Presence of oxidized cholesterol in caveolae uncouples active platelet-derived growth factor receptors from tyrosine kinase substrates.
Platelet-derived growth factor receptor beta (PDGFRbeta) in fibroblasts is concentrated in caveolae where it controls the tyrosine phosphorylation of multiple proteins. Caveolae are enriched in cholesterol and sphingolipids, but the role of these lipids in PDGFR signal transduction is unknown. We report that introduction of cholest-4-en-3-one into caveolae membranes uncouples PDGFR autophosphorylation from tyrosine phosphorylation of neighboring proteins. Cholest-4-en-3-one appears to interfere with the normal interaction between PDGFR and its partners. The results suggest that tightly packed caveolae lipids form a membrane platform that functions as a lipid scaffold for organizing the molecular interactions of multiple signaling pathways. (+info)
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.
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. (+info)
Scavenger receptor BI transfers major lipoprotein-associated phospholipids into the cells.
The phospholipids of lipoproteins can be transferred to cells by an endocytosis-independent uptake pathway. We analyzed the role of scavenger receptor BI (SR-BI) for the selective cellular phospholipid import. Human monocytes rapidly acquired the pyrene (py)-labeled phospholipids sphingomyelin (SM), phosphatidylcholine, and phosphatidylethanolamine from different donors (low and high density lipoproteins (LDL, HDL), lipid vesicles). The anti-SR-BI antibody directed against the extracellular loop of the membrane protein lowered the cellular import of the phospholipids by 40-80%. The phospholipid transfer from the lipid vesicles into the monocytes was suppressed by LDL, HDL, and apoprotein AI. Transfection of BHK cells with the cDNA for human SR-BI enhanced the cellular import of the vesicle-derived py-phospholipids by 5-6-fold. In the case of the LDL donors, transfer of py-SM to the transfected cells was stimulated to a greater extent than the uptake of the other py-phospholipids. Similar differences were not observed when the vesicles and HDL were used as phospholipid donors. The concentration of LDL required for the half-maximal phospholipid import was close to the previously reported apparent dissociation constant for LDL binding to SR-BI. The low activation energy of the SR-BI-mediated py-phospholipid import indicated that the transfer occurs entirely in a hydrophobic environment. Disruption of cell membrane caveolae by cyclodextrin treatment reduced the SR-BI-catalyzed incorporation of py-SM, suggesting that intact caveolae are necessary for the phospholipid uptake. In conclusion, SR-BI mediates the selective import of the major lipoprotein-associated phospholipids into the cells, the transfer efficiency being dependent on the structure of the donor lipoprotein. (+info)
Isoforms of caveolin-1 and caveolar structure.
The relationship between caveolin-1 isoforms alpha and beta and caveolar ultrastructure was studied. By immunofluorescence microscopy of human fibroblasts, caveolae were observed as dots positive for caveolin-1, but many dots labeled by an antibody recognizing both isoforms (anti-alphabeta) were not labeled by another antibody specific for the alpha isoform (anti-alpha). Immunogold electron microscopy of freeze-fracture replicas revealed caveolae of different depths, and indicated that anti-alpha labeled deep caveolae preferentially over shallow ones, whereas anti-alphabeta labeled both forms with an equivalent frequency and intensity. The presence of the beta isoform in deep caveolae was confirmed by labeling epitope-tagged beta-caveolin. When made to be expressed in HepG2 cells lacking endogenous caveolins, the alpha isoform formed caveolar depressions efficiently, but the beta isoform hardly did so. Caveolae were also formed in cells expressing the two isoforms, but their frequency was variable among cells of the same clone. Coexpression of caveolin-1 and caveolin-2 caused more efficient formation of deep caveolae than caveolin-1 alone. The result indicates that the two isoforms of caveolin-1 have a different potential for forming caveolae structure, and more importantly, that deep and shallow caveolae may be diversified in their molecular composition. (+info)
Ultrastructural and histochemical characterization of special muscle cells in the monkey small intestine.
