Altered localization of H-Ras in caveolin-1-null cells is palmitoylation-independent

Caveolin-1 is a palmitoylated protein involved in the formation of plasma membrane subdomains termed caveolae, intracellular cholesterol transport, and assembly and regulation of signaling molecules in caveolae. Caveolin-1 interacts via a consensus binding motif with several signaling proteins, including H-Ras. Ras oncogene products function as molecular switches in several signal transduction pathways regulating cell growth and differentiation. Post-translational modifications, including palmitoylation, are critical for the membrane targeting and function of H-Ras. Subcellular localization regulates the signaling pathways engaged by H-Ras activation. We show here that H-Ras is localized at the plasma membrane in caveolin-1-expressing cells but not in caveolin-1-deficient cells. Since palmitoylation is required for trafficking of H-Ras from the endomembrane system to the plasma membrane, we tested whether the altered localization of H-Ras in caveolin-1-null cells is due to decreased H-Ras palmitoylation. Although the palmitoylation profiles of cultured embryo fibroblasts isolated from wild type and caveolin-1 gene-disrupted mice differed, suggesting that caveolin-1, or caveolae, play a role in the palmitate incorporation of a subset of palmitoylated proteins, the palmitoylation of H-Ras was not decreased in caveolin-1-null cells. We conclude that the altered localization of H-Ras in caveolin-1-deficient cells is palmitoylation-independent. This article shows two important new mechanisms by which loss of caveolin-1 expression may perturb intracellular signaling, namely the mislocalization of signaling proteins and alterations in protein palmitoylation.     

Palmitoylation of caveolin-1 in endothelial cells is post-translational but irreversible

Caveolin-1 is a palmitoylated protein involved in assembly of signaling molecules in plasma membrane subdomains termed caveolae and in intracellular cholesterol transport. Three cysteine residues in the C terminus of caveolin-1 are subject to palmitoylation, which is not necessary for caveolar targeting of caveolin-1. Protein palmitoylation is a post-translational and reversible modification that may be regulated and that in turn may regulate conformation, membrane association, protein-protein interactions, and intracellular localization of the target protein. We have undertaken a detailed analysis of [(3)H]palmitate incorporation into caveolin-1 in aortic endothelial cells. The linkage of palmitate to caveolin-1 was hydroxylamine-sensitive and thus presumably a thioester bond. However, contrary to expectations, palmitate incorporation was blocked completely by the protein synthesis inhibitors cycloheximide and puromycin. In parallel experiments to show specificity, palmitoylation of aortic endothelial cell-specific nitric-oxide synthase was unaffected by these reagents. Inhibitors of protein trafficking, brefeldin A and monensin, blocked caveolin-1 palmitoylation, indicating that the modification was not cotranslational but rather required caveolin-1 transport from the endoplasmic reticulum and Golgi to the plasma membrane. In addition, immunophilin chaperones that form complexes with caveolin-1, i.e. FK506-binding protein 52, cyclophilin A, and cyclophilin 40, were not necessary for caveolin-1 palmitoylation because agents that bind immunophilins did not inhibit palmitoylation. Pulse-chase experiments showed that caveolin-1 palmitoylation is essentially irreversible because the release of [(3)H]palmitate was not significant even after 24 h. These results show that [(3)H]palmitate incorporation is limited to newly synthesized caveolin-1, not because incorporation only occurs during synthesis but because the continuous presence of palmitate on caveolin-1 prevents subsequent repalmitoylation.     

The subcellular localization of 3-phosphoinositide-dependent protein kinase is controlled by caveolin-1 binding

