COPURIFICATION AND DIRECT INTERACTION OF RAS WITH CAVEOLIN, AN INTEGRAL MEMBRANE-PROTEIN OF CAVEOLAE MICRODOMAINS - DETERGENT-FREE PURIFICATION OF CAVEOLAE MEMBRANES

Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane prote in, is a principal structural component of caveolae membranes in vivo, G protein alpha subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G p rotein alpha subunits, including G(s), G(o), and G(i2). Mutational or pharmacologic activation of G, subunits prevents the interaction of ca veolin with G(alpha) proteins, indicating that inactive G(alpha) subun its preferentially interact with caveolin, Here, we show that caveolin interacts with another well characterized signal transducer, Ras, Usi ng a detergent-free procedure for purification of caveolin-rich membra ne domains and a polyhistidine tagged form of caveolin, we find that H as and other classes of lipid-modified signaling molecules co-fraction ate and co-elute with caveolin, The association of Ras with caveolin w as further evaluated using two distinct in vitro binding assays, Wild- type H-Ras interacted with glutathione S-transferase (GST)-caveolin fu sion proteins but not with GST alone. Using a battery of GST fusion pr oteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41-amino acid membrane proximal region of the cytosoli c N-terminal domain of caveolin, In addition, reconstituted caveolin-r ich membranes (prepared with purified recombinant caveolin and purifie d lipids) interacted with a soluble form of wild-type H-Ras but failed to interact with mutationally activated soluble H-Ras (G12V), Thus, a single amino acid change (G12V) that constitutively activates Ras pre vents or destabilizes this interaction, These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid mem branes and (ii) membrane-bound caveolin preferentially interacts with inactive Ras proteins, In direct support of these in vitro studies, we also show that recombinant overexpression of caveolin in intact cells is sufficient to functionally recruit a nonfarnesylated mutant of Ras (C186S) onto membranes, overcoming the normal requirement for lipid m odification of has. Taken together, these observations suggest that ca veolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within c aveolin-rich microdomains of the plasma membrane.