Reliable expression and purification of highly insoluble transmembrane domains.

A general procedure for the reliable preparation of insoluble transmembrane domains has been developed. Improved expression schemes were developed by expressing the transmembrane domains of caveolin proteins 1, 2, and 3 as a fusion to the Trp leader protein. This construct readily formed inclusion bodies during overexpression, allowing high levels of protein to be achieved. Cleavage of the transmembrane domain away from the Trp leader carrier protein was performed with cyanogen bromide. The transmembrane domains were then purified using reverse-phase high-performance liquid chromatography with a C4 column and were eluted with a mixture of 1-butanol and acetic acid. Using this method, the 39-42 amino acid transmembrane domains from caveolin proteins 1, 2, and 3 were successfully purified to homogeneity. Further verification of this method was successfully done with Rfbp(18-51), another insoluble transmembrane domain.

A novel role for a YXXPhi motif in directing the caveolin-dependent sorting of membrane-spanning proteins.

Previous studies showed that the sequence between amino acids 38 and 63 of the chicken AE1-4 anion exchanger is sufficient to direct basolateral sorting and recycling to the Golgi when fused to a cytoplasmic tailless F(c)RII B2 receptor. Further characterization of the recycling pathway has indicated that the chimera F(c)38-63 colocalizes with caveolin 1 in the basolateral membrane of MDCK cells, and in early endosomes following its internalization from the cell surface. Studies using small interfering RNA (siRNA) and dominant-negative mutants revealed that F(c)38-63 endocytosis is primarily caveolin-dependent and clathrin-independent. The endocytosis of the chimera is also dependent upon cholesterol and dynamin. Co-precipitation studies indicated that caveolin 1 associates with F(c)38-63. Mutation of the tyrosine or leucine residues in the cytoplasmic sequence Y(47)VEL of F(c)38-63 disrupts this interaction and inhibits the endocytosis of the chimera. Additional analyses revealed that AE1-4 also associates with caveolin 1. Mutation of the leucine in the Y(47)VEL sequence of AE1-4 disrupts this interaction, and blocks the recycling of this transporter from the basolateral membrane to the Golgi. The Y(47)VEL tetrapeptide matches the sequence of a YXXPhi motif, and our results indicate a novel role for this motif in directing caveolin-dependent sorting.

Novel role of ARF6 in vascular endothelial growth factor-induced signaling and angiogenesis.

Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that VEGF stimulates a Rac1-dependent NAD(P)H oxidase to produce reactive oxygen species (ROS) that are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. The small GTPase ARF6 is involved in membrane trafficking and cell motility; however, its roles in VEGF signaling and physiological responses in ECs are unknown. In this study, we show that overexpression of dominant-negative ARF6 [ARF6(T27N)] almost completely inhibits VEGF-induced Rac1 activation, ROS production, and VEGFR2 autophosphorylation in ECs. Fractionation of caveolae/lipid raft membranes demonstrates that ARF6, Rac1, and VEGFR2 are localized in caveolin-enriched fractions basally. VEGF stimulation results in the release of VEGFR2 from caveolae/lipid rafts and caveolin-1 without affecting localization of ARF6, Rac1, or caveolin-1 in these fractions. The egress of VEGFR2 from caveolae/lipid rafts is contemporaneous with the tyrosine phosphorylation of caveolin-1 (Tyr14) and VEGFR2 and with their association with each other. ARF6(T27N) significantly inhibits both VEGF-induced responses. Immunofluorescence studies show that activated VEGFR2 and phosphocaveolin colocalize at focal complexes/adhesions after VEGF stimulation. Both overexpression of ARF6(T27N) and mutant caveolin-1(Y14F), which cannot be phosphorylated, block VEGF-stimulated EC migration and proliferation. Moreover, ARF6 expression is markedly upregulated in association with an increase in capillary density in a mouse hindlimb ischemia model of angiogenesis. Thus, ARF6 is involved in the temporal-spatial organization of caveolae/lipid rafts- and ROS-dependent VEGF signaling in ECs as well as in angiogenesis in vivo.

Bovine caveolin-2 cloning and effects of shear stress on its localization in bovine aortic endothelial cells.

Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Normally, cells that express caveolin-1 also express caveolin-2, but this has not been demonstrated in bovine aortic endothelial cells (BAECs). Here, we show that BAECs express caveolin-2, which localizes in caveolae with caveolin-1. We have cloned the bovine caveolin-2 gene and after comparison with known protein sequences (human, murine, rat, and canine) have found divergent immunogenic regions (amino acid [aa] 21 to aa 50 and aa 79 to 88), which may explain the inability to detect caveolin-2 in different cell types. We developed a bovine caveolin-2-specific antibody to examine this protein's expression and localization in BAECs. We used differential gradient centrifugations and immunoprecipitation to show that bovine caveolin-2 and caveolin-1 form a hetero-oligomer in plasma membrane caveolae. Using immunocytochemistry we show that a pool of caveolin-2 also colocalizes with the cis-Golgi in static culture, but unlike caveolin-1, this Golgi associated pool is maintained after 1 day of shear exposure. Therefore, the interaction of caveolin-2 with caveolin-1 could play an important role in caveolae biogenesis and shear stimulated mechano-signal transduction.

The caveolin scaffolding domain modifies 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor binding properties by inhibiting phospholipase A2 activity.

Activation of the enzyme phospholipase (PLA 2) has been proposed to be part of the molecular mechanism involved in the alteration of 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptor responsiveness during long term changes in synaptic plasticity (long term potentiation). This study assesses the effect of the caveolin-1 scaffolding domain (CSD) on the activity of the regulatory enzyme PLA2. Caveolin-1 is a 22-kDa cholesterol-binding membrane protein known to inhibit the activity of most of its interacting partners. Our results show that the calcium-dependent cytosolic form of PLA2 (cPLA2) and caveolin-1 co-localized in mouse primary hippocampal neuron cultures and that they were co-immunoprecipitated from mouse hippocampal homogenates. A peptide corresponding to the scaffolding domain of caveolin-1 (Cav-(82-101)) dramatically inhibited cPLA2 activity in purified hippocampal synaptoneurosomes. Activation of endogenous PLA2 activity with KCl or melittin increased the binding of [3H]AMPA to its receptor. This effect was almost completely abolished by the addition of the CSD peptide to these preparations. Moreover, we demonstrated that the inhibitory action of the CSD peptide on AMPA receptor binding properties is specific (because a scrambled version of this peptide failed to have any effect) and that it is mediated by an inhibition of PLA2 enzymatic activity (because the CSD peptide failed to have an effect in membrane preparations lacking endogenous PLA2 activity). These results raised the possibility that caveolin-1, via the inhibition of cPLA2 enzymatic activity, may interfere with synaptic facilitation and long term potentiation formation in the hippocampus.

The cardiac sodium-calcium exchanger associates with caveolin-3.

The cardiac Na/Ca exchanger's (NCX1) role in calcium homeostasis during myocardial contractility makes it a possible target of signaling factors regulating inotropy. Caveolae, structured invaginations of the plasmalemma, are known to concentrate a wide variety of signaling factors. The predominant coat proteins of caveolae, caveolins, dock to and regulate the activity of these signaling factors and other proteins through interaction with their scaffolding domain. In this study we investigated the interaction of NCX1 with caveolin proteins. Western blots of bovine cardiac sarcolemmal vesicles revealed the presence of caveolin-1, -2, and -3. Immunoprecipitation of detergent-solubilized vesicle proteins with either NCX1 or caveolin-3 antibodies indicated that NCX1 coprecipitates with caveolin-3, but not with caveolin-1 and -2. Functional disruption of caveolae, by beta-cyclodextrin treatment of vesicles, diminished coprecipitation of caveolin-3 and NCX1 activity. NCX1 has five potential caveolin-binding motifs, two of which are in the transporter's exchange inhibitory peptide (XIP) domain. The presence of 50 mM XIP peptide enhanced coprecipitation of caveolin-3 with NCX1 independent of calcium concentration. We conclude that NCX1 associates specifically with caveolin-3. Partitioning of NCX1 in caveolae has implications for temporal and spatial regulation of excitation-contraction and -relaxation coupling in cardiac myocytes.

Isolation and characterization of detergent-resistant microdomains responsive to NCAM-mediated signaling from growth cones.