3-Phosphoinositide-dependent protein kinase 1 (PDK1), a member of the serine/threonine kinase family, has been demonstrated to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that PDK1 is associated with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of the caveolae membranes in COS-1. First, we noted the presence of two potential caveolin-1 binding motifs (¹⁴¹FFVKLYFTF¹⁴⁹ and ²⁹⁹YDFPEKFF³⁰⁶) in the PDK1 catalytic domain. Using a pull-down approach, we observed that PDK1 interacts physically with caveolin-1 both in vivo and in vitro. Second, we detected the co-localization of PDK1 and caveolin-1 via confocal microscopy. The localization of PDK1 to the plasma membrane was disrupted by caveolin binding. Third, in transient transfection assays, interaction with caveolin-1 induced a substantial reduction in the in vivo serine/threonine phosphorylation of PDK1, whereas the caveolin-1 binding site mutant (¹⁴¹FFVKLYFTF¹⁴⁹ and ²⁹⁹YDFPEKFF³⁰⁶ change to ¹⁴¹AFVKLAFTA¹⁴⁹ and ²⁹⁹ADAPEFLA³⁰⁶) did not. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82-101) functionally suppressed the self-phosphorylation and kinase activities of purified recombinant PDK1 protein. Thus, our observations indicated that PDK1 binds to caveolin-1 through its caveolin-binding motifs, and also that the protein-protein interaction between PDK1 and caveolin-1 regulates PDK1 self-phosphorylation, kinase activity, and subcellular localization.     

The subcellular localization of 3-phosphoinositide-dependent protein kinase is controlled by caveolin-1 binding

3-Phosphoinositide-dependent protein kinase 1 (PDK1), a member of the serine/threonine kinase family, has been demonstrated to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that PDK1 is associated with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of the caveolae membranes in COS-1. First, we noted the presence of two potential caveolin-1 binding motifs ((141)FFVKLYFTF(149) and (299)YDFPEKFF(306)) in the PDK1 catalytic domain. Using a pull-down approach, we observed that PDK1 interacts physically with caveolin-1 both in vivo and in vitro. Second, we detected the co-localization of PDK1 and caveolin-1 via confocal microscopy. The localization of PDK1 to the plasma membrane was disrupted by caveolin binding. Third, in transient transfection assays, interaction with caveolin-1 induced a substantial reduction in the in vivo serine/threonine phosphorylation of PDK1, whereas the caveolin-1 binding site mutant ((141)FFVKLYFTF(149) and (299)YDFPEKFF(306) change to (141)AFVKLAFTA(149) and (299)ADAPEFLA(306)) did not. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82-101) functionally suppressed the self-phosphorylation and kinase activities of purified recombinant PDK1 protein. Thus, our observations indicated that PDK1 binds to caveolin-1 through its caveolin-binding motifs, and also that the protein-protein interaction between PDK1 and caveolin-1 regulates PDK1 self-phosphorylation, kinase activity, and subcellular localization.     

Cellular localization and interaction of ABCA1 and caveolin-1 in aortic endothelial cells after HDL incubation

The goal of this study was to investigate the cellular localization and the interaction between caveolin-1 and ABCA1 in cholesterol-loaded aortic endothelial cells after HDL incubation. Immunofluorescence confocal microscopy showed that ABCA1 was found primarily on the cell surface, whereas caveolin-1 was revealed on the cell surface and in the cytoplasm. The HDL appeared to colocalize with ABCA1 and caveolin-1 on the cell surface. No free HDL was revealed in the cytoplasm. The HDL was colocalized neither with early endosome marker (CD71) nor with late endosome marker (LAMP2). The chemical cross-linking and immunoprecipitation analysis revealed that ABCA1 binds directly to both HDL and caveolin-1, whereas HDL does not bind directly to caveolin-1. The studies provide evidence for a direct interaction between ABCA1 and HDL, ABCA1 and caveolin-1, but not HDL and caveolin-1, indicating that ABCA1 may act as a structural platform between HDL and caveolin-1 on the cell surface during cellular cholesterol efflux.     

ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells

Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.     

Flotillin-1 stabilizes caveolin-1 in intestinal epithelial cells

Flotillins and caveolins represent two types of resident proteins associated with lipid rafts in mammalian cells, however, their possible cross-talk in regulating lipid raft functions remains poorly understood. In this report, we observed that siRNA-mediated down-regulation of flotillin-1 expression which disrupted lipid raft-mediated endocytosis of BODIPY FL C₅-lactosylceramide also substantially decreased caveolin-1 level in SK-CO15 human intestinal epithelial cells. The decrease in caveolin-1 expression appeared to be specific for flotillin-1 knock-down and was not observed after down-regulation of flotillin-2. The decrease in caveolin-1 level in flotillin-1-depleted cells was not due to suppression of its mRNA synthesis and was not mimicked by cholesterol depletion of SK-CO15 cells. Furthermore, flotillin-1 dependent down-regulation of caveolin-1 was reversed after cell exposure to lysosomal inhibitor, chloroquine but not proteosomal inhibitor, MG262. Our data suggest that flotillin-1 regulates caveolin-1 level by preventing its lysosomal degradation in intestinal epithelial cells.     

