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

Abstract

The GnRH receptor (GnRH-R) belongs to the rhodopsin/β-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 Gq/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 Gs and Gq/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 Gs-, Gi/o-, or Gq/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 α1B-adrenergic (Gq/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 Gs protein). Transfection of loop 3i from the D1A -dopamine receptor (coupled to the Gs protein) produced a selective attenuation (40%) in Gs-mediated cellular responses. In contrast, receptor/G protein coupling appeared unaffected by expression of loop 3i domains derived from two receptors coupled to Gi/o proteins (M2Ach-muscarinic andα 2A-adrenergic receptors). These data indicate that the third intracellular loop of the rat GnRH-R is involved in receptor Gq/11 protein coupling and/or selectivity, and in the GGH31′ cell line, this loop is also involved in signal transduction mediated through the Gs protein pathway.