Signal transduction mechanism of the seabream growth hormone secretagogue receptor

We have recently cloned the full-length cDNAs of the two growth hormone secretagogue receptor (GHSR) subtypes from a teleost species, the black seabream (Acanthopagrus schlegeli) [Mol. Cell. Endocrinol. 214 (2004) 81], namely sbGHSR-1a and sbGHSR-1b. Functional expression of these two receptor constructs in human embryonic kidney 293 (HEK293) cells indicated that stimulation of sbGHSR-1a by growth hormone secretagogues (GHS) could evoke increases in intracellular Ca2+ concentration ([Ca2+]i), whereas sbGHSR-1b appeared to play an inhibitory role on the signal transduction activity of sbGHSR-1a. In the present study, we have further investigated the signal transduction mechanism of sbGHSR-1a. The peptide GHS GHRP-6 and the non-peptide GHS L163,540 were able to trigger a receptor specific and phospholipase C (PLC)-dependent elevation of [Ca2+]i in HEK293 cells stably expressing sbGHSR-1a. This GHS-induced calcium mobilization was also dependent on protein kinase C activated L-type calcium channel opening. It was found that sbGHSR-1a could function in an agonist-independent manner as it exhibited a high basal activity of inositol phosphate production in the absence of GHS, indicating that the fish receptor is constitutively active. In addition, the extracellular signal-regulated kinases 1 and 2 (ERK1/2) were found to be activated upon stimulation of sbGHSR-1a by GHRP-6. This observation provides direct evidence in the coupling of sbGHSR-1a to ERK1/2 activation. Neither Gs nor Gi proteins are coupled to the receptor, as GHS did not induce cAMP production nor inhibit forskolin-stimulated cAMP accumulation in the sbGHSR-1a bearing cells. Furthermore, the ability of the GHSR antagonist D-Lys3-GHRP-6 to inhibit basal PLC and basal ERK1/2 activity suggests that this compound is an inverse agonist. In summary, the sbGHSR-1a appears to couple through the G(q/11)-mediated pathway to activate PLC, resulting in increased IP3 production and Ca2+ mobilization from both intracellular and extracellular stores. Moreover, sbGHSR-1a may trigger multiple signal transduction cascades to exert its physiological functions.