It is still largely unclear how cell adhesion molecule (CAM)-mediated signaling evokes responses from the growth cone cytoskeleton. Here we used TX-114 extraction of growth cones followed by equilibrium gradient centrifugation to isolate subfractions of detergent-resistant microdomains (DRMs) that could be structurally and functionally distinguished on the basis of localization and activation of components of CAM-mediated signaling pathways. DRMs enriched in cholesterol, caveolin, NCAM140, GPI-linked NCAM120, fyn, and GAP-43, all conventional markers of microdomains or rafts, were located in areas 2 and 3 of the gradient. Coimmunoprecipitation of specific components of CAM signaling pathways by GAP-43 then identified distinct subpopulations of DRMs. GAP-43 from area 2 DRMs coprecipitated GPI-linked NCAM120 and was inactive, i.e., PKC phosphorylation had not been stimulated. In contrast the GAP-43 from area 3 DRMs coprecipitated both transmembrane NCAM140 and caveolin and was active, i.e., highly phosphorylated by PKC. A different subset of DRMs from both area 2 and area 3 contained fyn that could not be coprecipitated with GAP-43 antibodies. In this case area 2 DRMs contained activated fyn that was phosphorylated on Y415. In contrast area 3 DRMs contained inactive fyn. Hence fyn and GAP-43, both targets of NCAM signaling, are located in distinct populations of DRMs, and their activated forms are reciprocally distributed on the gradient. A detergent-resistant membrane fraction recovered from area 4 was enriched in NCAM140, phosphorylated GAP-43, and actin, but not cholesterol, caveolin, or fyn. Immunoelectron microscopy revealed that phosphorylated GAP-43 was localized where the membranes and F-actin interacted. Our results provide evidence for NCAM-mediated signaling in DRMs and suggest that the DRMs responsible for fyn and PKC/GAP-43-mediated NCAM signaling are structurally distinct and differentially distributed in growth cones.

Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation.

Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis.

The association of pericytes (PCs) to newly formed blood vessels has been suggested to regulate endothelial cell (EC) proliferation, survival, migration, differentiation, and vascular branching. Here, we addressed these issues using PDGF-B-- and PDGF receptor-beta (PDGFR-beta)--deficient mice as in vivo models of brain angiogenesis in the absence of PCs. Quantitative morphological analysis showed that these mutants have normal microvessel density, length, and number of branch points. However, absence of PCs correlates with endothelial hyperplasia, increased capillary diameter, abnormal EC shape and ultrastructure, changed cellular distribution of certain junctional proteins, and morphological signs of increased transendothelial permeability. Brain endothelial hyperplasia was observed already at embryonic day (E) 11.5 and persisted throughout development. From E 13.5, vascular endothelial growth factor-A (VEGF-A) and other genes responsive to metabolic stress became upregulated, suggesting that the abnormal microvessel architecture has systemic metabolic consequences. VEGF-A upregulation correlated temporally with the occurrence of vascular abnormalities in the placenta and dilation of the heart. Thus, although PC deficiency appears to have direct effects on EC number before E 13.5, the subsequent increased VEGF-A levels may further abrogate microvessel architecture, promote vascular permeability, and contribute to formation of the edematous phenotype observed in late gestation PDGF-B and PDGFR-beta knock out embryos.

Heterologous desensitization mediated by G protein-specific binding to caveolin.

We examined the notion that sequestration of G protein subunits by binding to caveolin impedes G protein reassociation and leads to transient, G protein-specific desensitization of response in dispersed smooth muscle cells. Cholecystokinin octapeptide (CCK-8) and substance P (SP) were used to activate G(q/11), cyclopentyl adenosine (CPA) was used to activate G(i3), and acetylcholine (ACh) was used to activate both G(q/11) and G(i3) via m3 and m2 receptors, respectively. CCK-8 and SP increased only Galpha(q/11), and CPA increased only Galpha(i3) in caveolin immunoprecipitates; caveolin and other G proteins were not increased. ACh increased both Galpha(q/11) and Galpha(i3) in a time- and concentration-dependent fashion: only Galpha(q/11) was increased in the presence of an m2 antagonist, and only Galpha(i3) was increased in the presence of an m3 antagonist. To determine whether transient G protein binding to caveolin affected subsequent responses mediated by the same G protein, PLC-beta activity was measured in cells stimulated sequentially with two different agonists that activate either the same or a different G protein. After treatment of the cells with ACh and an m2 antagonist, the phospholipase C-beta (PLC-beta) response to CCK-8 and SP, but not CPA, was decreased; conversely, after treatment of the cells with ACh and an m3 antagonist, the PLC-beta response to CPA, but not CCK-8 or SP, was decreased. Similarly, after treatment with CCK-8 or SP, the PLC-beta response mediated by G(q/11) only was decreased, whereas after treatment with CPA, the PLC-beta response mediated by G(i3) only was decreased. A caveolin-binding Galpha(q/11) fragment blocked the binding of activated Galpha(q/11) but not Galpha(i3) to caveolin-3 and prevented desensitization of the PLC-beta response mediated only by other G(q/11)-coupled receptors. A caveolin-binding Galpha(i3) fragment had the reverse effect. Thus, transient binding of receptor-activated G protein subunits to caveolin impedes reassociation of the heterotrimeric species and leads to desensitization of response mediated by other receptors coupled to the same G protein.