Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains (original) (raw)
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Journal of Biological Chemistry
Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein α subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein α subunits, including G, G, and G. Mutational or pharmacologic activation of G subunits prevents the interaction of caveolin with G proteins, indicating that inactive G subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro b...
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
Journal of Biological Chemistry, 2000
Caveolins form interlocking networks on the cytoplasmic face of caveolae. The cytoplasmically directed N and C termini of caveolins are separated by a central hydrophobic segment, which is believed to form a hairpin within the membrane. Here, we report that the caveolin scaffolding domain (CSD, residues 82-101), and the C terminus (residues 135-178) of caveolin-1 are each sufficient to anchor green fluorescent protein (GFP) to membranes in vivo. We also show that the first 16 residues of the C terminus (i.e. residues 135-150) are necessary and sufficient to attach GFP to membranes. When fused to the caveolin-1 C terminus, GFP co-localizes with two trans-Golgi markers and is excluded from caveolae. In contrast, the CSD targets GFP to caveolae, albeit less efficiently than full-length caveolin-1. Thus, caveolin-1 contains at least two membrane attachment signals: the CSD, dictating caveolar localization, and the C terminus, driving trans-Golgi localization. Additionally, we find that caveolin-1 oligomer/oligomer interactions require the distal third of the caveolin-1 C terminus. Thus, the caveolin-1 C-terminal domain has two separate functions: (i) membrane attachment (proximal third) and (ii) protein/protein interactions (distal third). Caveolae are flask-shaped invaginations of the plasma membrane that are found in most cell types. However, caveolae are most abundant in endothelial cells, adipocytes, epithelial cells, fibroblasts, and myocytes (1). These structures participate in three main areas of cell physiology: endocytosis (2), cholesterol traffic (3), and signal transduction (4). They are coated on their cytoplasmic face by a family of proteins, the caveolins. Three mammalian caveolin genes (caveolin-1,-2, and-3) have been identified and characterized (5). Whereas caveolin-1 and-2 have overlapping tissue distributions (6), caveolin-3 is limited to muscle and neuroglial cells (7-10). Although expression of caveolin-1 or-3 is sufficient to form caveolae in cells lacking these structures (11-14), caveolins are
Expression of Caveolin-1 Is Required for the Transport of Caveolin-2 to the Plasma Membrane
Journal of Biological Chemistry, 1999
Caveolins-1 and-2 are normally co-expressed, and they form a hetero-oligomeric complex in many cell types. These caveolin hetero-oligomers are thought to represent the assembly units that drive caveolae formation in vivo. However, the functional significance of the interaction between caveolins-1 and-2 remains unknown. Here, we show that caveolin-1 co-expression is required for the transport of caveolin-2 from the Golgi complex to the plasma membrane. We identified a human erythroleukemic cell line, K562, that expresses caveolin-2 but fails to express detectable levels of caveolin-1. This allowed us to stringently assess the effects of recombinant caveolin-1 expression on the behavior of endogenous caveolin-2. We show that expression of caveolin-1 in K562 cells is sufficient to reconstitute the de novo formation of caveolae in these cells. In addition, recombinant expression of caveolin-1 allows caveolin-2 to form high molecular mass oligomers that are targeted to caveolae-enriched membrane fractions. In striking contrast, in the absence of caveolin-1 expression, caveolin-2 forms low molecular mass oligomers that are retained at the level of the Golgi complex. Interestingly, we also show that expression of caveolin-1 in K562 cells dramatically up-regulates the expression of endogenous caveolin-2. Northern blot analysis reveals that caveolin-2 mRNA levels remain constant under these conditions, suggesting that the expression of caveolin-1 stabilizes the caveolin-2 protein. Conversely, transient expression
Caveolins: structure and function in signal transduction
Cellular & molecular biology letters, 2004
The caveolin family proteins are typically associated with microdomains that are found in the plasma membrane of numerous cells. These microdomains are referred to as/called caveolae. Caveolins are small proteins (18-24 kDa) that have a hairpin loop conformation with both the N and C termini exposed to the cytoplasm. Apart from having a structural function within caveolae, these proteins have the capacity to bind cholesterol as well as a variety of proteins, such as receptors, Src-like kinases, G-proteins, H-Ras, MEK/ERK kinases and nitric oxide synthases, which are involved in signal transduction processes. Considerable data allow the assumption to be made that the majority of the interactions with signaling molecules hold them in an inactive or repressed state. The activity of caveolins seems to be dependent on its specific post-translation modifications. It is suggested that caveolins fulfill a role in the modulation of cellular signaling cascades.
The Journal of Cell Biology, 1994
Caveolae are 50-100-nm membrane microdomains that represent a subcompartment of the plasma membrane. Previous morphological studies have implicated caveolae in (a) the transcytosis of macromolecules (including LDL and modified LDLs) across capillary endothelial cells, (b) the uptake of small molecules via a process termed potocytosis involving GPI-linked receptor molecules and an unknown anion transport protein, (c) interactions with the actin-based cytoskeleton, and (d) the compartmentalization of certain signaling molecules, including G-protein coupled receptors. Caveolin, a 22-kD integral membrane protein, is an important structural component of caveolae that was first identified as a major v-Src substrate in Rous sarcoma virus transformed cells. This finding initially suggested a relationship between caveolin, transmembrane signaling, and cellular transformation. We have recently developed a procedure for isolating caveolin-rich membrane domains from cultured cells. To facilitat...
Caveolae as Organizers of Pharmacologically Relevant Signal Transduction Molecules
Annual Review of Pharmacology and Toxicology, 2008
Caveolae, a subset of membrane (lipid) rafts, are flask-like invaginations of the plasma membrane that contain caveolin proteins, which serve as organizing centers for cellular signal transduction. Caveolins (-1, -2, and -3) have cytoplasmic N and C termini, palmitolylation sites, and a scaffolding domain that facilitates interaction and organization of signaling molecules so as to help provide coordinated and efficient signal transduction. Such signaling components include upstream entities (e.g., G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and steroid hormone receptors) and downstream components (e.g., heterotrimeric and low-molecularweight G proteins, effector enzymes, and ion channels). Diseases associated with aberrant signaling may result in altered localization or expression of signaling proteins in caveolae. Caveolin-knockout mice have numerous abnormalities, some of which may reflect the impact of total body knockout throughout the life span. This review provides a general overview of caveolins and caveolae, signaling molecules that localize to caveolae, the role of caveolae/caveolin in cardiac and pulmonary pathophysiology, pharmacologic implications of caveolar localization of signaling molecules, and the possibility that caveolae might serve as a therapeutic target.
The role of caveolae and the caveolins in mammalian physiology
Undergraduate Res, 2002
Caveolae are 50-100 nm invaginations of the plasma membrane that have captured the interest of scientists for many decades. However, the wide-ranging and physiologically important roles of these curious structures have only recently been addressed. Among the important milestones in the understanding of caveolae is the discovery of a family of proteins that are intimately involved in caveolar function (the caveolins). It has become clear now that caveolae and their caveolin "marker proteins" are involved in a variety of cellular processes including endocytosis, lipid homeostasis, signal transduction, and tumorigenesis. In this review, we will highlight the current view of caveolae in cell biology and discuss the relevance of these structures to mammalian physiology.
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.