Receptor-proximal effectors mediating GnRH actions in the goldfish pituitary: Involvement of G protein subunits and GRKs

In goldfish (Carassius auratus), two endogenous isoforms of gonadotropin-releasing hormone (GnRH) stimulate luteinizing hormone (LH) and growth hormone (GH) secretion. These isoforms, GnRH2 and GnRH3, act on a shared population of cell-surface GnRH receptors (GnRHRs) expressed on both gonadotrophs and somatotrophs, and can signal through unique, yet partially overlapping, suites of intracellular effectors, in a phenomenon known as functional selectivity or biased signalling. In this study, G-protein alpha (G alpha) subunits were targeted with two inhibitors, YM-254890 and BIM-46187, to ascertain the contribution of specific G-protein subunits in GnRH signalling. Results with the G alpha q/11-specific inhibitor YM-254890 on primary cultures of goldfish pituitary cells revealed the use of these subunits in GnRH control of both LH and GH release, as well as GnRH-induced elevations in phospho-ERK levels. Results with the pan-G alpha inhibitor BIM-46187 matched those using YM254890 in LH release but GH responses differed, indicating additional, non-G alpha q/11 subunits may be involved in somatotrophs. BIM-46187 also elevated unstimulated LH and GH release suggesting that G alpha subunits regulate basal hormone secretion. Furthermore, G-protein-coupled receptor kinase (GRK2/3) inhibition reduced LH responses to GnRH2 and GnRH3, and selectively enhanced GnRH2-stimulated GH release, indicating differential use of GRK2/3 in GnRH actions on gonadotrophs and somatotrophs. These findings in a primary untransformed system provide the first direct evidence to establish G alpha q/11 as an obligate driver of GnRH signalling in goldfish pituitary cells, and additionally describe the differential agonist- and cell type-selective involvement of GRK2/3 in this system.