SUN-225 - Syndecans Act as GPCR Accessory Proteins Modulating MC4R Signaling

Syndecans (SDCs) are a family of heparan sulfate proteoglycans. Overexpression of SDC1 in the mouse brain leads to hyperphagia and obesity, whereas SDC3 knockout mice show resistance to high-fat diet induced obesity. In HEK293 cells, full-length AgRP, an MC4R antagonist, showed increased potency in suppressing MC4R activity in the presence of SDC3. Therefore, SDC3 was proposed to enhance AgRP binding to MC4R. However, whether SDCs are able to modulate MC4R activity independently of AgRP has not been investigated. Additionally, it is unknown whether SDCs interact with mature post-translationally cleaved AgRP. In this study, we explored 1) whether SDCs modulate MC4R signaling independently of AgRP and 2) whether SDCs contribute to the antagonistic effects of mature post-translationally cleaved AgRP on MC4R.
Analysis of human hypothalamic single-cell RNA sequencing data showed neuronal co-expression of MC4R with SDC2 and SDC3 at a ratio of 1:0.8 and 1:1, respectively. We therefore assessed the impact of SDC2 and SDC3 at a 1:1 ratio on α-MSH-induced MC4R cAMP responsiveness in HEK293 cells, which lack AgRP. At a 1:1 ratio with MC4R, SDC2 and SDC3 suppressed Emax by 29±3% (P < 0.0001) and 24±1.5% (P < 0.0001), respectively. A previous mouse study showed that fasting causes hypothalamic SDC3 protein levels to increase >4-fold. To mimic this hunger state, we next investigated the effect of SDC2 and SDC3 on α-MSH-induced cAMP production using an MC4R:SDC ratio of 1:5. At this ratio, SDC2 and SDC3 suppressed Emax by 56±4% (P < 0.0001) and 36±4% (P < 0.0001), respectively. Additionally, at a 1:5 ratio SDC2 and SDC3 suppressed the constitutive activity of MC4R by 44±8% (P < 0.005) and 32±8% (P < 0.05), respectively.
Next, we assessed whether SDCs modulate the antagonistic effects of mature post-translationally cleaved AgRP on MC4R signaling. Stimulation with 0.1 µM AgRP suppressed Emax by 65±4% (P < 0.0001). SDC2 and SDC3 enhanced this antagonistic effect of AgRP by 70±6% (P < 0.0001) and 32±6% (P < 0.005), respectively.
In conclusion, these results suggest that SDCs modulate MC4R signaling both independently from AgRP through suppressing basal and α-MSH stimulated MC4R activity and in concert with AgRP. The dose-dependency of this effect could be paralleled in vivo upon fasting when hypothalamic SDC levels are increased. Together with results from our previous study, which shows that SDCs potentiate the downstream ghrelin-induced GHSR response, we propose a function for SDCs as GPCR accessory proteins to modulate appetite.