Altered mitochondrial function and metabolic inflexibility associated with loss of caveolin-1

Caveolin-1 is a major structural component of raft structures within the plasma membrane and has been implicated as a regulator of cellular signal transduction with prominent expression in adipocytes. Here, we embarked on a comprehensive characterization of the metabolic pathways dysregulated in caveolin-1 null mice. We found that these mice display decreased circulating levels of total and high molecular weight adiponectin and a reduced ability to change substrate use in response to feeding/fasting conditions. Caveolin-1 null mice are extremely lean but retain muscle mass despite lipodystrophy and massive metabolic dysfunction. Hepatic gluconeogenesis is chronically elevated, while hepatic steatosis is reduced. Our data suggest that the complex phenotype of the caveolin-1 null mouse is caused by altered metabolic and mitochondrial function in adipose tissue with a subsequent compensatory response driven mostly by the liver. This mouse model highlights the central contributions of adipose tissue for system-wide preservation of metabolic flexibility.     

H-Ras is a negative regulator of alpha3beta1 integrin expression in ECV304 endothelial cells

We have examined the role of Ras in integrin expression in ECV304 endothelial cells. Among the integrins examined in stable ECV304 transfectants expressing dominant active H-Ras (DAR-ECV), expression of alpha3beta1 integrin showed a prominent reduction in all the DAR-ECV clones when compared to the parental ECV304 cells. This implies that H-Ras negatively regulates the expression of alpha3beta1 integrin in ECV304 cells. When treated with inhibitors of the Ras downstream pathway (LY294002, PD98059, SB203580), the expression of alpha3beta1 integrin was up-regulated most significantly by LY294002, suggesting that among the downstream pathways of Ras, phosphatidylinositol 3-kinase is a major determinant. With the application of blocking antibody to alpha3beta1 integrin (2 - 2 x 10(4) nM), migration of ECV304 cells was enhanced to maximal (18%) at 20 nM. These results suggest that migration of endothelial cells could be modulated by H-Ras via alteration of the expression levels of alpha3beta1 integrin.     

Changes in membrane cholesterol affect caveolin-1 localization and ICC-pacing in mouse jejunum

Pacing of mouse is dependent on the spontaneous activity of interstitial cells of Cajal in the myenteric plexus (ICC-MP). These ICC, as well as intestinal smooth muscle, contain small membrane invaginations called caveolae. Caveolae are signaling centers formed by insertions of caveolin proteins in the inner aspect of the plasma membrane. Caveolins bind signaling proteins and thereby negatively modulate their signaling. We disrupted caveolae by treating intestinal segments with methyl beta-clodextrin (CD) to remove cholesterol or with water-soluble cholesterol (WSC) to load cholesterol. Both of these treatments reduced pacing frequencies, and these effects were reversed by the other agent. These treatments also inhibited paced contractions, but complete reversal was not observed. To evaluate the specificity of the effects of CD and WSC, additional studies were made of their effects on responses to carbamoyl choline and to stimulation of cholinergic nerves. Neither of these treatments affected these sets of responses compared with their respective time controls. Immunochemical and ultrastructural studies showed that caveolin 1 was present in smooth muscle membranes and ICC-MP. CD depleted both caveolin 1 and caveolae, whereas WSC increased the amount of caveolin 1 immunoreactivity and altered its distribution but failed to increase the number of caveolae. The effects of each agent were reversed in major part by the other. We conclude that signaling through caveolae may play a role in pacing by ICC but does not affect responses to acetylcholine from nerves or when added exogenously.     

