Disruption of Microtubules Reveals Two Independent Apical Targeting Mechanisms for G-protein-coupled Receptors in Polarized Renal Epithelial Cells*
- From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
Abstract
G-protein-coupled receptors demonstrate differing trafficking itineraries in polarized Madin-Darby canine kidney (MDCK II) cells. The α2A adrenergic receptor (α2AAR) is directly delivered to the basolateral subdomain; the A1 adenosine receptor (A1AdoR) is apically enriched in its targeting; and the α2BAR subtype is randomly delivered to both domains but selectively retained basolaterally (Keefer, J. R., and Limbird, L. E. (1993) J. Biol. Chem. 268, 11340–11347; Saunders, C., Keefer, J. R., Kennedy, A. P., Wells, J. N., and Limbird, L. E. (1996) J. Biol. Chem. 271, 995–1002; Wozniak, M., and Limbird, L. E. (1996) J. Biol. Chem. 271, 5017–5024). The present studies explore the role of the polarized cytoskeleton in localization of G-protein-coupled receptors in MDCK II cells. Nocodazole or colchicine, which disrupt microtubules, did not perturb lateral localization of α2AR subtypes but led to a relocalization the A1AdoR to the basolateral surface, revealed by immunocytochemical and metabolic labeling strategies. Conversely, the apical component of the random delivery of α2BAR was not affected by these agents, suggesting microtubule-dependent and -independent apical targeting mechanisms for G-protein-coupled receptors in polarized cells. Apparent rerouting of the apically targeted A1AdoR was selective for microtubule-disrupting agents, since cytochalasin D, which disrupts actin polymerization, did not alter A1AdoR or α2BAR localization or targeting. These data suggest that multiple apical targeting mechanisms exist for G-protein-coupled receptors and that microtubule-disrupting agents serve as tools to probe their different trafficking mechanisms.
Footnotes
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↵* This work was supported by National Institutes of Health Grant DK 43879 (to L. E. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Recipient of a postdoctoral fellowship in pharmacology-morphology from the Pharmaceutical Research and Manufacturers of America Foundation.
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↵§ To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, MRBI 464, Nashville, TN 37209-6600. Tel.: 615-343-3538; Fax: 615-343-1084; E-mail: lee.limbird{at}mcmail.vanderbilt.edu.
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↵1 The abbreviations used are: GPCR, G-protein-coupled receptor(s); α2AR, α2adrenergic receptor(s); A1AdoR, A1 adenosine receptor(s); MDCK, Madin-Darby canine kidney; WT, wild-type; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; BFA, brefeldin A; [3H]DPCPX, 8-cyclopentyl-1,3-di-[2,3-3H]propylxanthine; BFA, brefeldin A; EGF, epidermal growth factor.
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↵2 Studies in our laboratory indicate that the intracellular pool of α2BAR decreased when the cells were grown 24 h in the absence of serum as evidenced immunocytochemically.
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- Received March 17, 1997.
- Revision received May 20, 1997.
