Molecular characterization of growth hormone secretagogue receptor in black seabream Acanthopagrus schlegeli

Growth hormone secretagogues (GHS) are small molecular weight compounds that possess potent stimulatory effect on growth hormone (GH) secretion. The identification of the endogenous GHS, ghrelin, changes the classical model that interaction between growth hormone-releasing hormone (GHRH) and somatostatin (SST) is the main regulatory mechanism for GH secretion. Because of the limited studies of this novel system in non-mammalian vertebrates and also to study the potential applications of GHS in aquaculture, a systematic investigation of this newly discovered system was carried out in an extensively cultured finfish, the black seabream Acanthopagrus schlegeli.; Synthetic GHS were found to be potent stimulators of GH secretion in seabream pituitary cells in vitro. This GH releasing effect, however, was independent of GH transcription. Based on these results, two alternatively spliced forms of the growth hormone secretagogue receptor (GHSR), namely sbGHSR-1a and sbGHSR-1b, were identified from the seabream pituitary. The sbGHSR-1a is a seven-transmembrane domain receptor whereas the sbGHSR-1b contains only five putative transmembrane helixes. These two receptor subtypes could be found in different regions within the central nervous system (CNS) of the fish. The 1a receptor has the highest expression in the pituitary and hypothalamus, corroborating with its GH secretion controlling function. On the other hand, the 1b receptor expression pattern is very different in that it has the highest expression in the telencephalon. The wide and differential distribution of the two receptor subtypes within the CNS indicates that the yet unidentified ligand for the receptor is probably a multifunctional hormone in this fish species. The two receptor cDNAs were subcloned into a mammalian expression vector and were successfully expressed in cultured human embryonic kidney (HEK) 293 cells and Chinese hamster ovary (CHO) cells for functional characterization. A fluorescent based high-throughput screening method for characterizing the actions of various GHS on GHSR was developed. It was found that the spiropiperidine GHS L163,540 has both the highest potency and efficacy in activating the sbGHSR-1a. Once the sbGHSR-1a is activated, a phospholipase C (PLC) and protein kinase C (PKC) dependent intracellular Ca2+ concentration elevation was observed. Furthermore, the receptor activation is not associated with any adenylate cyclase (AC) and protein kinase A (PKA) activity. The sbGHSR-1b, however, does not seem to give any detectable functional activity when challenged with various GHS. Interestingly, the sbGHSR-1b appears to play a regulatory role on the function of sbGHSR-1a. Co-expression of both receptor subtypes on cultured cells decreased the functional activity of sbGHSR-1a. It is thus hypothesized that heterodimerization occurred once the two receptors were co-expressed and this changed the original pharmacology of sbGHSR-1a.