Regulation of pancreatic cancer cell migration and invasion by RhoC GTPase and caveolin-1.

Division of Hematology-Oncology, Department of Internal Medicine, The University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan 48109, USA. liming@yahoo.com

BACKGROUND: In the current study we investigated the role of caveolin-1 (cav-1) in pancreatic adenocarcinoma (PC) cell migration and invasion; initial steps in metastasis. Cav-1 is the major structural protein in caveolae; small Omega-shaped invaginations within the plasma membrane. Caveolae are involved in signal transduction, wherein cav-1 acts as a scaffolding protein to organize multiple molecular complexes regulating a variety of cellular events. Recent evidence suggests a role for cav-1 in promoting cancer cell migration, invasion and metastasis; however, the molecular mechanisms have not been described. The small monomeric GTPases are among several molecules which associate with cav-1. Classically, the Rho GTPases control actin cytoskeletal reorganization during cell migration and invasion. RhoC GTPase is overexpressed in aggressive cancers that metastasize and is the predominant GTPase in PC. Like several GTPases, RhoC contains a putative cav-1 binding motif. RESULTS: Analysis of 10 PC cell lines revealed high levels of cav-1 expression in lines derived from primary tumors and low expression in those derived from metastases. Comparison of the BxPC-3 (derived from a primary tumor) and HPAF-II (derived from a metastasis) demonstrates a reciprocal relationship between cav-1 expression and p42/p44 Erk activation with PC cell migration, invasion, RhoC GTPase and p38 MAPK activation. Furthermore, inhibition of RhoC or p38 activity in HPAF-II cells leads to partial restoration of cav-1 expression. CONCLUSION: Cav-1 expression inhibits RhoC GTPase activation and subsequent activation of the p38 MAPK pathway in primary PC cells thus restricting migration and invasion. In contrast, loss of cav-1 expression leads to RhoC-mediated migration and invasion in metastatic PC cells.

PMID: 15969750 [PubMed - indexed for MEDLINE]

PMCID: PMC1173138

Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes.

Division of Cell Biology and Diabetes Research Centre, Faculty of Health Sciences, Linköping University, SE58185 Linköping, Sweden.

Caveolae, the specialized invaginations of plasma membranes, formed sealed vesicles with outwards-orientated cytosolic surface after isolation from primary human adipocytes. This morphology allowed differential, vectorial identification of proteins at the opposite membrane surfaces by proteolysis and MS. Extracellular-exposed caveolae-specific proteins CD36 and copper-containing amine oxidase were concealed inside the vesicles and resisted trypsin treatment. The cytosol-orientated caveolins were efficiently digested by trypsin, producing peptides amenable to direct MS sequencing. Isolation of peripheral proteins associated with the cytosolic surface of caveolae revealed a set of proteins that contained nuclear localization signals, leucine-zipper domains and PEST (amino acid sequence enriched in proline, glutamic acid, serine and threonine) domains implicated in regulation by proteolysis. In particular, PTRF (polymerase I and transcript release factor) was found as a major caveolae-associated protein and its co-localization with caveolin was confirmed by immunofluorescence confocal microscopy. PTRF was present at the surface of caveolae in the intact form and in five different truncated forms. Peptides (44 and 45 amino acids long) comprising both the PEST domains were sequenced by nanospray-quadrupole-time-of-flight MS from the full-length PTRF, but were not found in the truncated forms of the protein. Two endogenous cleavage sites corresponding to calpain specificity were identified in PTRF; one of them was in a PEST domain. Both cleavage sites were flanked by mono- or diphosphorylated sequences. The phosphorylation sites were localized to Ser-36, Ser-40, Ser-365 and Ser-366 in PTRF. Caveolae of human adipocytes are proposed to function in targeting, relocation and proteolytic control of PTRF and other PEST-domain-containing signalling proteins.

PMID: 15242332 [PubMed - indexed for MEDLINE]

PMCID: PMC1134064

Caveolin-1 regulates cellular trafficking and function of the glucagon-like Peptide 1 receptor.

Division of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine, E1140 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, Pennsylvania 15261, USA.

