Abstract

Previous studies in cultured, polarized Madin-Darby canine kidney II (MDCKII) renal epithelial cells have demonstrated that the apical steady-state localization and delivery of the A₁ adenosine receptor is modified by disruption of the microtubule network with colchicine, whereas the basolateral localization and trafficking of the α₂-adrenergic receptors (α₂AR) are not; instead, the binding capacity of the α_2BAR, but not α_2AAR or α_2CAR subtypes, is increased in a time-dependent fashion. The present studies explore the molecular basis for this α_2BAR subtype-selective phenomenon. Colchicine selectively increased α_2BAR density at the cell surface, as determined by confocal microscopy, receptor binding, and surface biotinylation studies. The colchicine-induced increase in the functional density of the α_2BAR requires the third intracellular loop because the α_2BAR loop deletion (α_2BAR▵i3) mutant did not show an increased receptor density after colchicine treatment. Furthermore, the colchicine-mediated increase in α_2BAR density is manifest only in polarized cells because colchicine treatment of nonpolarized MDCKII renal epithelial cells as well as simian kidney COSM6 and human embryonic kidney HEK293 cells did not effect an increase in α_2BAR density. Colchicine-dependent increases in α_2BAR density did not depend on functional coupling to G proteins, however, because pretreatment with pertussis toxin did not eliminate the effect of colchicine. These data indicate that microtubule-dependent regulation of α_2BAR density at the basolateral surface of polarized MDCKII cells requires the third intracellular loop of α_2BAR but not functional α_2BAR-G protein coupling.