title: Identification of Ser153 in ICL2 of the gonadotropin-releasing hormone (GnRH) receptor as a phosphorylation-independent site for inhibition of Gq coupling.
abstract: 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 alphaT3-1 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 alphaT3-1 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.
authors.affiliation: Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel.
authors.forename: Sharon
authors.initials: S
authors.lastname: Shacham
authors.forename: Maya N
authors.initials: MN
authors.lastname: Cheifetz
authors.forename: Mati
authors.initials: M
authors.lastname: Fridkin
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Pawson
authors.forename: Robert P
authors.initials: RP
authors.lastname: Millar
authors.forename: Zvi
authors.initials: Z
authors.lastname: Naor
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D017931:DNA Primers / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000737:chemistry / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry; D006868:Hydrolysis; D015151:Immunoblotting; D007295:Inositol Phosphates / Q000737:chemistry; D007700:Kinetics; D020935:MAP Kinase Signaling System; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011966:Receptors, LHRH / Q000737:chemistry*; D012694:Serine / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
chemical_list: D017931:DNA Primers; D000939:Epitopes; D007295:Inositol Phosphates; D010455:Peptides; D011966:Receptors, LHRH; D013912:Threonine; D018698:Glutamic Acid; D012694:Serine; D048049:Extracellular Signal-Regulated MAP Kinases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000409:Alanine