High-resolution proton NMR measures mobile lipids associated with Triton-resistant membrane domains in haematopoietic K562 cells lacking or expressing caveolin-1.

High-resolution proton NMR spectra of intact tumour cells generally exhibit intense signals due to isotropically mobile lipids (MLs) of still uncertain nature and origin. NMR studies performed on intact wild-type and caveolin-1-infected haematopoietic K562 cells showed that, under our experimental conditions, part of the ML signals are due to lipid complexes resistant to extraction in Triton X-100 at 4 degrees C. This evidence suggests that a portion of NMR-visible lipid structures are compatible with Triton-resistant membrane rafts and therefore biophysically distinct from NMR-visible Triton-soluble lipid bodies. Similarly to lipid rafts and caveolae, the organization of the Triton-insoluble ML domains could be compromised by treatment with beta-octylglucoside or methyl-beta-cyclodextrin. Exposure to exogenous sphingomyelinase caused an increase in ML NMR visibility, indicating the possible involvement of ceramides in ML formation. The mobility of these lipids was found to be temperature sensitive, suggesting a transition in cells going from 4 degrees C to 25-37 degrees C. These new results are here discussed in the light of possible contributions of plasma membrane microdomains to NMR-visible ML signals.

Organization of plasma membrane functional rafts upon T cell activation.

Raft microdomains have been shown to play a key role in T cell activation. We found that in human T lymphocytes the formation of functional rafts at the plasma membrane was induced by T cell priming. In resting T cells from peripheral blood Lck and the raft glycosphingolipid GM1 resided in intracellular membranes. T cell activation induced synthesis of GM1 and effector cells showed very high levels of this lipid, which became predominantly plasma membrane associated. TCR triggering also induced targeting of the cytosolic Lck to the plasma membrane. Thus, effector cells acquire an improved signaling machinery by increasing the amount of rafts at the plasma membrane. The fact that, when compared with naive T cells, memory T cells showed higher GM1 levels suggests that raft lipid synthesis may be developmentally regulated and tune T cell responsiveness.

Biotinylation and analysis of membrane-bound and soluble proteins.

Coupling of a biotin moiety to proteins allows sensitive detection of those proteins with a variety of commercially available avidin or streptavidin conjugates. This unit presents protocols for biotinylation of cell-surface proteins and proteins in solution. A major advantage of these techniques is that they do not require use of radioisotopes (although use of radioiodinated streptavidin is optional for biotin detection).

Caveolin-3 directly interacts with the C-terminal tail of beta -dystroglycan. Identification of a central WW-like domain within caveolin family members.

Caveolin-3, the most recently recognized member of the caveolin gene family, is muscle-specific and is found in both cardiac and skeletal muscle, as well as smooth muscle cells. Several independent lines of evidence indicate that caveolin-3 is localized to the sarcolemma, where it associates with the dystrophin-glycoprotein complex. However, it remains unknown which component of the dystrophin complex interacts with caveolin-3. Here, we demonstrate that caveolin-3 directly interacts with beta-dystroglycan, an integral membrane component of the dystrophin complex. Our results indicate that caveolin-3 co-localizes, co-fractionates, and co-immunoprecipitates with a fusion protein containing the cytoplasmic tail of beta-dystroglycan. In addition, we show that a novel WW-like domain within caveolin-3 directly recognizes the extreme C terminus of beta-dystroglycan that contains a PPXY motif. As the WW domain of dystrophin recognizes the same site within beta-dystroglycan, we also demonstrate that caveolin-3 can effectively block the interaction of dystrophin with beta-dystroglycan. In this regard, interaction of caveolin-3 with beta-dystroglycan may competitively regulate the recruitment of dystrophin to the sarcolemma. We discuss the possible implications of our findings in the context of Duchenne muscular dystrophy.

Characterization of caveolae from rat heart: localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation.

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse alpha- and beta-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states.

Extramitochondrial porin: facts and hypotheses.

