnRHR, thus enabling further activation. GnRH does not induce receptor phosphorylation in T3-1 cells. Because the phosphomimetic ICL2-S153E peptide did not mimic the stimulatory effect of the ICL2 peptide, the inhibitory effect of Ser¹⁵³ operates through a phosphorylation-independent mechanism.

Received for publication, January 10, 2005 , and in revised form, May 26, 2005.

^* This work was supported by the Adams Super Center for Brain Studies at Tel Aviv University and by Tel Aviv University. 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.

Present address: Predix Pharmaceuticals Ltd., Ramat-Gan, Israel.

^** To whom correspondence should be addressed. Tel.: 44-131-242-6216; Fax: 44-131-242-6231; E-mail: z.naor{at}hrsu.mrc.ac.uk.

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