The Three 
-Adrenergic Receptor Subtypes Achieve Basolateral Localization in Madin-Darby Canine Kidney II Cells via Different Targeting
Mechanisms (*)
- From the Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600
- ¶ To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University, School of Medicine, Nashville, TN 37232-6600. Tel.: 615-343-3533; Fax: 615-343-1084.
Abstract
The present studies examined the localization of the α
- and α
-adrenergic receptor (AR) subtypes in polarized Madin-Darby canine kidney cells (MDCK II) and the mechanisms by which this
is achieved. Previously we demonstrated that the α
AR subtype is directly delivered to lateral subdomain of MDCK II cells. Surface biotinylation strategies demonstrated that
the α
AR, like the α
AR, achieves 85-90% basolateral localization at steady-state. However, in contrast to the α
AR, this polarization occurs after initial random insertion of the α
AR into both apical and basolateral surfaces followed by selective retention on the lateral subdomain (t
on the apical surface is 15-30 min; t
on the basolateral surface is 8-10 h). The α
AR also is enriched on the basolateral surface at steady-state and, like the α
AR, is directly delivered there. Morphological evaluation of the epitope-tagged α
AR, α
AR, and α
AR subtypes by laser confocal microscopy not only corroborated the biochemically-defined basolateral localization of all three
α
AR subtypes but also revealed that the α
AR uniquely exists in an intracellular compartment(s) as well. Immunofluorescence due to intracellular α
AR partially overlaps that due to calnexin, a marker for endoplasmic reticulum, as well as that due to mannosidase II, a marker
for the trans-Golgi network. Taken together, the present findings demonstrate that the α
AR, α
AR, and α
AR subtypes, which possess highly homologous structures and ultimately achieve similar polarization to the lateral surface
of MDCK II cells, nonetheless manifest distinct trafficking itineraries.
Footnotes
-
↵§ Supported by a postdoctoral fellowship from the National Kidney Foundation.
-
↵* This work was supported in part by National Institutes of Health Research Grant DK 43879. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- α
AR -
α
-adrenergic receptor
- MDCK
-
Madin-Darby canine kidney
- [
I]Rau-AzPEC -
17α-hydroxy-20α-yohimban-16β-(N-(4-azido-3[
I]-iodo)-phenethyl)-carboxamide
- HA
-
hemagglutinin
- PCR
-
polymerase chain reaction
- bp
-
base pair(s)
- PBS
-
phosphate-buffered saline
- PAGE
-
polyacrylamide gel electrophoresis
- BSA
-
bovine serum albumin
- WT
-
wild-type
- MOPS
-
4-morpholinepropanesulfonic acid
- FITC
-
fluorescein isothiocyanate.
- α
-
- Received November 7, 1995.
- Revision received December 13, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