Mitochondrial porin, or VDAC, is a pore-forming protein abundant in the outer mitochondrial membrane. Several publications have reported extramitochondrial localizations as well, but the evidence was considered insufficient by many, and the presence of porin in nonmitochondrial cellular compartments has remained in doubt for a long time. We have now obtained new data indicating that the plasma membrane of hematopoietic cells contains porin, probably located mostly in caveolae or caveolae-like domains. Porin was purified from the plasma membrane of intact cells by a procedure utilizing the membrane-impermeable labeling reagent NH-SS-biotin and streptavidin affinity chromatography, and shown to have the same properties as mitochondrial porin. A channel with properties similar to that of isolated VDAC was observed by patch-clamping intact cells. This review discusses the evidence supporting extramitochondrial localization, the putative identification of the plasma membrane porin with the "maxi" chloride channel, the hypothetical mechanisms of sorting porin to various cellular membrane structures, and its possible functions.

Porin is present in the plasma membrane where it is concentrated in caveolae and caveolae-related domains.

Mitochondrial porin, or voltage-dependent anion channel, is a pore-forming protein first discovered in the outer mitochondrial membrane. Later investigations have provided indications for its presence also in other cellular membranes, including the plasma membrane, and in caveolae. This extra-mitochondrial localization is debated and no clear-cut conclusion has been reached up to now. In this work, we used biochemical and electrophysiological techniques to detect and characterize porin within isolated caveolae and caveolae-like domains (low density Triton-insoluble fractions). A new procedure was used to isolate porin from plasma membrane. The outer surface of cultured CEM cells was biotinylated by an impermeable reagent. Low density Triton-insoluble fractions were prepared from the labeled cells and used as starting material to purify a biotinylated protein with the same electrophoretic mobility and immunoreactivity of mitochondrial porin. In planar bilayers, the porin from these sources formed slightly anion-selective pores with properties indistinguishable from those of mitochondrial porin. This work thus provides a strong indication of the presence of porin in the plasma membrane, and specifically in caveolae and caveolae-like domains.

Glycosphingolipid domains on cell plasma membrane.

In this study we analyzed by immunofluorescence, laser confocal microscopy, immunoelectron microscopy and label fracture technique the ganglioside distribution on the plasma membrane of several different cell types: human peripheral blood lymphocytes (PBL), Molt-4 lymphoid cells, and NIH 3T3 fibroblasts, which mainly express monosialoganglioside GM3, and murine NS20Y neuroblastoma cells, which have been shown to express a high amount of monosialoganglioside GM2. Our observations showed an uneven distribution of both GM3 and GM2 on the plasma membrane of all cells, confirming the existence of ganglioside-enriched microdomains on the cell surface. Interestingly, in lymphoid cells the clustered immunolabeling appeared localized over both the microvillous and the nonvillous portions of the membrane. Similarly, in cells growing in monolayer, the clusters were distributed on both central and peripheral regions of the cell surface. Therefore, glycosphingolipid clusters do not appear confined to specific areas of the plasma membrane, implying general functions of these domains, which, as structural components of a cell membrane multimolecular signaling complex, may be involved in cell activation and adhesion, signal transduction and, when associated to caveolae, in endocytosis of specific molecules.

Expression of caveolin-1 is required for the transport of caveolin-2 to the plasma membrane. Retention of caveolin-2 at the level of the golgi complex.

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 of caveolin-2 in CHO cells is sufficient to up-regulate endogenous caveolin-1 expression. Thus, the formation of a hetero-oligomeric complex between caveolins-1 and -2 stabilizes the caveolin-2 protein product and allows caveolin-2 to be transported from the Golgi complex to the plasma membrane.

Phorbol ester-induced disruption of the CD4-Lck complex occurs within a detergent-resistant microdomain of the plasma membrane. Involvement of the translocation of activated protein kinase C isoforms.

