Mutual regulation of NPC1 and A2A-adenosine receptors

The adenosine A2A receptor is a member of the large family of G protein coupled receptors (GPCR`s). Its cognate G protein is Gs , thus its canonical signalling pathway is the elevation of cAMP and the regulation of cAMP- dependent effectors (protein kinase A = PKA; cyclic nucleotide-gated channels, EPAC1 etc.). In addition, the A 2A- receptor can also engage signal-ling cascades in a manner independent of heterotrimeric G proteins, e.g. via ARNO and ARF6. The receptor appears to be confined in membrane microdomains and this may account for restricted collision coupling. Recently, we have shown that coupling of the A2A-receptor to Gs is contingent on plasma membrane cholesterol: depletion of membrane cholesterol abrogated cAMP accumulation but did not affect ARF6- dependent signalling. Niemann Pick type C disease is a rare but fatal disorder and is characterized by accumulation of cholesterol in peripheral tissues and in the brain, including the striatum. Most patients suffer from progressive neurodegeneration. NPC disease has been mainly attributed to a gene defect in npc1: the gene encodes a cholesterol sensing lipid transporter and regulates trafficking of cholesterol from endosomal compartments to the plasma membrane. Accordingly, the cholesterol content and the lipid composition of the plasma membrane are altered in Niemann Pick disease. Moreover, NPC1 expression is regulated by cAMP and CREB. In my working hypothesis underlying the current project I postulate that A2A receptors regulate NPC1 expression and that NPC1 expression regulates signal transfer through A2A-receptor-dependent pathways. This is likely to occur in the striatum, a brain region enriched in A2A receptors. I also posit that striatal A2A receptor signaling is altered in Niemann Pick disease. Apart from activating adenylyl cyklase, A 2A receptor agonists also promote phosphorylation of ERK1/2 by recruitment of ARNO/ARF6. This is of interest, because activated ARF6 was observed to reverse cholesterol accumulation in cellular NPC1 models. The aims of the project are to (i) verify that NPC1 expression can be enhanced upon A 2A receptor activation and (ii) to examine the extent by which variation in NPC1 levels (raised expression levels or absence of the protein) affect A2A-receptor mobility (and possibly receptor trafficking) and downstream signalling pathways in cellular models of NPC1. (iii) I will also specifically explore, if signalling via ARF6 can mitigate the abnormal lipid distribution seen in NPC1. These experiments are designed to generate insights in the mutual regulation of NPC1 and the A 2A-receptor. They exploit a perturbation in membrane lipid composition (arising from an -admittedly tragic - experiment by nature) to explore how GPCR signaling is regulated by cholesterol-enriched membrane domains. Last but not least they may sdhed some light on the pathophysiology of Niemann Pick type C disease.