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The Third Intracellular Loop of the Rat Gonadotropin-Releasing Hormone Receptor Couples the Receptor to G_s- and G_q/11-Mediated Signal Transduction Pathways: Evidence from Loop Fragment Transfection in GGH₃ Cells¹

Oregon Regional Primate Research Center (A.U.-A., D.S., V.A., J.V., S.B., J.A.J., P.M.C.), Beaverton, Oregon 97006; and the Department of Physiology and Pharmacology, Oregon Health Sciences University (P.M.C.), Portland, Oregon 97201

Address all correspondence and requests for reprints to: Dr. P. Michael Conn, Oregon Regional Primate Research Center, Beaverton, Oregon 97006.

The GnRH receptor (GnRH-R) belongs to the rhodopsin/ß-adrenergic family of G protein-coupled receptors. The intracellular domains of these receptors, particularly the regions closest to the plasma membrane in intracellular loops 2 (2i) and 3 (3i) as well as some regions located in the membrane-proximal end of the COOH-terminus, are frequently important sites for G protein coupling and specificity determination. Although studies in mouse and human GnRH-R have identified loop 2i as a critical determinant for coupling the receptor to the G_q/11-mediated signal transduction pathway, given the functional similarity among the members of this particular G protein-coupled receptor subfamily and the fact that the GnRH-R lacks the typical intracellular COOH-terminal domain of its superfamily (a potential site for G protein coupling), we investigated the possibility that loop 3i of this receptor also participates in GnRH-R coupling to G proteins. GGH₃1' cells, a pituitary-derived cell line that expresses a functional rat GnRH-R coupled to both G_s and G_q/11 proteins, were transiently transfected with a plasmid DNA containing a complementary DNA (cDNA) coding for the entire loop 3i of the GnRH-R as well as with other expression plasmids containing cDNAs encoding loop 3i of other G_s-, G_i/o-, or G_q/11-coupled receptors. The effects of coexpression of these loops with the wild-type GnRH-R on inositol phosphate (IP) production, cAMP accumulation, and PRL release were then examined. Transfection of GGH₃1' cells with the cDNA for loop 3i of the rat GnRH-R (efficiency, 35–45%) maximally inhibited buserelin-stimulated IP turnover by 20% as well as cAMP accumulation and PRL secretion by 30%. This attenuation in cellular responses to a GnRH agonist was statistically significant (P < 0.05) compared with the responses exhibited by GGH₃1' cells transfected with a control plasmid and stimulated with the same GnRH agonist. Transfection of minigenes coding for loop 3i of the M₁Ach-muscarinic and the _1B-adrenergic (G_q/11-coupled) receptors resulted in 25–55% inhibition of maximal GnRH-evoked IP turnover. Paradoxically, loop 3i from the M₁Ach-muscarinic receptor also maximally inhibited GnRH agonist-stimulated cAMP accumulation and PRL release by 40% (both effects mediated through activation of the G_s protein). Transfection of loop 3i from the D_1A -dopamine receptor (coupled to the G_s protein) produced a selective attenuation (40%) in G_s-mediated cellular responses. In contrast, receptor/G protein coupling appeared unaffected by expression of loop 3i domains derived from two receptors coupled to G_i/o proteins (M₂Ach-muscarinic and _2A-adrenergic receptors). These data indicate that the third intracellular loop of the rat GnRH-R is involved in receptor G_q/11 protein coupling and/or selectivity, and in the GGH₃1' cell line, this loop is also involved in signal transduction mediated through the G_s protein pathway.

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