Recent studies have highlighted the existence of discrete microdomains at the cell surface that are distinct from caveolae. The function of these microdomains remains unknown. However, recent evidence suggests that they may participate in a subset of transmembrane signaling events. In hematopoietic cells, these low density Triton-insoluble (LDTI) microdomains (also called caveolae-related domains) are dramatically enriched in signaling molecules, such as cell surface receptors (CD4 and CD55), Src family tyrosine kinases (Lyn, Lck, Hck, and Fyn), heterotrimeric G proteins, and gangliosides (GM1 and GM3). Human T lymphocytes have become a well established model system for studying the process of phorbol ester-induced down-regulation of CD4. Here, we present evidence that phorbol 12-myristate 13-acetate (PMA)-induced down-regulation of the cell surface pool of CD4 occurs within the LDTI microdomains of T cells. Localization of CD4 in LDTI microdomains was confirmed by immunoelectron microscopy. PMA-induced disruption of the CD4-Lck complex was rapid (within 5 min), and this disruption occurred within LDTI microdomains. Because PMA is an activator of protein kinase C (PKC), we next evaluated the possible roles of different PKC isoforms in this process. Our results indicate that PMA induced the rapid translocation of cytosolic PKCs to LDTI microdomains. We identified PKCalpha as the major isoform involved in this translocation event. Taken together, our results support the hypothesis that LDTI microdomains represent a functionally important plasma membrane compartment in T cells.

Mutational analysis of caveolin-induced vesicle formation. Expression of caveolin-1 recruits caveolin-2 to caveolae membranes.

Caveolae are vesicular organelles with a characteristic uniform diameter in the range of 50-100 nm. Although recombinant expression of caveolin-1 is sufficient to drive caveolae formation, it remains unknown what controls the uniform diameter of these organelles. One hypothesis is that specific caveolin-caveolin interactions regulate the size of caveolae, as caveolin-1 undergoes two stages of self-oligomerization. To test this hypothesis directly, we have created two caveolin-1 deletion mutants that lack regions of caveolin-1 that are involved in directing the self-assembly of caveolin-1 oligomers. More specifically, Cav-1 delta61-100 lacks a region of the N-terminal domain that directs the formation of high molecular mass caveolin-1 homo-oligomers, while Cav-1 deltaC lacks a complete C-terminal domain that is required to allow caveolin homo-oligomers to interact with each other, forming a caveolin network. It is important to note that these two mutants retain an intact transmembrane domain. Our current results show that although Cav-1 delta61-100 and Cav-1 deltaC are competent to drive vesicle formation, these vesicles vary widely in their size and shape with diameters up to 500-1000 nm. In addition, caveolin-induced vesicle formation appears to be isoform-specific. Recombinant expression of caveolin-2 under the same conditions failed to drive the formation of vesicles, while caveolin-3 expression yielded caveolae-sized vesicles. These results are consistent with the previous observation that in transformed NIH 3T3 cells that lack caveolin-1 expression, but continue to express caveolin-2, no morphologically distinguishable caveolae are observed. In addition, as caveolin-2 alone exists mainly as a monomer or homo-dimer, while caveolins 1 and 3 exist as high molecular mass homo-oligomers, our results are consistent with the idea that the formation of high molecular mass oligomers of caveolin are required to regulate the formation of uniform caveolae-sized vesicles. In direct support of this notion, regulated induction of caveolin-1 expression in transformed NIH 3T3 cells was sufficient to recruit caveolin-2 to caveolae membranes. The ability of caveolin-1 to recruit caveolin-2 most likely occurs through a direct interaction between caveolins 1 and 2, as caveolins 1 and 2 are normally co-expressed and interact with each other to form high molecular mass hetero-oligomers containing both caveolins 1 and 2.

Expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion neurons: caveolin-2 is up-regulated in response to cell injury.

