Vol. 57, Issue 1, 44-52, January 2000

Microtubule-Dependent Regulation of _2B Adrenergic Receptors in Polarized MDCKII Cells Requires the Third Intracellular Loop but Not G Protein Coupling

Christine Saunders¹ and Lee E. Limbird

Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee

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 ₂-adrenergic receptors (₂AR) are not; instead, the binding capacity of the _2BAR, but not _2AAR or _2CAR subtypes, is increased in a time-dependent fashion. The present studies explore the molecular basis for this _2BAR subtype-selective phenomenon. Colchicine selectively increased _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 _2BAR requires the third intracellular loop because the _2BAR loop deletion (_2BARi3) mutant did not show an increased receptor density after colchicine treatment. Furthermore, the colchicine-mediated increase in _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 _2BAR density. Colchicine-dependent increases in _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 _2BAR density at the basolateral surface of polarized MDCKII cells requires the third intracellular loop of _2BAR but not functional _2BAR-G protein coupling.