The ultrastructure, three-dimensional arrangement, and histochemical features of special muscle cells in the monkey small intestine were investigated. The cells formed a special layer separated from the main part by a connective tissue space along the submucosal surface of the circular muscle coat. Scanning electron microscopy using alkali maceration demonstrated this inner sublayer to be a continuous thin sheet consisting of irregularly-shaped muscle cells equipped with many cytoplasmic projections and caveolae. Other ultrastructural features included direct contact with interstitial cells, due to their close association with nerve fibers of the deep muscular plexus. Histochemical examination revealed significant alkaline phosphatase activity and immunoreactivity for vascular smooth muscle alpha actin in these muscle cells, whereas the ordinary circular muscle cells were immunopositive for enteric smooth muscle gamma actin. These findings suggest that the special muscle cells play an important role in regulating the radial stretch of the monkey small intestinal wall. (+info)
Bradykinin activates the Janus-activated kinase/signal transducers and activators of transcription (JAK/STAT) pathway in vascular endothelial cells: localization of JAK/STAT signalling proteins in plasmalemmal caveolae.
Bradykinin (BK) is an important physiological regulator of endothelial cell function. In the present study, we have examined the role of the Janus-activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in endothelial signal transduction through the BK B2 receptor (B2R). In cultured bovine aortic endothelial cells (BAECs), BK activates Tyk2 of the JAK family of tyrosine kinases. Activation results in the tyrosine phosphorylation and subsequent nuclear translocation of STAT3. BK also activates the mitogen-activated p44 and p42 protein kinases, resulting in STAT3 serine phosphorylation. Furthermore, Tyk2 and STAT3 form a complex with the B2R in response to BK stimulation. Under basal conditions, Tyk2, STAT3 and the B2R are localized either partially or entirely in endothelial plasmalemmal caveolae. Following BK stimulation of BAECs, however, the B2R and STAT3 are translocated out of caveolae. Taken together, these data suggest that BK activates the JAK/STAT pathway in endothelial cells and that JAK/STAT signalling proteins are localized in endothelial caveolae. Moreover, caveolar localization of the B2R and STAT3 appears to be regulated in an agonist-dependent manner. (+info)
Differential targeting of beta -adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway.
Differential modes for beta(1)- and beta(2)-adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdomains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in cardiomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in beta(2)-ARs, Galpha(i), protein kinase A RIIalpha subunits, caveolin-3, and flotillins (caveolin functional homologues); beta(1)-ARs, m(2)-muscarinic cholinergic receptors, Galpha(s), and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RIalpha subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface beta(2)-ARs localize to caveolae in cardiomyocytes and cardiac fibroblasts (with markedly different beta(2)-AR expression levels), indicating that the fidelity of beta(2)-AR targeting to caveolae is maintained over a physiologic range of beta(2)-AR expression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of beta(2)-ARs (but not beta(1)-ARs) in caveolae. Other studies show co-immunoprecipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. However, caveolin is not required for adenylyl cyclase targeting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Nevertheless, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accumulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domain-specific differences in the stoichiometry of individual elements in the beta-AR signaling cascade represent important modifiers of cAMP-dependent signaling in the heart. (+info)
Downmodulation of caveolin-1 expression in human ovarian carcinoma is directly related to alpha-folate receptor overexpression.
Caveolin (cav-1) and the GPI-anchored alpha-folate receptor (alphaFR) are membrane proteins both found associated to caveolar structures. Several studies in tumor cells independently reported cav-1 downregulation and alphaFR overexpression. Here we analysed the expression of the two molecules in normal and tumor ovarian samples derived from fresh specimens and from cultured cell lines. Whereas normal ovary surface epithelial cells displayed only cav-1 expression, ovarian tumor surgical samples and cell lines (COR, IGROV1, OVCAR3 and OVCA432) displayed high alphaFR and low-level or no cav-1 expression, except those cell lines (SKOV3 and SW626) with the lower alphaFR expression. SKOV3, but not two alphaFR-negative non-ovarian cell lines, exhibited down-regulation of cav-1 expression following stable alphaFR cDNA transfection. Conversely, cav-1 transfection in IGROV1 cells led to downregulated alphaFR expression, together with formation of caveolar structures and reduction of growth capability. Moreover, cav-1 expression was induced in IGROV1 cells by transfection with intracellular anti-alphaFR antibodies to downmodulate alphaFR expression. In cav-1 transfected cells, transcriptional activity of the alphaFR-specific promoter P1 was reduced by 70% and an additional specific DNA-protein complex was identified by gel-shift assay, indicating that cav-1 expression influences alphaFR gene transcription. Together these results support the notion that alphaFR and cav-1 protein expression is reciprocally regulated in ovary cancer cells. (+info)