Role for the third intracellular loop in cell surface stabilization of the α2A-adrenergic receptor

Previous studies have shown that alpha(2A)-adrenergic receptor (alpha(2A)-AR) retention at the basolateral surface of polarized MDCKII cells involves its third intracellular (3i loop). The present studies examining mutant alpha(2A)-ARs possessing short deletions of the 3i loop indicate that no single region can completely account for the accelerated surface turnover of the Delta 3i alpha(2A)-AR, suggesting that the entire 3i loop is involved in basolateral retention. Both wild-type and Delta 3i loop alpha(2A)-ARs are extracted from polarized Madin-Darby canine kidney (MDCK) cells with 0.2% Triton X-100 and with a similar concentration/response profile, suggesting that Triton X-100-resistant interactions of the alpha(2A)-AR with cytoskeletal proteins are not involved in receptor retention on the basolateral surface. The indistinguishable basolateral t(1/2) for either the wild-type or nonsense 3i loop alpha(2A)-AR suggests that the stabilizing properties of the alpha(2A)-AR 3i loop are not uniquely dependent on a specific sequence of amino acids. The accelerated turnover of Delta 3i alpha(2A)-AR cannot be attributed to alteration in agonist-elicited alpha(2A)-AR redistribution, because alpha(2A)-ARs are not down-regulated in response to agonist. Taken together, the present studies show that stabilization of the alpha(2A)-AR on the basolateral surface of MDCKII cells involves multiple mechanisms, with the third intracellular loop playing a central role in regulating these processes.