Tuberin is a component of lipid rafts and mediates caveolin-1 localization: role of TSC2 in post-Golgi transport

Mutations of the TSC2 gene lead to the development of hamartomas in tuberous sclerosis complex. Their pathology exhibits features indicative of defects in cell growth, proliferation, differentiation, and migration. We have previously shown that tuberin, the TSC2 protein, resides in multiple subcellular compartments and as such may serve multiple functions. To further characterize the microsomal pool of tuberin, we found that it cofractionated with caveolin-1 in a low-density, Triton X-100-resistant fraction (i.e., lipid rafts) and regulated its localization. In cells lacking tuberin, most of the endogenous caveolin-1 was displaced from the plasma membrane to a Brefeldin-A-sensitive, post-Golgi compartment distinct from the endosome and lysosome. Correspondingly, there was a paucity of caveolae at the plasma membrane of Tsc2-/- cells. Reintroduction of TSC2, but not a disease-causing mutant, reversed the caveolin-1 localization to the membrane. Exogenously expressed caveolin-1-GFP and vesicular stomatitis virus G protein, VSVG-GFP in the Tsc2-/- cells failed to be transported to the plasma membrane and were retained in distinct post-Golgi vesicles. Our data suggest a role of tuberin in regulating post-Golgi transport without apparent effects on protein sorting. The presence of mislocalized proteins in Tsc2-/- cells may contribute to the abnormal signaling and cellular phenotype of tuberous sclerosis.     

Expression of caveolin-1 enhances cholesterol efflux in hepatic cells

HepG2 cells were stably transfected with human caveolin-1 (HepG2/cav cells). Transfection resulted in expression of caveolin-1 mRNA, a high abundance of caveolin-1 protein, and the formation of caveolae on the plasma membrane. Cholesterol efflux from HepG2/cav cells was 280 and 45% higher than that from parent HepG2 cells when human plasma and human apoA-I, respectively, were used as acceptors. The difference in efflux was eliminated by treatment of cells with progesterone. There was no difference in cholesterol efflux to cyclodextrin. Cholesterol efflux from plasma membrane vesicles was similar for the two cell types. Transfection led to a 40% increase in the amount of plasma membrane cholesterol in cholesterol-rich domains (caveolae and/or rafts) and a 67% increase in the rate of cholesterol trafficking from intracellular compartments to these domains. Cholesterol biosynthesis in HepG2/cav cells was increased by 2-fold, and cholesterol esterification was reduced by 50% compared with parent HepG2 cells. The proliferation rate of transfected cells was significantly lower than that of non-transfected cells. Transfection did not affect expression of ABCA1 or the abundance of ABCA1 protein, but decreased secretion of apoA-I. We conclude that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to cholesterol-rich domains in the plasma membrane.     

Altered arachidonate distribution in macrophages from caveolin-1 null mice leading to reduced eicosanoid synthesis

In this work we have studied the effect of caveolin-1 deficiency on the mechanisms that regulate free arachidonic acid (AA) availability. The results presented here demonstrate that macrophages from caveolin-1-deficient mice exhibit elevated fatty acid incorporation and remodeling and a constitutively increased CoA-independent transacylase activity. Mass spectrometry-based lipidomic analyses reveal stable alterations in the profile of AA distribution among phospholipids, manifested by reduced levels of AA in choline glycerophospholipids but elevated levels in ethanolamine glycerophospholipids and phosphatidylinositol. Furthermore, macrophages from caveolin-1 null mice show decreased AA mobilization and prostaglandin E(2) and LTB(4) production upon cell stimulation. Collectively, these results provide insight into the role of caveolin-1 in AA homeostasis and suggest an important role for this protein in the eicosanoid biosynthetic response.     

Regulation of SGLT expression and localization through Epac/PKA-dependent caveolin-1 and F-actin activation in renal proximal tubule cells

This study demonstrated that exchange proteins directly activated by cAMP (Epac) and protein kinase A (PKA) by 8-bromo (8-Br)-adenosine 3',5'-cyclic monophosphate (cAMP) stimulated [(14)C]-α-methyl-D-glucopyranoside (α-MG) uptake through increased sodium-glucose cotransporters (SGLTs) expression and translocation to lipid rafts in renal proximal tubule cells (PTCs). In PTCs, SGLTs were colocalized with lipid raft caveolin-1 (cav-1), disrupted by methyl-β-cyclodextrin (MβCD). Selective activators of Epac or PKA, 8-Br-cAMP, and forskolin stimulated expressions of SGLTs and α-MG uptake in PTCs. In addition, 8-Br-cAMP-induced PKA and Epac activation increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB), which were involved in expressions of SGLTs. Furthermore, 8-Br-cAMP stimulated SGLTs translocation to lipid rafts via filamentous actin (F-actin) organization, which was blocked by cytochalasin D. In addition, cav-1 and SGLTs stimulated by 8-Br-cAMP were detected in lipid rafts, which were blocked by cytochalasin D. Furthermore, 8-Br-cAMP-induced SGLTs translocation and α-MG uptake were attenuated by inhibition of cav-1 activation with cav-1 small interfering RNA (siRNA) and inhibition of F-actin organization with TRIO and F-actin binding protein (TRIOBP). In conclusion, 8-Br-cAMP stimulated α-MG uptake via Epac and PKA-dependent SGLTs expression and trafficking through cav-1 and F-actin in PTCs.     

