Caveolins, a Family of Scaffolding Proteins for Organizing "Preassembled Signaling Complexes" at the Plasma Membrane (original) (raw)

1998, Journal of Biological Chemistry

Caveolae are vesicular invaginations of the plasma membrane. The chief structural proteins of caveolae are the caveolins. Caveolins form a scaffold onto which many classes of signaling molecules can assemble to generate preassembled signaling complexes. In addition to concentrating these signal transducers within a distinct region of the plasma membrane, caveolin binding may functionally regulate the activation state of caveolae-associated signaling molecules. Because the responsibilities assigned to caveolae continue to increase, this review will focus on: (i) caveolin structure/function and (ii) caveolae-associated signal transduction. Studies that link caveolae to human diseases will also be considered. The Caveolin Gene Family: Caveolin-1,-2, and-3 Molecular cloning has identified three distinct caveolin genes (1-6), caveolin-1, caveolin-2, and caveolin-3. Two isoforms of caveolin-1 (Cav-1␣ and Cav-1␤) are derived from alternate initiation during translation. Caveolin-1 and-2 are most abundantly expressed in adipocytes, endothelial cells, and fibroblastic cell types, whereas the expression of caveolin-3 is muscle-specific. Caveolin proteins interact with themselves to form homo-and hetero-oligomers (7-9), which directly bind cholesterol (10) and require cholesterol for insertion into model lipid membranes (10, 11). Caveolin oligomers may also interact with glycosphingolipids (12). These protein-protein and protein-lipid interactions are thought to be the driving force for caveolae formation (7). In addition, the caveolin gene family is structurally and functionally conserved from worms (Caenorhabditis elegans) to man (13), supporting the idea that caveolins play an essential role. Caveolin-1 assumes an unusual topology. A central hydrophobic domain (residues 102-134) is thought to form a hairpin-like structure within the membrane. As a consequence, both the N-terminal domain (residues 1-101) and the C-terminal domain (residues 135-178) face the cytoplasm. A 41-amino acid region of the N-terminal domain (residues 61-101) directs the formation of caveolin homooligomers (7), whereas the 44-amino acid C-terminal domain acts as a bridge to allow these homo-oligomers to interact with each other, thereby forming a caveolin-rich scaffold (14). Recent co-immunoprecipitation and dual labeling experiments directly show that caveolin-1 and-2 form a stable hetero-oligomeric complex and are strictly co-localized (9). Caveolin-2 localization corresponds to caveolae membranes as visualized by immunoelectron microscopy (9). Thus, caveolin-2 may function as an "accessory protein" in conjunction with caveolin-1. Caveolin-interacting Proteins A number of studies support the hypothesis that caveolin proteins provide a direct means for resident caveolae proteins to be sequestered within caveolae microdomains. These caveolin-interacting proteins include G-protein ␣ subunits, Ha-Ras, Src family tyrosine kinases, endothelial NOS, 1 EGF-R and related receptor tyrosine kinases, and protein kinase C isoforms (11, 15-18, 20-32). Heterotrimeric G-proteins-G-proteins are dramatically enriched within caveolae membranes, where caveolin-1 directly interacts with the ␣ subunits of G-proteins (18). Mutational or pharmacological activation of G s ␣ prevents its co-fractionation with caveolin-1 and blocks its direct interaction with caveolin-1 in vitro, indicating that the inactive GDP-bound form of G s ␣ preferentially interacts with caveolin-1. G-protein binding activity is located within a 41-amino acid region of the cytoplasmic N-terminal domain of caveolin-1 (residues 61-101). A polypeptide derived from this region of caveolin-1 (residues 82-101) effectively suppresses the basal GTPase activity of purified G-proteins by inhibiting GDP/ GTP exchange. In contrast, the analogous region of caveolin-2 possesses GTPase-activating protein activity with regard to heterotrimeric G-proteins (3). However, both of these activities (GDI and GAP) actively hold or place G-proteins in the inactive GDP-liganded conformation (3). Ha-Ras-Ha-Ras and Src family tyrosine kinases also directly interact with caveolin-1 (20, 23). Using a detergent-free procedure and a polyhistidine-tagged form of caveolin-1 for affinity purification of caveolin-rich membranes, G-proteins, Src family kinases, and Ha-Ras were all found to co-fractionate and co-elute with caveolin-1. Wild-type Ha-Ras also interacted with recombinant caveolin-1 in vitro. Ras binding activity was localized to a 41-amino acid membrane-proximal region (61-101) of the cytosolic N-terminal domain of caveolin-1, i.e. the same caveolin-1 region responsible for interacting with G-protein ␣ subunits. Reconstituted caveolinrich membranes interacted with a soluble recombinant form of wild-type Ha-Ras but failed to interact with mutationally activated soluble Ha-Ras (G12V) (23). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents this interaction. Recombinant overexpression of caveolin in intact cells was sufficient to functionally recruit a non-farnesylated mutant of Ras (C186S) onto membranes (23). This is consistent with the hypothesis that direct interaction with caveolin-1 promotes the sequestration of inactive Ha-Ras within caveolae microdomains. Src Family Tyrosine Kinases-Caveolin-1 interacts with wildtype Src (c-Src) but does not form a stable complex with mutationally activated Src (v-Src) (20). Thus, caveolin prefers the inactive conformation of G␣ subunits, Ha-Ras and c-Src. Deletion mutagenesis indicates that the Src-interacting domain of caveolin is located within residues 61-101. A caveolin peptide derived from this region (residues 82-101) functionally suppressed the autoactivation of purified recombinant c-Src tyrosine kinase and a related Src family kinase, Fyn. Co-expression of caveolin-1 with c-Src shows that caveolin-1 dramatically suppresses the tyrosine kinase activity of c-Src. Thus, it appears that caveolin-1 functionally interacts with wild-type c-Src via caveolin residues 82-101. Endothelial Nitric Oxide Synthase (eNOS)-Several independent co-immunoprecipitation and domain-mapping studies demonstrate that eNOS interacts directly with caveolin-1 residues 82-101 (30, 34, 36-38). In support of these data, recombinant co-expres