Inhibiting Intracellular α2C-Adrenoceptor Surface Translocation Using Decoy Peptides: Identification of an Essential Role of the C-Terminus in Receptor Trafficking.

The G protein-coupled α2-adrenoceptor subtype C (abbreviated α2C-AR) has been implicated in peripheral vascular conditions and diseases such as cold feet-hands, Raynaud's phenomenon, and scleroderma, contributing to morbidity and mortality. Microvascular α2C-adrenoceptors are expressed in specialized smooth muscle cells and mediate constriction under physiological conditions and the occlusion of blood supply involving vasospastic episodes and tissue damage under pathological conditions. A crucial step for receptor biological activity is the cell surface trafficking of intracellular receptors, triggered by cAMP-Epac-Rap1A GTPase signaling, which involves protein-protein association with the actin-binding protein filamin-2, mediated by critical amino acid residues in the last 14 amino acids of the receptor carboxyl (C)-terminus. This study assessed the role of the C-terminus in Rap1A GTPase coupled receptor trafficking by domain-swapping studies using recombinant tagged receptors in transient co-transfections and compared with wild-type receptors using immunofluorescence microscopy. We further tested the biological relevance of the α2C-AR C-terminus, when introduced as competitor peptides, to selectively inhibit intracellular α2C-AR surface translocation in transfected as well as in microvascular smooth muscle cells expressing endogenous receptors. These studies contribute to establishing proof of principle to target intracellular α2C-adrenoceptors to reduce biological activity, which in clinical conditions can be a target for therapy.