Binding activity of H-Ras is necessary for in vivo inhibition of ASK1 activity

H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals. In this study, we found that H-Ras interacted with ASK1 to cause the inhibition of both ASK1 activity and ASKl-induced apoptosis in vivo, which was reversed only partially by addition of RafS621 A, an antagonist of Raf, whereas MEK inhibitor, PD98059, and PI3K inhibitor, LY294002, did not disturb the inhibitory effect of H-Ras on ASK-1-induced apoptosis. Furthermore, by means of immunoprecipitate and kinase assays, we demonstrated that the interaction between H-Ras and ASK1 as well as the inhibition of ASKI activity were dependent on the binding activity of H-Ras. These results suggest that a novel mechanism may be involved in H-Rasmediated cell survival in addition to the well established MEK/ERK and PI3K/Akt kinase-dependent enhancement of cell survival.     

Integrin-linked kinase complexes with caveolin-1 in human neuroblastoma cells

Integrin-linked kinase (ILK) and caveolin-1 (cav-1) are implicated in the pathogenesis of cancer. Overexpression of ILK leads to altered expression of cell cycle regulators, a decreased level of cell adhesion to the extracellular matrix, a decreased level of apoptosis, in vitro phosphorylation of Akt, and tumor formation in nude mice. Conversely, cav-1 expression is frequently downregulated in many forms of cancer. We examined whether ILK and cav-1 interact in SHEP human neuroblastoma cells because ILK is present in caveolae-enriched membranes and contains a putative cav-binding domain. SHEP cells were stably transfected with vector, wild-type ILK (ILK-wt), kinase-deficient ILK (ILK-kd), or mutant cav-binding domain ILK (ILK-mutCavbd). Control SHEP cells and ILK transfectants express high levels of ILK and cav-1. Immunoprecipitation with anti-cav-1 co-immunoprecipitates a 59 kDa protein that is immunoreactive with the anti-ILK antibody, and this interaction is partially prevented in cells expressing ILK-mutCavbd. Cav-1 and ILK partially colocalize in SHEP cells, also supporting these data. Last, affinity chromatography with a biotinylated cav-scaffolding domain peptide precipitates ILK-wt but not ILK-mutCavbd. These data suggest that the cav-binding domain of ILK and the cav-scaffolding domain of cav-1 mediate complex formation in human neuroblastoma cells.     

Cellular apoptosis is associated with increased caveolin-1 expression in macrophages

Macrophage apoptosis is an important factor in determining the efficiency of the immune response, atherosclerotic lesion stability, and clearance of aged cells by phagocytosis. The involvement of caveolin-1 in the regulation of apoptosis has been previously suggested in fibroblasts and epithelial cells. Here we show that treatment of thioglycollate-elicited mouse peritoneal macrophages with various unrelated apoptotic agents, including simvastatin, camptothecin, or glucose deprivation, is associated with a specific and large increase in caveolin-1 expression. In contrast, caveolin-2 levels remain unaffected. Induction of apoptosis was measured by changes in cell morphology, annexin V-labeling, and DNA fragmentation. We demonstrate that caveolin-1 in macrophages is present in lipid rafts and colocalizes with phosphatidylserine (PS) at the cell surface of apoptotic macrophages. Our data suggest that caveolin-1 increase is an early event, closely accompanied by PS externalization and independent of caspase activation and nuclear DNA fragmentation. The increase in caveolin-1 levels does not require new protein synthesis, as cycloheximide does not prevent the apoptosis-mediated increase in caveolin-1 levels. We propose that increased levels of caveolin-1 characterize the apoptotic phenotype of macrophages. Caveolin-1 may be involved in the efficient externalization of PS at the surface of the apoptotic cells.     

