The dually acylated NH2-terminal domain of G(i1 alpha) is sufficient to target a green fluorescent protein reporter to caveolin-enriched plasma membrane domains - Palmitoylation of caveolin-1 is required for the recognition of dually acylated G-protein alpha subunits in vivo

Here we investigate the molecular mechanisms that govern the targeting of G -protein cr subunits to the plasma membrane. For this purpose, we used G(i1 alpha), as a model dually acylated G-protein, We fused full-length G(i1 al pha) or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green f luorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of G(i1 alpha) are sufficient to target GFP to caveolin-enriched domains of the plasma membran e in vivo, as demonstrated by cofractionation and co-immunoprecipitation wi th caveolin-1, Interestingly, when dual acylation of this 32-amino acid dom ain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin -rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only pa rtially partitioned into caveolin-containing fractions. However, both nonac ylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveo lin-1, Taken together, these results indicate that lipid modification of th e NH2-terminal of G(i1 alpha) is essential for targeting to its correct des tination and interaction with caveolin-1, Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co- immunoprecipitate these dually acylated GFP-G-protein fusions, Thus, dual a cylation of the NH2-terminal domain of G(i1 alpha) and palmitoylation of ca veolin-1 are both required to stabilize and perhaps regulate this reciproca l interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its in teraction with signaling molecules.