Agonist-dependent trafficking of α2-adrenoceptor subtypes:: dependence on receptor subtype and employed agonist

Many G protein-coupled receptors (GPCRs) are internalized from the plasma membrane after agonist exposure. Previously, marked agonist-induced internalization of human alpha(2A)- and alpha(2B)-adrenergic receptors (AR) was observed in transfected neuronal rat pheochromocytoma (PC12) cells; alpha(2A)- and alpha(2B)-AR were internalized into partly distinct intracellular vesicles (Olli-Lahdesmaki et al., J. Neurosci. 19, 9281 - 9288, 1999). In this paper, the extent of alpha(2)-AR internalization was quantitated in human embryonic kidney (HEK-293) and PC12 cells by combined application of cell surface biotinylation and ELISA methods, which allow measurement of protein trafficking in intact, differentiated and undifferentiated cells. Significant subtype-specific (but not cell type-dependent) trafficking of human a2-AR was observed by quantitation and immunocytochemistry. Agonist-induced sequestration of alpha(2B)-AR was markedly reduced after blocking the formation of clathrin-coated vesicles by hyperosmotic sucrose pretreatment. The sequestration of alpha(2A)-AR was partly inhibited after sucrose pretreatment but could be further reduced after inhibiting the formation of both clathrin-coated and caveolin vesicles by combined pretreatment with hyperosmotic sucrose and filipin. Differences were also observed in the recycling of alpha(2A)- and alpha(2B)-AR. The extent of maximal agonist-induced sequestration in PC12 cells was not directly dependent on relative agonist efficacy.