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)

Biochemistry. 2003 Jul 8;42(26):7892-903. doi: 10.1021/bi027162n.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cattle
  • Caveolin 1
  • Caveolins / metabolism*
  • Cell Membrane / chemistry
  • Cholesterol / chemistry
  • Cholesterol / metabolism*
  • Cyclodextrins / pharmacology*
  • Darkness
  • Guanosine 5'-O-(3-Thiotriphosphate) / pharmacology*
  • Immunoblotting
  • Light
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Peptide Fragments / metabolism
  • Precipitin Tests
  • Protein Binding
  • Retina / metabolism
  • Rod Cell Outer Segment / drug effects*
  • Rod Cell Outer Segment / metabolism
  • Sequence Homology, Amino Acid
  • Subcellular Fractions
  • Transducin / drug effects
  • Transducin / metabolism*
  • beta-Cyclodextrins*

Substances

  • Caveolin 1
  • Caveolins
  • Cyclodextrins
  • Peptide Fragments
  • beta-Cyclodextrins
  • methyl-beta-cyclodextrin
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Cholesterol
  • Transducin