Colocalization of beta-adrenergic receptors and caveolin within the plasma membrane.
The rapid amplification of beta-adrenergic receptor signaling involves the sequential activation of multiple signaling molecules ranging from the receptor to adenylyl cyclase. The prevailing view of the agonist-induced interaction between signaling molecules is based on random collisions between proteins that diffuse freely in the plasma membrane. The recent identification of G protein alpha- and betagamma-subunits in caveolae and their functional interaction with caveolin suggests that caveolae may participate in G protein-coupled signaling. We have investigated the potential interaction of beta-adrenergic receptors with caveolin under resting conditions. beta1- and beta2-adrenergic receptors were recombinantly overexpressed in COS-7 cells. Caveolae were isolated using the detergent-free sucrose gradient centrifugation method. beta1- and beta2-adrenergic receptors were localized in the same gradient fractions as caveolin, where Gsalpha- and betagamma-subunits were detected as well. Immunofluorescence microscopy demonstrated the colocalization of beta-adrenergic receptors with caveolin, indicating a nonrandom distribution of beta-adrenergic receptors in the plasma membrane. Using polyhistidine-tagged recombinant proteins, beta-adrenergic receptors were copurified with caveolin, suggesting that they were physically bound. Our results suggest that, in addition to clathrin-coated pits, caveolae may act as another plasma membrane microdomain to compartmentalize beta-adrenergic receptors.
Schwencke
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C
Cardiovascular & Pulmonary Research Institute, Allegheny University of the Health Sciences, Pittsburgh, Pennsylvania 15212, USA.
Okumura
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S
Yamamoto
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M
Geng
Y J
YJ
Ishikawa
Y
Y
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D051242:Caveolin 1; D022461:Caveolins; D002462:Cell Membrane / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D051242:Caveolin 1; D022461:Caveolins; D008565:Membrane Proteins; D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins