OR24-02 Identification of Membrane Proteins That Enhance the Responsiveness of the Ghrelin Receptor

Abstract Ghrelin, a 28-amino acid peptide gut hormone, occurs in acylated (AG) and unacylated (UAG) variants. AG is a GH secretagogue as well as being orexigenic and diabetogenic, acting via the growth hormone secretagogue receptor (GHSR1a) in the hypothalamus and pituitary. UAG counteracts these metabolic effects through unknown mechanisms. While screening for potential UAG receptor(s) we discovered previously uncharacterised interactions of AG and UAG with five cell membrane proteins (MPs), three of which are known to modulate metabolism. Here, we studied if two of these MPs (MP1 & 2) could modulate GHSR1a signalling by expressing their transgenes in HEK293 cells. As GHSR1a is coupled with calcium signalling via Gq proteins, aequorin luminescence was used to evaluate Ca2+ influx into the cells. Transfected cells were treated with either AG, UAG, or soluble parts of the MPs, or combinations thereof. MP2 markedly enhanced the efficacy (~5.5-fold), but not the potency, of AG-induced Ca2+ influx, whereas MP1 had no effect on Ca2+ influx. Neither MP1 nor MP2 overexpression altered cellular GHSR1a levels. In the absence of GHSR1a, MP2 was unable to stimulate an AG-induced Ca2+ influx. UAG treatment (100nM) had no effect on GHSR1a-mediated Ca2+ influx in the presence or absence of MP2. MP2 is post-translationally modified and we suspected this to be important for its activity. However, removal or blockade of these modifications had no effect on the ability of MP2 to enhance GHSR1a signalling. Moreover, incubating the cells with soluble ectodomain of MP2 did not alter its effect on GHSR1a signalling. Nevertheless, induction of ectodomain shedding with PMA (0.4-1µM) dose-dependently reduced the AG-induced Ca2+ response to 0.5-0.2 of control levels (DMSO) in MP2-GHSR1a co-transfected cells. Unlike MP2, which has a transmembrane and intracellular domain, MP1 is attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor and lacks an intracellular domain. Since MP1 is otherwise structurally similar to MP2, we suspected that the intracellular domain of MP2 may be important for its function. Therefore, we expressed chimeras of MP2 and MP1 in which the GPI linkage site and the transmembrane/intracellular domains were exchanged. The MP2 ectodomain with a GPI-anchor had similar stimulatory effects on GHSR1a signalling as full-length MP2, whereas the MP1 ectodomain with MP2 transmembrane and intracellular domain only enhanced GHSR1a signalling by approximately 3-fold. In conclusion, we have identified a membrane protein as a novel component of the ghrelin signalling pathway that markedly enhances the response of the ghrelin receptor to AG. Our current data suggest its ectodomain is important in mediating this effect. Studies are ongoing to fully delineate the mode of interaction and to determine the role of MP2 in ghrelin signalling in vivo.