OR0303 Syndecans Enhance Ghrelin-Induced GHSR1a Signaling

Abstract Disclosure: K. Prins: None. M. Huisman: None. R. Mies: None. P.J. Delhanty: None. J.A. Visser: None. Ghrelin is a gut hormone that enhances food intake and growth hormone secretion through its receptor, the G-protein coupled receptor (GPCR) GHSR1a. Recently, we showed that ghrelin interacts with syndecans (SDCs), a family of four membrane proteins that have been linked to obesity in genome-wide association studies. Here, we investigated whether SDCs impact ghrelin signaling at GHSR1a. We used HEK293 cells that expressed GHSR1a and SDC1, SDC2, SDC3, or SDC4 through transient transfection. GHSR1a mainly signals through the Gαq pathway, so we first explored the effects of SDCs on ghrelin-induced intracellular Ca2+ influx. Using a mitochondrial-targeted aequorin, this Ca2+ response can be measured seconds after ghrelin stimulation. We found that all SDCs increased the maximum Ca2+ response at least 4.2-fold (P<0.001), without affecting the EC50. This enhanced Ca2+ response was not caused by increased receptor availability: SDCs reduced cell membrane receptor expression by 51-85% (P<0.001). This low GHSR1a membrane availability contrasted with the increased Ca2+ response, and raised the question whether SDCs could be an alternate receptor for ghrelin. To test this, we transfected cells with SDC1 alone, but this did not permit a ghrelin-stimulated Ca2+ response, indicating that the ghrelin-induced Ca2+ response was GHSR1a-dependent. Moreover, the SDC-potentiated response was also Gαq-dependent, since GNAQ knockout cells transfected with GHSR1a and SDC1 expression plasmids failed to generate a Ca2+ response. However, using a Gq biosensor, we found that the level of activation of Gαq by ghrelin was not affected by SDC1 transfection, suggesting an effect of SDC1 either downstream of Gαq or on the quenching of GHSR. Thus, we investigated the interaction between GHSR1a and β-arrestin 2. Β-arrestin 2 downregulates GHSR function following activation by sterically hindering G-protein coupling and mediating receptor internalization. Four minutes after ghrelin stimulation, SDCs reduced β-arrestin 2 recruitment by 69-87% (P<0.001). SDC-transfected cells also reached a maximum β-arrestin 2 response 8-14 minutes later than GHSR1a only-transfected cells. Notably, these differences in β-arrestin 2 recruitment occurred minutes after the Ca2+ response. These findings suggest that the reduction in β-arrestin 2 recruitment does not cause the increased Ca2+ response in the presence of SDCs, and that the effect of SDCs is probably downstream of Gαq. Interestingly, the effects of SDCs mimic those of melanocortin receptor accessory protein 2 (MRAP2). MRAP2 also enhances the ghrelin-stimulated activation of the Gαq signaling pathway, independently of β-arrestin recruitment. Altogether, we showed that SDCs enhanced Ca2+ influx and reduced β-arrestin 2 recruitment to GHSR1a in response to ghrelin. This could be a novel mechanism through which SDCs affect metabolism and obesity. Presentation: Thursday, June 15, 2023