Ganglioside GD1a regulation of caveolin-1 and Stim1 expression in mouse FBJ cells:Augmented expression of caveolin-1 and Stim1 in cells with increased GD1a content

GD1a was previously shown responsible for regulating cell motility, cellular adhesiveness to vitronectin, phosphorylation of c-Met and metastatic ability of mouse FBJ osteosarcoma cells. To determine the particular molecules regulated by GD1a, FBJ cells were assessed for tumor-related gene expression by semi-quantitative RT-PCR. Caveolin-1 and stromal interaction molecule 1 (Stim1) expression in FBJ-S1 cells, rich in GD1a, were found to be 6 and 4 times as much, respectively, than in FBJ-LL cells devoid of GD1a. Enhanced production of caveolin-1 in protein was confirmed by Western blotting. A low-metastatic FBJ-LL cell variant, having high GD1a expression through β1-4GalNAcT-1 (GM2/GD2 synthase) cDNA transfection (Hyuga S, et al, Int J Cancer 83: 685-91, 1999), showed enhanced production of caveolin-1 and Stim1 in mRNA and protein, compared to mock-transfectant M5. Incubation of FBJ-M5 cells with exogenous GD1a augmented the expression of caveolin-1 in mRNA and protein and Stim1 in mRNA as well. Treatment of FBJ-S1 with fumonisin B1, an inhibitor of N-acylsphinganine synthesis, for 15 days caused the complete depletion of gangliosides and suppressed the expression of caveolin-1 and Stim1. St3gal5 siRNA transfected cells showed decreased expression of caveolin-1 and Stim1 mRNA, as well as St3gal5 mRNA. These findings clearly indicate ganglioside GD1a to be involved in the regulation of the transformation suppressor genes, caveolin-1 and Stim1. Moreover, treatment with GD1a of mouse melanoma B16 cells and human hepatoma HepG2 cells brought about elevated expression of caveolin-1 and Stim1. Li Wang and Shizuka Takaku are equal contributors to the present work     

Antibody-specific detection of caveolin-1 in subapical compartments of MDCK cells

Caveolin-1 is the major structural component of caveolae and is also found in the Golgi complex of many cell types. Occasionally, caveolin-1 has been observed in additional intracellular compartments, including recycling endosomes. Why caveolin-1 expression is detected at these sites only infrequently is not clear. In this study, we test the hypothesis that non-caveolar, non-Golgi pools of caveolin-1 display unique and/or fixation-dependent epitopes. We compared the ability of a panel of antibodies raised against various domains of caveolin-1 to detect distinct subcellular pools of the protein by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells, a cell line where the subcellular localization of caveolin-1 has been extensively characterized. We show that three antibodies directed to the N-terminus of caveolin-1 recognize a previously undetected pool of caveolin-1 in the subapical region of MDCK cells, a localization characteristic of endosomal recycling compartments. The antibodies vary in their ability to label caveolin-1 at the cell surface, and the epitopes detected by each are highly fixation dependent. Our findings suggest that no single caveolin antibody or staining condition is capable of detecting all the caveolin-1 in a cell simultaneously. Consequently, the subcellular distribution of caveolin-1 may be much broader than currently believed.     

Identification of caveolin-1 in lipoprotein particles secreted by exocrine cells

Caveolin-1 is a protein component (of relative molecular mass 22, 000) of the striated coat that decorates the cytoplasmic surface of caveolae membranes. Previous biochemical and molecular tests have indicated that caveolin-1 is an integral membrane protein that is co-translationally inserted into endoplasmic-reticulum membranes of fibroblast and epithelial cells such that its carboxy- and amino-terminal ends are in the cytoplasm. Here we identify caveolin-1 in the secretory pathway of exocrine cells. Secretion of caveolin-1 from pancreatic acinar cells and a transfected exocrine cell line, but not from Chinese hamster ovary cells, is stimulated by the secretagogues secretin, cholecystokinin and dexamethasone. The secreted caveolin-1 co-fractionates with apolipoproteins, indicating that it may be secreted in a complex with lipids.