The alpha 2A-adrenergic receptor is targeted directly to the basolateral membrane domain of Madin-Darby canine kidney cells independent of coupling to pertussis toxin-sensitive GTP-binding proteins.
The alpha 2-adrenergic receptor (alpha 2-AR) is a member of the seven transmembrane-spanning G-protein-coupled receptor superfamily. In the kidney, the alpha 2-AR is most abundant in the epithelial cells of the proximal tubule where it is important in enhancing Na+ reabsorption via the modulation of Na+/H+ exchange. Radioligand binding and physiological studies suggest that the alpha 2-AR residues primarily on the basolateral surface of these proximal tubule cells in vivo. To investigate the mechanisms underlying alpha 2-AR polarization in epithelial cells, we permanently expressed wild-type and an epitope-tagged version of the alpha 2A-AR in Madin-Darby canine kidney (MDCK) cells. Using a steady-state surface biotinylation assay, we observe that 80-90% of the alpha 2A-AR in MDCK cell clones is located on the basolateral membrane domain. Immunolocalization studies confirm the biotinylation results and demonstrate that the alpha 2A-AR is actually confined primarily to the lateral domain of the basolateral surface. Metabolic labeling experiments suggest that basolateral polarization of the alpha 2A-AR is achieved by direct targeting of the receptor to the basolateral domain. Targeting of the alpha 2A-AR to the basolateral surface is not perturbed by pertussis toxin-treatment of MDCK cells, suggesting that coupling of the alpha 2A-AR to GTP-binding proteins is not important for receptor polarization.
Department of Pharmacology, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232-6600.
J R
JR
Keefer
L E
LE
Limbird
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003000:Clonidine / Q000378:metabolism; D004285:Dogs; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007150:Immunohistochemistry; D007457:Iodine Radioisotopes; D007668:Kidney; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D037342:Pertussis Toxin; D011869:Radioligand Assay; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014162:Transfection; D014316:Tritium; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*; D015016:Yohimbine / Q000378:metabolism
D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D011943:Receptors, Adrenergic, beta; D013462:Sulfur Radioisotopes; D010566:Virulence Factors, Bordetella; D014316:Tritium; D015016:Yohimbine; D008715:Methionine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D003000:Clonidine