Mechanisms regulating the cell surface residence time of the alpha(2A)-adrenergic receptor

Despite considerable insights concerning the mechanisms regulating short-te rm agonist-mediated G protein-coupled receptor (GPCR) internalization, litt le is known about the mechanisms regulating GPCR surface residence over lon g periods of time. Herein, we experimentally evaluated mechanisms regulatin g the surface t(1/2) of various alpha(2A)-adrenergic receptor (alpha(2A)AR) structures. The Delta 3i alpha(2A)AR (lacking the third intracellular loop ), D79Na(2A)AR (impaired G protein coupling), and CAM alpha(2A)AR (enhanced G protein coupling) all exhibited a cell surface alpha(2A)AR turnover in C hinese hamster ovary cells that was faster than that of the wild type (WT). Cell surface receptor turnover could be slowed with Ligand occupancy of D7 9N alpha(2A)AR (agonist or antagonist) and CAM alpha(2A)AR (antagonist only ) but not the Delta 3i- or WT alpha(2A)AR. This selective ligand-induced su rface stabilization was paralleled by a dramatic Ligand-dependent receptor density upregulation for D79N- and CAM alpha(2A)AR structures. Receptors wh ich exhibited surface turnover and density that could be modulated by ligan d (D79N and CAM) also demonstrated structural instability, measured by a lo ss of radioligand binding capacity in detergent solution over time without parallel changes in receptor protein content. In contrast, the shorter surf ace t(1/2) Of the Delta 3i alpha(2A)AR, whose cell surface t(1/)2 and stead y state density were not altered by ligand occupancy, occurred in the conte xt of a structurally stable receptor in detergent solution. These results d emonstrate that changes in receptor structure which alter receptor-G protei n coupling (either an increase or decrease) are paralleled by structural in stability and ligand-induced surface stabilization. These studies also prov ide criteria for assessing the structural instability of the alpha(2A)AR th at can likely be generalized to all GPCRs.