Anales RANF

P11. P URINERGIC S IGNALING IN O THER F UNCTIONS P.101 HETERODIMERIZATION OF ADENOSINE RECEPTORS IN BROWN ADIPOSE TISSUE Gnad T. 1 , Navarro G. 2,3 , Franco R. 2,3 , Pfeifer A. 1 1 University of Bonn, Bonn, Germany 2 University of Barcelona, Barcelona, Spain 3 CiberNed. Instituto de Salud Carlos III, Madrid, Spain. Background: the purine nucleoside adenosine regulates brown adipose tissue (BAT) physiology by signalling via the abundantly expressed Gs-coupled A2A and A2B adenosine receptors. BAT is specialized in burning energy by uncoupling ATP synthesis due to its unique uncoupling protein 1 (UCP1) and adenosine activates human and murine brown adipocytes (BA) at low nanomolar concentrations (Gnad et al.; 2014). An increasing number of evidence suggests adenosine receptor homo- and heterodimer formation. Therefore, we analysed the role of A2B signalling and heterodimerization in the context of adenosine-mediated BA activation. Material and methods: to study the role and consequence of A2B heterodimerization, we used pharmacological inhibition as well BA/BAT deficient of A2B. Moreover, we applied biological resonance energy transfer (BRET) assays and proximity ligation assays (PLA). Results: adenosine-mediated BA activation was suppressed after pre-treatment with an A2B-specific antagonist. This complete dependence of adenosine signalling on A2B was not expected since adenosine can enhance murine and human BAT activity via the A2A receptor (Gnad et al.; 2014; Ruan et al.; 2018) and recent work showed that heterologously overexpressed A2B inhibits signalling of A2A (Hinz et al.; 2018). In line with the repressed adenosine signalling, A2A-mediated energy expenditure was fully blunted in adipose-specific A2B knockout mice. On a molecular level, BRET analysis showed specific molecular A2B/A2A interaction in murine and human BA. In situ PLA revealed co-localization of endogenous A2B and A2A receptors in murine BAT. To disrupt A2B/A2A heterodimerization, peptides derived from TM regions 5 and 6 of both receptors were applied, which abrogated adenosine-induced BA activation demonstrating the functional relevance of these regions/interactions. Vice versa, no differences in A2B-activated BA activation of A2A+/+ and A2A-/- BA could be detected. Conclusion: we describe the adenosine receptor A2B as pivotal centrepiece for adenosine-mediated brown adipocyte activation forming heterodimers with A2A receptors. Moreover, A2B signals independently of A2A, but is crucially required for A2A receptor signalling in BA/BAT.