The glucagon-like peptide 1 receptor (GLP-1R) mediates important effects on beta-cell function and glucose homeostasis and is one of the most promising therapeutic targets for type 2, and possibly type 1, diabetes. Yet, little is known regarding the molecular and cellular mechanisms that regulate its function. Therefore, we examined the cellular trafficking of the GLP-1R and the relation between receptor localization and signaling activity. In resting human embryonic kidney 293 and insulinoma MIN6 cells, a fully functional green fluorescent protein-tagged GLP-1R was localized both at the cell membrane and in highly mobile intracellular compartments. Real-time confocal fluorescence microscopy allowed direct visualization of constitutive cycling of the receptor. Overexpression of K44A-dynamin increased the number of functional receptors at the cell membrane. Immunoprecipitation, sucrose sedimentation, and microscopy observations demonstrated that the GLP-1R localizes in lipid rafts and interacts with caveolin-1. This interaction is necessary for membrane localization of the GLP-1R, because overexpression of a dominant-negative form of caveolin-1 (P132L-cav1) or specific mutations within the putative GLP-1R's caveolin-1 binding domain completely inhibited GLP-1 binding and activity. Upon agonist stimulation, the GLP-1R underwent rapid and extensive endocytosis independently from arrestins but in association with caveolin-1. Finally, GLP-1R-stimulated activation of ERK1/2, which involves transactivation of epidermal growth factor receptors, required lipid raft integrity. In summary, the interaction of the GLP-1R with caveolin-1 regulates subcellular localization, trafficking, and signaling activity. This study provides further evidence of the key role of accessory proteins in specifying the cellular behavior of G protein-coupled receptors.

PMID: 16931572 [PubMed - indexed for MEDLINE]

Cellular internalization of insulin-like growth factor binding protein-3: distinct endocytic pathways facilitate re-uptake and nuclear localization.

Division of Pediatric Endocrinology, Mattel Children's Hospital, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, USA.

Insulin-like growth factor binding protein-3 (IGFBP-3) is well established as a growth-inhibitory, apoptosis-inducing secreted molecule that acts via insulin-like growth factor (IGF)-independent as well as IGF-dependent pathways. Nuclear localization of IGFBP-3 has been observed and nuclear binding partners for IGFBP-3 demonstrated. However, little is known about the mechanism of IGFBP-3 internalization. We hypothesized that IGFBP-3 is first secreted then taken up again into cells and that its internalization could occur via binding to transferrin or caveolin. Incubation of cells with an IGFBP-3-neutralizing antibody demonstrated that nuclear translocation of endogenous IGFBP-3 requires IGFBP-3 secretion and re-uptake. Nuclear localization of exogenously added IGFBP-3 was rapid, occurring within 15 min, inhibited by co-incubation and extracellular sequestration with IGF-I, and dependent on the transferrin-binding C-terminal peptide region of IGFBP-3. Co-immunoprecipitation assays confirmed that IGFBP-3 binds transferrin but not directly to the transferrin receptor (TfR1); however, transferrin binds TfR1 and a ternary complex is formed. Specific binding to caveolin scaffolding docking sequence was confirmed utilizing radiolabeled IGFBP-3. Blocking TfR1-mediated endocytosis prevents both endogenous and exogenous IGFBP-3 re-uptake and inhibitors of caveolae formation also retard IGFBP-3 nuclear entry. Co-treatment with anti-transferrin receptor antibody and cholesterol depletion agents completely abolished endogenous and exogenous IGFBP-3 uptake. Suppression of IGFBP-3 internalization by TfR1 blockade inhibited IGFBP-3-induced apoptosis. Together, these data indicate that the actions of IGFBP-3 are mediated by internalization via distinct endocytic pathways.

PMID: 14576164 [PubMed - indexed for MEDLINE]

Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation: implications for epidermal growth factor signaling.

Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Korea.

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCalpha mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.

PMID: 12429840 [PubMed - indexed for MEDLINE]

PMCID: PMC133608

Connexin family members target to lipid raft domains and interact with caveolin-1.

