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J Biol Chem, Vol. 274, Issue 23, 16331-16336, June 4, 1999

Role for the Third Intracellular Loop in Cell Surface Stabilization of the _2A-Adrenergic Receptor

From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232

Previous studies have shown that _2A-adrenergic receptor (_2A-AR) retention at the basolateral surface of polarized MDCKII cells involves its third intracellular (3i loop). The present studies examining mutant _2A-ARs possessing short deletions of the 3i loop indicate that no single region can completely account for the accelerated surface turnover of the 3i_2A-AR, suggesting that the entire 3i loop is involved in basolateral retention. Both wild-type and 3i loop _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 _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 _2A-AR suggests that the stabilizing properties of the _2A-AR 3i loop are not uniquely dependent on a specific sequence of amino acids. The accelerated turnover of 3i _2A-AR cannot be attributed to alteration in agonist-elicited _2A-AR redistribution, because _2A-ARs are not down-regulated in response to agonist. Taken together, the present studies show that stabilization of the _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.

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