The three alpha 2-adrenergic receptor subtypes achieve basolateral localization in Madin-Darby canine kidney II cells via different targeting mechanisms.
The present studies examined the localization of the alpha2B- and alpha2C-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 alpha2AAR subtype is directly delivered to lateral subdomain of MDCK II cells. Surface biotinylation strategies demonstrated that the alpha2BAR, like the alpha2AAR, achieves 85-90% basolateral localization at steady-state. However, in contrast to the alpha2AAR, this polarization occurs after initial random insertion of the alpha2BAR into both apical and basolateral surfaces followed by selective retention on the lateral subdomain (t1/2 the apical surface is 15-30 min; t1/2 the basolateral surface is 8-10 h). The alpha2CAR also is enriched on the basolateral surface at steady-state and, like the alpha2AAR, is directly delivered there. Morphological evaluation of the epitope-tagged alpha2AAR, alpha2BAR, and alpha2CAR subtypes by laser confocal microscopy not only corroborated the biochemically-defined basolateral localization of all three alpha2AR subtypes but also revealed that the alpha2CAR uniquely exists in an intracellular compartment(s) as well. Immunofluorescence due to intracellular alpha2CAR 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 alpha2AAR, alpha2BAR, and alpha2CAR subtypes, which possess highly homologous structures and ultimately achieve similar polarization to the lateral surface of MDCK II cells, nonetheless manifest distinct trafficking itineraries.
Wozniak
M
M
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600, USA.
Limbird
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D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002999:Clone Cells; D017931:DNA Primers; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000939:Epitopes; D006056:Golgi Apparatus / Q000378:metabolism; D007150:Immunohistochemistry; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D018341:Receptors, Adrenergic, alpha-2 / Q000096:biosynthesis / Q000145:classification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D016324:Sequence Tagged Sites; D013997:Time Factors
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D018341:Receptors, Adrenergic, alpha-2; D011994:Recombinant Proteins