Department of Molecular Pharmacology and Albert Einstein Cancer Center and Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

Lipid rafts are cholesterol-sphingolipid-rich microdomains that function as platforms for membrane trafficking and signal transduction. Caveolae are specialized lipid raft domains that contain the structural proteins known as the caveolins. Connexins are a family of transmembrane proteins that self-associate to form cell-cell connections known as gap junctions and that are linked to cytosolic proteins, forming a protein complex or Nexus. To determine the extent to which these intracellular compartments intersect, we have systematically evaluated whether connexins are associated with lipid rafts and caveolin-1. We show that connexin 43 (Cx43) colocalizes, cofractionates, and coimmunoprecipitates with caveolin-1. A mutational analysis of Cx43 reveals that the hypothesized PDZ- and presumptive SH2/SH3-binding domains within the Cx43 carboxyl terminus are not required for this targeting event or for its stable interaction with caveolin-1. Furthermore, Cx43 appears to interact with two distinct caveolin-1 domains, i.e., the caveolin-scaffolding domain (residues 82-101) and the C-terminal domain (135-178). We also show that other connexins (Cx32, Cx36, and Cx46) are targeted to lipid rafts, while Cx26 and Cx50 are specifically excluded from these membrane microdomains. Interestingly, recombinant coexpression of Cx26 with caveolin-1 recruits Cx26 to lipid rafts, where it colocalizes with caveolin-1. This trafficking event appears to be unique to Cx26, since the other connexins investigated in this study do not require caveolin-1 for targeting to lipid rafts. Our results provide the first evidence that connexins interact with caveolins and partition into lipid raft domains and indicate that these interactions are connexin specific.

PMID: 11980479 [PubMed - indexed for MEDLINE]

Caveolin-1 interacts with a lipid raft-associated population of fatty acid synthase.

The Urological Diseases Research Center and Department of Surgery, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115, USA. dolores.divizio@childrens.harvard.edu

Fatty Acid Synthase (FASN), a cytoplasmic biosynthetic enzyme, is the major source of long-chain fatty acids, particularly palmitate. Caveolin-1 (Cav-1) is a palmitoylated lipid raft protein that plays a key role in signal transduction and cholesterol transport. Both proteins have been implicated in prostate cancer (PCa) progression, and Cav-1 regulates FASN expression in a mouse model of aggressive PCa. We demonstrate that FASN and Cav-1 are coordinately upregulated in human prostate tumors in a hormone-insensitive manner. Levels of FASN and Cav-1 protein expression discriminated between localized and metastatic cancers, and the two proteins exhibited analogous subcellular locations in a tumor subset. Endogenous FASN and Cav-1 were reciprocally co-immunoprecipitated from human and murine PCa cells, indicating that FASN forms a complex with Cav-1. FASN, a cytoplasmic enzyme, was induced to associate transiently with lipid raft membranes following alterations in signal transduction within the Src, Akt and EGFR pathways, suggesting that co-localization of FASN and Cav-1 is dependent on activation of upstream signaling mediators. A Cav-1 palmitoylation mutant, Cav-1(C133/143/156S), that prevents phosphorylation by Src, did not interact with FASN. When overexpressed in Cav-1-negative PCa cells, Cav-1(C133/143/156S) caused a reduction of both Src and Akt levels, as well as of their active, phosphorylated forms, in comparison with wild type Cav-1. These findings suggest that FASN and Cav-1 physically and functionally interact in PCa cells. They also imply that palmitoylation within this complex is involved in tumor growth and survival.

PMID: 18635971 [PubMed - indexed for MEDLINE]

Tissue factor induction by protease-activated receptor 1 requires intact caveolin-enriched membrane microdomains in human endothelial cells.

Monzino Cardiologic Centre IRCCS, and Department of Pharmacological Sciences, University of Milan, via Parea 4, Milan, Italy. cristina.banfi@unimi.it

BACKGROUND: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique mechanism of proteolytic activation. PARs regulate a broad range of cellular functions and are active in the pathogenesis of disorders characterized by chronic inflammation or activation of the coagulation cascade. Signaling through PAR1 and PAR2 shifts the endothelium towards a prothrombotic phenotype, thereby exacerbating the initial pathophysiologic condition. OBJECTIVES: This study aimed to analyze the localization of PARs in the cell membrane and how their compartmentalization affects tissue factor (TF) in human endothelial cells. METHODS: TF expression was determined by quantitative real-time polymerase chain reaction analysis and by activity assays. The interaction of PARs with caveolin was investigated through: (i) caveolin-1 gene knockdown performed by transfection with specific small interfering RNA (siRNA); (ii) caveolin-enriched membrane microdomain disruption; and (iii) coimmunoprecipitation assay. RESULTS: We have shown that PAR1, but not PAR2, is present in endothelial caveolin-enriched membrane microdomains, where it is bound to caveolin-1, and that these structures must be intact if PAR1-induced signaling is to increase TF activity. Cholesterol depletion of endothelial cells by cholesterol-sequestering agents caused the PAR1 to relocate to high-density membranes, and impaired the induction of TF (P < 0.01) without affecting the PAR2-mediated procoagulant effect. In addition, siRNA directed against caveolin-1 inhibited TF activation by PAR1 (P < 0.01 and P < 0.01, respectively). CONCLUSIONS: PAR1 localization in the caveolin-enriched membrane microdomain, bound to caveolin-1, represents a crucial requirement for TF induction in endothelial cells.

