Structural insights into the function of human caveolin 1 (original) (raw)
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A bioinformatics study was performed to predict and compare the structural and functional properties of human caveolins: caveolin-1, -2 and -3. The computed local physicochemical properties, predictions of their secondary structure elements and interacting partners of caveolin-2 and -3 were compared to the experimentally determined structural and functional properties of caveolin-1. These data combined with sequence alignments of the three caveolins allowed the functional domains of caveolin-2 and -3 to be predicted and characterised. The hydrophobic regions of these proteins are highly similar in sequences and physicochemical properties, which is in good agreement with their known membrane locations and functions. The most divergent in terms of sequences and properties are the C-terminal regions of the caveolins, suggesting that they might be responsible for their distinct predicted interactions, with direct consequences on signalling processes.
Journal of Biological Chemistry, 1997
Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting proteins within caveolae membranes. In this regard, caveolin co-purifies with a variety of lipid-modified signaling molecules, including G-proteins, Src-like kinases, Ha-Ras, and eNOS. Using several independent approaches, it has been shown that a 20-amino acid membrane proximal region of the cytosolic amino-terminal domain of caveolin is sufficient to mediate these interactions. For example, this domain interacts with G-protein ␣ subunits and Src-like kinases and can functionally suppress their activity. This caveolinderived protein domain has been termed the caveolinscaffolding domain. However, it remains unknown how the caveolin-scaffolding domain recognizes these molecules.
Journal of Biological Chemistry, 1998
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
Oligomeric structure of caveolin: implications for caveolae membrane organization
Proceedings of the National Academy of Sciences of the United States of America, 1995
A 22-kDa protein, caveolin, is localized to the cytoplasmic surface of plasma membrane specializations called caveolae. We have proposed that caveolin may function as a scaffolding protein to organize and concentrate signaling molecules within caveolae. Here, we show that caveolin interacts with itself to form homooligomers. Electron microscopic visualization of these purified caveolin homooligomers demonstrates that they appear as individual spherical particles. By using recombinant expression of caveolin as a glutathione S-transferase fusion protein, we have defined a region of caveolin's cytoplasmic N-terminal domain that mediates these caveolin-caveolin interactions. We suggest that caveolin homooligomers may function to concentrate caveolin-interacting molecules within caveolae. In this regard, it may be useful to think of caveolin homooligomers as "fishing lures" with multiple "hooks" or attachment sites for caveolin-interacting molecules.
Quantitative Proteomics of Caveolin-1-regulated Proteins
Molecular & Cellular Proteomics, 2010
Caveolae are organelles abundant in the plasma membrane of many specialized cells including endothelial cells (ECs), epithelial cells, and adipocytes, and in these cells, caveolin-1 (Cav-1) is the major coat protein essential for the formation of caveolae. To identify proteins that require Cav-1 for stable incorporation into membrane raft domains, a quantitative proteomics analysis using isobaric tagging for relative and absolute quantification was performed on rafts isolated from wild-type and Cav-1-deficient mice. In three independent experiments, 117 proteins were consistently identified in membrane rafts with the largest differences in the levels of Cav-2 and in the caveola regulatory proteins Cavin-1 and Cavin-2. Because the lung is highly enriched in ECs, we validated and characterized the role of the newly described protein Cavin-1 in several cardiovascular tissues and in ECs. Cavin-1 was highly expressed in ECs lining blood vessels and in cultured ECs. Knockdown of Cavin-1 reduced the levels of Cav-1 and-2 and weakly influenced the formation of high molecular weight oligomers containing Cav-1 and-2. Cavin-1 silencing enhanced basal nitric oxide release from ECs but blocked proangiogenic phenotypes such as EC proliferation, migration, and morphogenesis in vitro. Thus, these data support an important role of Cavin-1 as a regulator of caveola function in ECs.
Identification, sequence, and expression of caveolin-2 defines a caveolin gene …
Proceedings of the …
Caveolin, a 21to 24-kDa integral membrane protein, is a principal component of caveolae membranes. Caveolin interacts directly with heterotrimeric guanine nucleotide binding proteins (G proteins) and can functionally regulate their activity. Here, an 20-kDa caveolin-related protein, caveolin-2, was identified through microsequencing of adipocyte-derived caveolin-enriched membranes; caveolin was retermed caveolin-1. Caveolins 1 and 2 are similar in most respects. mRNAs for both caveolin-1 and caveolin-2 are most abundantly expressed in white adipose tissue and are induced during adipocyte differentiation. Caveolin-2 colocalizes with caveolin-1, indicating that caveolin-2 also localizes to caveolae. However, caveolin-1 and caveolin-2 differ in their functional interactions with heterotrimeric G proteins, possibly explaining why caveolin-1 and-2 are coexpressed within a single cell. Caveolae are plasma membrane specializations present in most cell types (1). They are most conspicuous in adipocytes were they represent up to 20% of the total plasma membrane surface area (2). Cytoplasmically oriented signaling molecules are concentrated within these structures, including heterotrimeric guanine nucleotide binding proteins (G proteins), Srclike kinases, protein kinase Ca and Ras-related GTPases (3-9). The caveolae signaling hypothesis states that caveolar localization of signaling molecules could provide a compartmental basis for integrating certain transmembrane signaling events (1). Caveolin, a 21to 24-kDa integral membrane protein, is the main component of caveolae membranes (10). Structurally, caveolin can be divided into three distinct regions: a hydrophilic cytosolic N-terminal domain, a membranespanning region, and a hydrophilic C-terminal domain (11). The C-terminal domain undergoes palmitoylation (Sacylation) on three cysteine residues (12), suggesting that both the membrane-spanning region and the C-terminal domain of caveolin are associated with the membrane. Recent evidence suggests that caveolin may function as a scaffolding protein for organizing and concentrating certain caveolin-interacting molecules within caveolae membranes (3, 13). Although caveolin is the product of a single gene, it encodes one mRNA but two caveolin isoforms that differ by 3 kDa and have been termed a-and ,B-caveolin (14). a-caveolin contains residues 1-178; methionine 32 acts as an internal translation initiation site to form ,B-caveolin (14). Both caveolin isoforms are targeted to caveolae (15), form homooligomers (16, 17), and interact with G proteins (13). However, a-and ,B-caveolin assume a distinct but overlapping subcellular distribution in intact cells (14) and only ,B-caveolin is phosphorylated on serine residues in vivo (15). These results suggest The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
American journal of physiology. Cell physiology, 2017
Caveolins (Cavs) are ~20 kDa scaffolding proteins that assemble as oligomeric complexes in lipid raft domains to form caveolae, flask-shaped plasma membrane (PM) invaginations. Caveolae ("little caves") require lipid-lipid, protein-lipid, and protein-protein interactions that can modulate the localization, conformational stability, ligand affinity, effector specificity, and other functions of proteins that are partners of Cavs. Cavs are assembled into small oligomers in the endoplasmic reticulum (ER), transported to the Golgi for assembly with cholesterol and other oligomers, and then exported to the PM as an intact coat complex. At the PM, cavins, ~50 kDa adapter proteins, oligomerize into an outer coat complex that remodels the membrane into caveolae. The structure of caveolae protects their contents (i.e., lipids and proteins) from degradation. Cellular changes, including signal transduction effects, can destabilize caveolae and produce cavin dissociation, restructuring...