Cholesterol-dependent association of caveolin-1 with the transducin alpha subunit in bovine photoreceptor rod outer segments: disruption by cyclodextrin and guanosine 5'-O-(3-thiotriphosphate).
Evidence suggests that caveolins, 21-24 kDa cholesterol-binding proteins that generally reside in specialized detergent-resistant membrane microdomains, act as signaling scaffolds. Detergent-resistant membranes isolated from rod outer segments (ROS) have been previously shown to contain the photoreceptor G-protein, transducin. In this report we show, by subcellular fractionation, that caveolin-1 is an authentic component of purified ROS. We demonstrate that caveolin-1 in ROS almost exclusively resides in low-buoyant-density, cholesterol-rich, detergent-resistant membranes that can be disrupted by cholesterol depletion using methyl-beta-cyclodextrin (MCD). Cholesterol depletion was also observed to extract a pool of transducin alpha (Talpha) from ROS membranes. Immunoprecipitation with anti-caveolin-1 revealed the association of Talpha in the absence of Tbetagamma. Treatment of ROS with MCD resulted in a 2-fold decrease in recovery of Talpha in anti-caveolin-1 immunoprecipitates. This interaction was also completely disrupted when ROS were exposed to light in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), a nonhydrolyzable GTP analogue. In addition, caveolin-1/Talpha association in the immune complex was disrupted by a peptide based on the primary sequence of the caveolin-1 scaffolding domain. Finally, we confirm the colocalization of caveolin-1 and Talpha in photoreceptors by immunofluorescence microscopy. These results strongly suggest that the association between Talpha and caveolin-1 occurs in cholesterol-rich, detergent-resistant membranes and is likely to be dependent upon the activation state of Talpha.
Elliott
Michael H
MH
Dean A. McGee Eye Institute, Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA. Michael-Elliott@ouhsc.edu
Fliesler
Steven J
SJ
Ghalayini
Abboud J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology*; D003624:Darkness; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D015151:Immunoblotting; D008027:Light; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D012160:Retina / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000187:drug effects* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D015293:Transducin / Q000187:drug effects / Q000378:metabolism*; D047392:beta-Cyclodextrins
D051242:Caveolin 1; D022461:Caveolins; D003505:Cyclodextrins; D010446:Peptide Fragments; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002784:Cholesterol; D015293:Transducin