PMID: 17848177 [PubMed - indexed for MEDLINE]

Agonist-modulated targeting of the EDG-1 receptor to plasmalemmal caveolae. eNOS activation by sphingosine 1-phosphate and the role of caveolin-1 in sphingolipid signal transduction.

Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

Plasmalemmal caveolae are membrane microdomains that are specifically enriched in sphingolipids and contain a wide array of signaling proteins, including the endothelial isoform of nitric-oxide synthase (eNOS). EDG-1 is a G protein-coupled receptor for sphingosine 1-phosphate (S1P) that is expressed in endothelial cells and has been implicated in diverse vascular signal transduction pathways. We analyzed the subcellular distribution of EDG-1 in COS-7 cells transiently transfected with cDNA constructs encoding epitope-tagged EDG-1. Subcellular fractionation of cell lysates resolved by ultracentrifugation in discontinuous sucrose gradients revealed that approximately 55% of the EDG-1 protein was recovered in fractions enriched in caveolin-1, a resident protein of caveolae. Co-immunoprecipitation experiments showed that EDG-1 could be specifically precipitated by antibodies directed against caveolin-1 and vice versa. The targeting of EDG-1 to caveolae-enriched fractions was markedly increased (from 51 +/- 11% to 93 +/- 14%) by treatment of transfected cells with S1P (5 microm, 60 min). In co-transfection experiments expressing EDG-1 and eNOS cDNAs in COS-7 cells, we found that S1P treatment significantly and specifically increased nitric-oxide synthase activity, with an EC(50) of 30 nm S1P. Overexpression of transfected caveolin-1 cDNA together with EDG-1 and eNOS markedly diminished S1P-mediated eNOS activation; caveolin overexpression also attenuated agonist-induced phosphorylation of EDG-1 receptor by >90%. These results suggest that the interaction of the EDG-1 receptor with caveolin may serve to inhibit signaling through the S1P pathway, even as the targeting of EDG-1 to caveolae facilitates the interactions of this receptor with ligands and effectors that are also targeted to caveolae. The agonist-modulated targeting of EDG-1 to caveolae and its dynamic inhibitory interactions with caveolin identify new points for regulation of sphingolipid-dependent signaling in the vascular wall.

PMID: 10921915 [PubMed - indexed for MEDLINE]

Caveolin-1 binding to endoplasmic reticulum membranes and entry into the regulated secretory pathway are regulated by serine phosphorylation. Protein sorting at the level of the endoplasmic reticulum.

Department of Molecular Pharmacology, Division of Endocrinology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

Caveolin-1 serves as the main coat protein of caveolae membranes, as an intracellular cholesterol shuttle, and as a regulator of diverse signaling molecules. Of the 12 residues conserved across all caveolin isoforms from all species examined to date, only Ser(80) and Ser(168) could serve as phosphorylation sites. We show here that mimicking chronic phosphorylation of Ser(80) by mutation to Glu (i.e. Cav-1(S80E)), blocks phosphate incorporation. However, Cav-1(S168E) is phosphorylated to the same extent as wild-type caveolin-1. Cav-1(S80E) targets to the endoplasmic reticulum membrane, remains oligomeric, and maintains normal membrane topology. In contrast, Cav-1(S80A), which cannot be phosphorylated, targets to caveolae membranes. Some exocrine cells secrete caveolin-1 in a regulated manner. Cav-1(S80A) is not secreted by AR42J pancreatic adenocarcinoma cells even in the presence of dexamethasone, an agent that induces the secretory phenotype. Conversely, Cav-1(S80E) is secreted to a greater extent than wild-type caveolin-1 following dexamethasone treatment. We conclude that caveolin-1 phosphorylation on invariant serine residue 80 is required for endoplasmic reticulum retention and entry into the regulated secretory pathway.