Caveolae are cholesterol/sphingolipid-rich microdomains of the plasma membrane that have been implicated in signal transduction and vesicular trafficking. Caveolins are a family of caveolae-associated integral membrane proteins. Caveolin-1 and -2 show the widest range of expression, whereas caveolin-3 expression is restricted to muscle cell types. It has been previously reported that little or no caveolin mRNA species are detectable in the brain by Northern blot analyses or in neuroblastoma cell lines. However, it remains unknown whether caveolins are expressed within neuronal cells. Here we demonstrate the expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion (DRG) neurons by using mono-specific antibody probes. In PC12 cells, caveolin-1 expression is up-regulated on day 4 of nerve growth factor (NGF) treatment, whereas caveolin-2 expression is transiently up-regulated early in the differentiation program and then rapidly down-regulated. Interestingly, caveolin-2 is up-regulated in response to the mechanical injury of differentiated PC12 cells; up-regulation of caveolin-2 under these conditions is strictly dependent on continued treatment with NGF. Robust expression of caveolin-1 and -2 is also observed along the entire cell surface of DRG neurons, including high levels on growth cones. These findings demonstrate that neuronal cells express caveolins.

Long-term cultures of human fetal liver cells: a three-dimensional experimental model for monitoring liver tissue development.

BACKGROUND/AIMS: The present study describes an embryonic-fetal liver culture system which allows morphogenetic interactions consistent with the development of the hepatocellular function. METHODS: Intact livers from 8-12-week embryos were soaked in an extracellular matrix at 4 degrees C and gently dissociated without any enzymatic treatment. The resulting spherical hepatic units were cultured in a chemically defined serum-free medium and seeded into an extracellular matrix layer. Adherent three-dimensional tissue specimens were examined at various times by light and electron microscopy to evaluate the maintenance of hepatocyte morphology. RESULTS: The liver cells were viable for over 4 months; erythropoietic burst colonies were detected for longer than 6 weeks. Parallel detection of bile salt production in the medium by high performance liquid chromatography proved liver tissue functionality. Bile salt composition revealed predominance of taurine-conjugates rather than glycine. Maximum bile salt concentration (approximately 3 months) coincided with structural and ultrastructural observations indicating a marked decline in hematopoiesis, well-defined biliary canaliculi and formation of an organ-like structure. CONCLUSIONS: This three-dimensional culture system recapitulates fetal liver development with: (i) initial proliferation of both fetal erythropoietic and hepatic cells and (ii) subsequent shut-off of erythropoiesis and a shift to a more advanced stage of hepatocyte function, such as bile salt secretion.

Interaction of a receptor tyrosine kinase, EGF-R, with caveolins. Caveolin binding negatively regulates tyrosine and serine/threonine kinase activities.

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We and others have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting signaling molecules within caveolae membranes. In this regard, it has been shown that a 20-amino acid membrane-proximal region of the cytosolic NH2-terminal domain of caveolin is sufficient to mediate the interaction of caveolin with signaling proteins, namely G-proteins, Src-like kinases, eNOS, and H-Ras. This caveolin-derived protein domain has been termed the caveolin-scaffolding domain. Binding of the caveolin-scaffolding domain functionally suppresses the activity of G-protein alpha subunits, eNOS, and Src-like kinases, suggesting that caveolin binding may also play a negative regulatory role in signal transduction. Here, we report the direct interaction of caveolin with a growth factor receptor, EGF-R, a known caveolae-associated receptor tyrosine kinase. Two consensus caveolin binding motifs have been previously defined using phage display technology. One of these motifs is present within the conserved kinase domains of most known receptor tyrosine kinases (termed region IX). We now show that this caveolin binding motif within the kinase domain of the EGF-R can mediate the interaction of the EGF-R with the scaffolding domains of caveolins 1 and 3 but not with caveolin 2. In addition, the scaffolding domains of caveolins 1 and 3 both functionally inhibit the autophosphorylation of the EGF-R kinase in vitro. Importantly, this caveolin-mediated inhibition of the EGF-R kinase could be prevented by the addition of an EGF-R-derived peptide that (i) contains a well conserved caveolin binding motif and (ii) is located within the kinase domain of the EGF-R and most known receptor tyrosine kinases. Similar results were obtained with protein kinase C, a serine/threonine kinase, suggesting that caveolin may function as a general kinase inhibitor. The implications of our results are discussed within the context of caveolae-mediated signal transduction. In this regard, caveolae-coupled signaling might explain how linear signaling pathways can branch and interconnect extensively, forming a signaling module or network.

