Dominant-negative caveolin inhibits H-Ras function by disrupting cholesterol-rich plasma membrane domains.
The plasma membrane pits known as caveolae have been implicated both in cholesterol homeostasis and in signal transduction. CavDGV and CavKSY, two dominant-negative amino-terminal truncation mutants of caveolin, the major structural protein of caveolae, significantly inhibited caveola-mediated SV40 infection, and were assayed for effects on Ras function. We find that CavDGV completely blocked Raf activation mediated by H-Ras, but not that mediated by K-Ras. Strikingly, the inhibitory effect of CavDGV on H-Ras signalling was completely reversed by replenishing cell membranes with cholesterol and was mimicked by cyclodextrin treatment, which depletes membrane cholesterol. These results provide a crucial link between the cholesterol-trafficking role of caveolin and its postulated role in signal transduction through cholesterol-rich surface domains. They also provide direct evidence that H-Ras and K-Ras, which are targeted to the plasma membrane by different carboxy-terminal anchors, operate in functionally distinct microdomains of the plasma membrane.
Roy
S
S
Queensland Cancer Fund Laboratory of Experimental Oncology, Department of Pathology, University of Queensland Medical School, Brisbane, Australia.
Luetterforst
R
R
Harding
A
A
Apolloni
A
A
Etheridge
M
M
Stang
E
E
Rolls
B
B
Hancock
J F
JF
Parton
R G
RG
D016475:3T3 Cells; D000818:Animals; D051242:Caveolin 1; D022461:Caveolins; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D005822:Genetic Vectors; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D013539:Simian virus 40; D014162:Transfection
C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D008563:Membrane Lipids; D008565:Membrane Proteins; D011994:Recombinant Proteins; D002784:Cholesterol; D016283:Proto-Oncogene Proteins p21(ras)