PMID: 11078729 [PubMed - indexed for MEDLINE]

Localization of low density lipoprotein receptor-related protein 1 to caveolae in 3T3-L1 adipocytes in response to insulin treatment.

Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa K1Y 4W7, Ontario, Canada..

The insulin-induced translocation of low density lipoprotein receptor-related protein 1 (LRP1) from intracellular membranes to the cell surface in 3T3-L1 adipocytes was differentiation-dependent and did not occur in 3T3-L1 fibroblasts. Prompted by findings that the plasma membrane of 3T3-L1 adipocytes was rich in caveolae, we determined whether LRP1 became caveolae-associated upon insulin stimulation. The caveolae domain was isolated by the well characterized detergent solubilization and sucrose density ultracentrifugation methodology. Under basal conditions, only a trace amount of LRP1 was caveolae-associated despite the markedly elevated caveolin-1 and caveolae after adipocytic cell differentiation. Upon insulin treatment, the amount of LRP1 associated with caveolae was increased by 4-fold within 10 min, which was blocked completely by pretreatment with wortmannin prior to insulin. The caveolar localization of LRP1 in adipocytes was specific to insulin; treatment with platelet-derived growth factor-bb isoform did not promote but rather decreased caveolar localization of LRP1 below basal levels. The insulin-induced caveolar localization of LRP1 was also observed in 3T3-L1 fibroblasts where translocation of LRP1 from intracellular membranes to the cell surface was absent, suggesting that association of LRP1 with caveolae was achieved, at least in part, through lateral transmigration along the plane of plasma membranes. Immunocytochemistry studies revealed partial co-localization of LRP1 (either endogenous LRP1 or an epitope-tagged minireceptor) with caveolin-1 in cells treated with insulin, which was confirmed by co-immunoprecipitation of LRP1 with caveolin-1 in cells treated with insulin but not platelet-derived growth factor-bb. These results suggest that the localization of LRP1 to caveolae responds selectively to extracellular signals.

PMID: 14593097 [PubMed - indexed for MEDLINE]

Activated epidermal growth factor receptor induces integrin alpha2 internalization via caveolae/raft-dependent endocytic pathway.

College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, USA.

Elevated expression or activity of the epidermal growth factor (EGF) receptor is common in ovarian cancer and is associated with poor patient prognosis. Our previous studies demonstrated that expression of the constitutively active mutant form of the EGF receptor (EGFRvIII) in ovarian cancer cells led to reduction in integrin alpha2 surface expression, defects in cell spreading, and disruption of focal adhesions. Inhibition of EGFRvIII catalytic activity reversed the response, suggesting that EGF receptor activation regulates integrin alpha2. In this study we found that EGF treatment resulted in a transient loss of integrin alpha2 from the cell surface. Before EGF stimulation, integrin alpha2 and EGF receptors were associated based on biochemical and immuno-colocalization approaches. After EGF treatment, EGF receptor and integrin alpha2 were internalized and segregated into different compartments. Integrin alpha2, but not EGF receptor, was associated with caveolin-1 and GM1 (Gal_1,3GalNAc_1,4(Neu5Ac-_ 2,3)Gal_1,4Glc_1,1-ceramide) gangliosides, suggesting caveolae-mediated endocytosis. Moreover, integrin alpha2 was subsequently targeted to the Golgi apparatus and the endoplasmic reticulum. Together, these findings demonstrate that activated EGF receptor transiently modulates integrin alpha2 cell surface expression and stimulates integrin alpha2 trafficking via caveolae/raft-mediated endocytosis, representing a novel mechanism by which the EGF receptor may regulate integrin-mediated cell behavior.

PMID: 17179151 [PubMed - indexed for MEDLINE]

Translocation of endothelial nitric-oxide synthase involves a ternary complex with caveolin-1 and NOSTRIN.

Institute of Biochemistry II, University of Frankfurt Medical School, D-60590 Frankfurt, Germany.

