An arginine‐rich region mediates trans Golgi retention of α 2C ‐adrenoceptors

Smooth muscle α 2 ‐adrenoceptor (α 2 ‐AR) subtypes A and C are Gi coupled receptors and mediators of vasoconstriction. Functional α 2A ‐ARs are expressed on the plasma membrane (PM) whereas α 2C ‐ARs are retained in the trans Golgi and are silent. The mechanism of α 2C ‐AR intracellular retention remains undefined. After moderate cooling, functional α 2C ‐ARs are quickly mobilized to the PM. In this study we investigated the role of a C‐terminus arginine‐rich motif (R 454–458 )in α 2C ‐AR, which is absent in α 2A ‐ARs, in subcellular distribution of the receptors. Mutation of arginines to alanines (R 454–458 →A 454–458 ) spatially rescued hemagluttinin (HA)‐tagged α 2C ‐ARs to the PM (3.0±0.6 fold increase, n=4, P < 0.05; quantitated by live cell labeling and immunoprecipitation analysis). In contrast to wild‐type α 2C ‐ARs, activation of mutant α 2C ‐ARs inhibited cAMP accumulation (19.5±2.2 to 7.6±0.9 pmol, n=3, P <0.001). Creation of an arginine‐rich region in α 2A ‐AR C‐terminus by site‐directed mutagenesis (R 442–446 ) led to trans Golgi retention and inability to inhibit accumulation of cAMP compared to the control wild‐type α 2A ‐ARs. However, cooling to 28°C failed to rescue the α 2A ‐AR‐R 442–446 in contrast to the control wild‐type α 2C ‐ARs. Thus, the arginine‐rich motif is responsible for retention of α 2C ‐ARs in the trans Golgi compartment. The mechanism whereby cooling counters this retention signal is unknown.