Molecular characterization and dynamics of hepatitis C virus replication in human fetal hepatocytes infected in vitro.

The molecular features of hepatitis C virus (HCV) replication in human fetal hepatocytes (HFHs) were addressed in this study. Using a competitive reverse-transcription polymerase chain reaction (RT-PCR) assay for the quantitation of HCV-RNA molecules, the highest level of viral replication was detected 30 days' postinfection. At this time point, viral particles of 41 to 45 nm in diameter accumulated in the cell cytoplasm. Their density in cell extracts and culture medium was distributed between heavy (1.180-1.360 g/cm3) and light fractions (1.105-1.050 g/cm3) of a sucrose gradient, while, in the serum inoculum, they had a positive fraction at 1.180 g/cm3. In infected HFHs, minus-strand HCV RNA was observed in fractions displaying a sedimentation coefficient of 28 S to 18 S, while plus-strand HCV RNA showed a peak restricted to the 21 S fraction; the HCV RNA of serum inoculum had a sedimentation coefficient of 38 to 40 S, which revealed the presence of HCV RNA of unique positive polarity. The 21 S RNA fraction of cell extracts was resistant to 20 minutes of RNase I digestion, while the same incubation time totally inactivated a comparable amount of HCV RNA purified from the serum inoculum, revealing the presence of completely and/or partially double-stranded HCV-RNA molecules in the infected cells. Detection in HFHs of replicative forms and replicative intermediates suggests that the dynamic profile of HCV replication in these cells is similar to that described in other flaviviruses.

Targeting of a G alpha subunit (Gi1 alpha) and c-Src tyrosine kinase to caveolae membranes: clarifying the role of N-myristoylation.

Many signaling molecules contain the consensus protein sequence Met-Gly at their N-termini that specifies N-myristoylation. Additionally, some of these proteins contain a cysteine at position-3 (Met-Gly-Cys) that can undergo palmitoylation. As many acylated proteins [G-protein subunits (alpha and beta gamma); c-Src and Src-family tyrosine kinases; H-Ras and Ras-related GTPases; endothelial nitric oxide synthase] are known to be targeted to caveolae membranes, it has been suggested that acylation is required or greatly facilitates this targeting event. However, it remains unclear whether myristoylation of Src-family kinases is necessary or sufficient for caveolar targeting. Our current study aims at clarifying the role of myristoylation in caveolar targeting using well-characterized acylation mutants of two model proteins, namely Gi1 alpha and c-Src. Here, we have used: i) detergent-free subcellular fractionation and ii) acylation mutants of Gi1 alpha and c-Src to systematically evaluate the relative contribution of myristoylation and palmitoylation to their caveolar targeting. Myristoylation (G2A) and palmitoytation (C3S) mutants of Gi1 alpha were poorly targeted to caveolae-enriched membrane fractions, while approximately 35% of total wild-type Gi1 alpha co-fractionated with caveolin, a caveolar marker protein. Similarly, a myristoylation minus mutant of c-Src was quantitatively excluded from caveolae. In contrast to a previous study, we conclude that myristoylation of Gi1 alpha and c-Src proteins is required for their correct caveolar targeting. However, the caveolar targeting of Gi1 alpha is dramatically augmented approximately 4-fold by palmitoylation. Our current studies are directly supported by the earlier in vivo observation that N-terminal myristoylation of v-Src is required for v-Src to phosphorylate caveolin on tyrosine residues in intact cells.