Recently, we characterized a novel endothelial nitric-oxide synthase (eNOS)-interacting protein, NOSTRIN (for eNOS-trafficking inducer), which decreases eNOS activity upon overexpression and induces translocation of eNOS away from the plasma membrane. Here, we show that NOSTRIN directly binds to caveolin-1, a well-established inhibitor of eNOS. Because this interaction occurs between the N terminus of caveolin (positions 1-61) and the central domain of NOSTRIN (positions 323-434), it allows for independent binding of each of the two proteins to eNOS. Consistently, we were able to demonstrate the existence of a ternary complex of NOSTRIN, eNOS, and caveolin-1 in Chinese hamster ovary (CHO)-eNOS cells. In human umbilical vein endothelial cells (HUVECs), the ternary complex assembles at the plasma membrane upon confluence or thrombin stimulation. In CHO-eNOS cells, NOSTRIN-mediated translocation of eNOS involves caveolin in a process most likely representing caveolar trafficking. Accordingly, trafficking of NOSTRIN/eNOS/caveolin is affected by altering the state of actin filaments or cholesterol levels in the plasma membrane. During caveolar trafficking, NOSTRIN functions as an adaptor to recruit mediators such as dynamin-2 essential for membrane fission. We propose that a ternary complex between NOSTRIN, caveolin-1, and eNOS mediates translocation of eNOS, with important implications for the activity and availability of eNOS in the cell.

PMID: 16807357 [PubMed - indexed for MEDLINE]

PMCID: PMC1593164

The immunogenic CBD1 peptide corresponding to the caveolin-1 binding domain in HIV-1 envelope gp41 has the capacity to penetrate the cell membrane and bind caveolin-1.

UPR 2228 CNRS, UFR Biomédicale-Université René Descartes, 45 rue des Saints Pères, 75270 Paris Cedex 06, France.

The potential caveolin-1 binding domain (CBD), referred to as CBD1 and CBD2, is highly conserved in the transmembrane envelope glycoprotein of various HIV-1 and HIV-2 isolates, respectively. However, HIV-1 neutralizing antibodies raised against the synthetic CBD1 peptide (SLEQIWNNMTWMQWDK) do not cross-react with the CBD2 peptide (SLTPDWNNMTWQEWER) and have no effect on HIV-2 infection. Here we show that the CBD2 peptide is not immunogenic under similar immunization conditions as the CBD1 peptide. Moreover, the CBD1 but not the CBD2 peptide has the capacity to bind caveolin-1 in crude cell extracts thus suggesting the existence of structural and/or conformational differences between CBD1 and CBD2. Accordingly, circular dichroism spectroscopy and fluorimetry analysis indicated that CBD1 but not CBD2 could adopt a defined secondary structure and form a complex with a peptide corresponding to the caveolin-1 scaffolding domain, which is the site of interaction of caveolin-1 with various proteins. In line with these observations, CBD1 but not CBD2 binds cells and forms large aggregates at the plasma membrane by colocalizing with cytofacial caveolin-1. This latter is dependent on the lipid raft integrity of the plasma membrane. Supporting that the ability to penetrate into plasma membranes is sustained by folding at the interface, CBD1 but not CBD2 has the capacity to insert into lipid monolayers, penetrate into artificial membranes and adopt a beta-sheet conformation in presence of lipid vesicles. These structural determinants and membrane partitioning properties could account for the immunogenicity of the CBD1 peptide in various animals.

PMID: 18054388 [PubMed - indexed for MEDLINE]

Serine 23 and 36 phosphorylation of caveolin-2 is differentially regulated by targeting to lipid raft/caveolae and in mitotic endothelial cells.

Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri 65212, USA. sowag@health.missouri.edu

In the present study, using a combination of reconstituted systems and endothelial cells endogenously expressing caveolins, we show that phosphorylation of caveolin-2 at serines 23 and 36 can be differentially regulated by caveolin-1 mediated subcellular targeting to lipid raft/caveolae and in endothelial cells synchronized in mitosis. Detergent insolubility and sucrose flotation gradient experiments revealed that serine 23 phosphorylation of caveolin-2 preferably occurs in detergent-resistant membranes (DRMs), while serine 36 phosphorylation takes place in non-DRMs. Furthermore, immunofluorescence microscopy studies determined that in the presence of caveolin-1, serine 23-phosphorylated caveolin-2 mostly localizes to plasma membrane, while serine 36-phosphorylated caveolin-2 primarily resides in intracellular compartments. To directly address the role of caveolin-1 in regulating phosphorylation of endogenous caveolin-2, we have used the siRNA approach. The specific knockdown of caveolin-1 in endothelial cells decreases caveolin-2 phosphorylation at serine 23 but not at serine 36. Thus, upregulation of serine 23 phosphorylation of caveolin-2 depends on caveolin-1-driven targeting to plasma membrane lipid rafts and caveolae. Interestingly, although serine 36 phosphorylation does not seem to be regulated in endothelial cells by caveolin-1, it can be selectively upregulated in endothelial cells synchronized in mitosis. The latter data suggests a possible involvement of serine 36-phosphorylated caveolin-2 in modulating mitosis.

