Mutations at the consensus phosphorylation sites in the third intracellular loop of the rat gonadotropin-releasing hormone receptor: Effects on receptor ligand binding and signal transduction

In this study, site-directed mutagenesis of potential phosphorylation sites (Thr(238), Ser(253), and Thr(264)) for protein kinase C and C-terminal por tion (Ala(260)-Leu(265)) Of the third intracellular loop of the rat GnRH re ceptor (rGnRHR) was performed to assess the significance of these regions i n the function of the GnRHR. Mutation at one or all of the three potential phosphorylation sites had differential effects on receptor ligand binding. Mutation of Ser(253) or Thr(264) to Al, did not significantly affect the re ceptor-binding affinity but decreased the number of measurable binding site s. Mutation of Thr(238) to Ala or triple mutation of Thr(238), Ser(253), an d Thr(264) impaired or abolished receptor-binding affinity, Mutations of th e potential phosphorylation sites affected receptor-mediated inositol phosp holipid (IP) production and correlated with alterations in receptor binding after mutation, but they did not significantly affect receptor-mediated cA MP production or cAMP-mediated prolactin release. In addition, mutation of Ser(253) or Thr(264) to Ala did not affect the GnRH-provoked desensitizatio n in terms of GnRH agonist-stimulated IP production. Deletion of the C-term inal portion (Ala(260)-Leu(265)) Of the third intracellular loop of the rGn RHR, including a potential phosphorylation site (Thr(264)), abolished the r eceptor-binding affinity and receptor-mediated signal transduction. Replace ment of the deleted C-terminal portion with a C-terminal portion (Ala-Ala-A rg-Thr-Leu-Ser) of the third intracellular loop of the G(q/11)-coupled rat M-1 muscarinic acetylcholine receptor did not restore receptor function, Th ese results suggest that the potential phosphorylation sites or the region around the phosphorylation site of the third intracellular loop of the GnRH R is important for the structural integrity and expression of the receptor but that phosphorylation at these sites is not required for desensitization .