title: Microtubule-dependent regulation of alpha(2B) adrenergic receptors in polarized MDCKII cells requires the third intracellular loop but not G protein coupling.
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 A(1) adenosine receptor is modified by disruption of the microtubule network with colchicine, whereas the basolateral localization and trafficking of the alpha(2)-adrenergic receptors (alpha(2)AR) are not; instead, the binding capacity of the alpha(2B)AR, but not alpha(2A)AR or alpha(2C)AR subtypes, is increased in a time-dependent fashion. The present studies explore the molecular basis for this alpha(2B)AR subtype-selective phenomenon. Colchicine selectively increased alpha(2B)AR 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 alpha(2B)AR requires the third intracellular loop because the alpha(2B)AR loop deletion (alpha(2B)ARtriangle upi3) mutant did not show an increased receptor density after colchicine treatment. Furthermore, the colchicine-mediated increase in alpha(2B)AR 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 alpha(2B)AR density. Colchicine-dependent increases in alpha(2B)AR 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 alpha(2B)AR density at the basolateral surface of polarized MDCKII cells requires the third intracellular loop of alpha(2B)AR but not functional alpha(2B)AR-G protein coupling.
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: C
authors.initials: C
authors.lastname: Saunders
authors.forename: L E
authors.initials: LE
authors.lastname: Limbird
mesh_terms: D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D004285:Dogs; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008870:Microtubules / Q000378:metabolism*; D011487:Protein Conformation; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism
chemical_list: C547771:ADRA2B protein, human; D018341:Receptors, Adrenergic, alpha-2; D011956:Receptors, Cell Surface; D019204:GTP-Binding Proteins; D003078:Colchicine