PMID: 18081315 [PubMed - indexed for MEDLINE]

The tetraspan protein EMP2 regulates expression of caveolin-1.

Molecular Biology Institute, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, USA.

Caveolin-1 is the primary component of caveolae and functions in a variety of intracellular activities, including membrane trafficking and signal transduction. EMP2 (epithelial membrane protein 2) is a tetraspan protein recently identified as a novel regulator of caveolin-1 expression. In this study, we analyzed the mechanism of EMP2-mediated caveolin-1 regulation. In NIH 3T3 cells and in the human retinal pigment epithelium cell line (ARPE-19), EMP2 regulates caveolin-1 transcription and more substantially its protein levels. EMP2-mediated down-regulation of caveolin-1 does not affect caveolin-1 translational efficiency, phosphorylation, or proteasome-mediated degradation. Analysis of caveolin-1 protein half-life indicates the EMP2-mediated loss of caveolin-1 occurs rapidly. Protease inhibition and laser confocal microscopy associates this fate with specific intracellular compartmentalization, including early lysosomal delivery. These findings elucidate a new mechanism of caveolin-1 regulation and define an additional role for EMP2 as a key regulator of cell membrane composition.

PMID: 17609206 [PubMed - indexed for MEDLINE]

Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization, efficient signaling, and interaction with beta-arrestin 2.

Institute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Gerbiceva 60, SI-1000, Ljubljana, Slovenia.

This study was focused on the relationship between the plasma-membrane localization of neurokinin-1 receptor (NK1-R) and its endocytic and signaling properties. First, we employed electron paramagnetic resonance (EPR) to study the domain structure of HEK-293 cells and NK1-R microlocalization. EPR spectra and the GHOST condensation routine demonstrated that NK1-R was distributed in a well-ordered domain of HEK-293 cells possibly representing lipid raft/caveolae microdomains, whereas the impairment of caveolae changed the NK1-R plasma-membrane distribution. Internalization and second messenger assays combined with bioluminescence resonance energy transfer were employed subsequently to evaluate the functional importance of the NK1-R microlocalization in lipid raft/caveolae microdomains. The internalization pattern was delineated through the use of dominant-negative mutants (DNM) of caveolin-1 S80E (Cav1 S80E), dynamin-1 K44A (Dyn K44A), and beta-arrestin (beta-arr 319-418) and by means of cell lines that expressed various endogenous levels of beta-arrestins. NK1-R displayed rapid internalization that was substantially reduced by DNMs of dynamin-1 and beta-arrestin and even more profoundly in cells lacking both beta-arrestin1 and beta-arrestin2. These internalization data were highly suggestive of the predominant use of the clathrin-mediated pathway by NK1-R, even though NK1-R tended to reside constitutively in lipid raft/caveolae microdomains. Evidence was also obtained that the proper clustering of the receptor in these microdomains was important for effective agonist-induced NK1-R signaling and for its interaction with beta-arrestin2.

PMID: 17713785 [PubMed - indexed for MEDLINE]

Interactions of STAT3 with caveolin-1 and heat shock protein 90 in plasma membrane raft and cytosolic complexes. Preservation of cytokine signaling during fever.

Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York 10595, USA.