Evidence for the existence of ganglioside-enriched plasma membrane domains in human peripheral lymphocytes.

In human peripheral blood lymphocytes (PBL) monosialoganglioside GM3 appears to be the major ganglioside on the cell plasma membrane. We have analyzed the expression and distribution pattern of GM3 molecules on the lymphocyte plasma membrane by flow cytometry, immunofluorescence, and immunoelectron microscopy, using an anti-GM3 monoclonal antibody. Both CD4+ and CD8+ T lymphocyte subpopulations showed substantial GM3 expression, as determined by thin-layer chromatography and flow cytometric analysis. A clustered distribution of GM3 molecules on the cell surface, revealed by immunofluorescence and immunogold electron microscopy, clearly indicated the presence of GM3 molecule-enriched plasma membrane domains. To better define these domains, we analyzed the ganglioside and protein composition of buoyant low-density Triton-insoluble (LDTI) lymphocyte fractions. The results show that GM3 is enriched approximately 20-fold in LDTI fraction, as compared with total cell lysates. In addition, CD4 and lck molecules are selectively recovered in the same LDTI fraction isolated from human PBL. These findings, together with the observation that anti-CD4 co-immunoprecipitated GM3, support the hypothesis of a possible GM3-CD4 interaction and suggest a role for gangliosides as structural components of the membrane multimolecular signaling complex involved in T-cell activation, antigen recognition, and other dynamic lymphocytic plasma membrane functions.

Signal transduction and glycophosphatidylinositol-linked proteins (lyn, lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton-insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes.

Src-family nonreceptor protein tyrosine kinases (NRPTK) are associated with cell surface receptors in large detergent-resistant complexes: in epithelial cells, yes is selectively located in vesicle structures containing caveolin ("caveolae"). These formations are typically also endowed with glycophosphatidylinositol (GPI)-anchored proteins. In the present study, we observed lck, lyn, src, hck, CD4, CD45, G proteins, and CD55 (decay-accelerating factor) expression in the buoyant low-density Triton-insoluble (LDTI) fraction of selected leukemic cell lines and granulocytes. We provide a detailed analysis of the two most highly expressed NRPTK, p53/p56lyn and p56lck, which are involved in the transduction of signals for proliferation and differentiation of monocytes/B lymphocytes and T lymphocytes, respectively. We show that lyn is selectively recovered in LDTI complexes isolated from human leukemic cell lines (promyelocytic [HL-60], erythroid [K562] and B-lymphoid [697]) and from normal human granulocytes, and that lck is recovered from LDTI fractions of leukemic T- and B-lymphoid cell lines (CEM, 697). In LDTI fractions of leukemic cells, lck and lyn are enriched 100-fold as compared with the total cell lysates. Analysis of these fractions by electron microscopy shows the presence of 70- to 200-nm vesicles: lyn and lck are homogenously distributed in the vesicles, as revealed by an immunogold labeling procedure. These novel results propose a role for these vesicles in signal transduction mechanisms of normal and neoplastic hematopoietic cells. In support of this hypothesis, we further observed that molecules participating in B- and T-cell receptor activation cofractionate in the LDTI fractions, CD45/lyn (B cells) and CD45/lck/CD4 (T cells).

Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains.

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 alpha subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein alpha subunits, including G(s), G(o), and G(i2). Mutational or pharmacologic activation of G alpha subunits prevents the interaction of caveolin with G proteins, indicating that inactive G alpha 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 binding assays. Wild-type H-Ras interacted with glutathione S-transferase (GST)-caveolin fusion proteins but not with GST alone. Using a battery of GST fusion proteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41-amino acid membrane proximal region of the cytosolic N-terminal domain of caveolin. In addition, reconstituted caveolin-rich membranes (prepared with purified recombinant caveolin and purified 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 prevents or destabilizes this interaction. These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid membranes 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 modification of Ras. Taken together, these observations suggest that caveolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within caveolin-rich microdomains of the plasma membrane.