Interleukin-6 (IL-6) initiates STAT3 signaling in plasma membrane rafts with the subsequent transit of Tyr-phosphorylated STAT3 (PY-STAT3) through the cytoplasmic compartment to the nucleus in association with accessory proteins. We initially identified caveolin-1 (cav-1) as a candidate STAT3-associated accessory protein due to its co-localization with STAT3 and PY-STAT3 in flotation raft fractions, and heat shock protein 90 (HSP90) due to its inclusion in cytosolic STAT3-containing 200-400-kDa complexes. Subsequent immunomagnetic bead pullout assays showed that STAT3, PY-STAT3, cav-1, and HSP90 interacted in plasma membrane and cytoplasmic complexes derived from uninduced and stimulated Hep3B cells. This was a general property of STAT3 in that these interactions were also observed in alveolar epithelial type II-like cells, lung fibroblasts, and pulmonary arterial endothelial cells. Exposure of Hep3B cells to the raft disrupter methyl-beta-cyclodextrin for 1-10 min followed by IL-6 stimulation for 15 min preferentially inhibited the appearance of PY-STAT3 in the cav-1-enriched sedimentable cytoplasmic fraction, suggesting that these complexes may represent a trafficking intermediate immediately downstream from the raft. Because IL-6 is known to function in the body in the context of fever, the possibility that HSP90 may help preserve IL-6-induced STAT3 signaling at elevated temperature was investigated. Geldanamycin, an HSP90 inhibitor, markedly inhibited IL-6-stimulated STAT3 signaling in Hep3B hepatocytes cultured overnight at 39.5 degrees C as evaluated by DNA-shift assays, trafficking of PY-STAT3 to the nucleus, cross-precipitation of HSP90 by anti-STAT3 polyclonal antibody, and reporter/luciferase construct experiments. Taken together, the data show that IL-6/raft/STAT3 signaling is a chaperoned pathway that involves cav-1 and HSP90 as accessory proteins and suggest a mechanism for the preservation of this signaling during fever.

PMID: 12235142 [PubMed - indexed for MEDLINE]

Annexin A6-induced inhibition of cytoplasmic phospholipase A2 is linked to caveolin-1 export from the Golgi.

Departament de Biologia Cellular, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain.

The molecular mechanisms regulating the exit of caveolin from the Golgi complex are not fully understood. Cholesterol and sphingolipid availability affects Golgi vesiculation events and involves the activity of cytoplasmic phospholipase A(2) (cPLA(2)). We recently demonstrated that high expression levels of annexin A6 (AnxA6) perturb the intracellular distribution of cellular cholesterol, thereby inhibiting caveolin export from the Golgi complex. In the present study we show that in Chinese hamster ovary cells overexpressing AnxA6, sequestration of cholesterol in late endosomes, leading to reduced amounts of cholesterol in the Golgi, inhibits cPLA(2) activity and its association with the Golgi complex. This correlates with the blockage of caveolin export from the Golgi in cells treated with methyl arachidonyl fluorophosphonate, a Ca(2+)-dependent cPLA(2) inhibitor. AnxA6-mediated down-regulation of cPLA(2) activity was overcome upon the addition of exogenous cholesterol or transfection with small interfering RNA targeting AnxA6. These findings indicate that AnxA6 interferes with caveolin transport through the inhibition of cPLA(2).

PMID: 18245088 [PubMed - indexed for MEDLINE]

Overexpression of caveolin-1 increases plasma membrane fluidity and reduces P-glycoprotein function in Hs578T/Dox.

National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

Cholesterol is a key lipid in mediating the enzyme activity or signaling pathway of many proteins on the plasma membrane in mammalian cells. In this report, we demonstrate for the first time that after overexpressing caveolin-1, the plasma membrane cholesterol level was decreased by about 12% and 30% for doxorubicin-sensitive and doxorubicin-resistant Hs578T breast cancer cells, respectively. However, the total cholesterol level in both cell lines was increased by about 10%. By measuring fluorescence and flow cytometry using the fluorescence dyes 1,6-diphenyl-1,3,5-hexatriene and Merocyanine 540, we found that overexpressing caveolin-1 resulted in a similar increase in membrane fluidity and loosening of lipid packing density as cholesterol depletion by 1 mM methyl-beta-cyclodextrin (MbetaCD) or 2-hydroxypropyl-beta-cyclodextrin (HbetaCD). Moreover, we found that the transport activity of P-gp was significantly inhibited by 1 mM MbetaCD or HbetaCD, which is also similar to the inhibitory effect of caveolin-1 overexpression. Our data demonstrate for the first time that the reduction of the plasma membrane cholesterol level induced by overexpressing caveolin-1 may indirectly inhibit P-gp transport activity by increasing plasma membrane fluidity.

PMID: 15240128 [PubMed - indexed for MEDLINE]