Caveolae Internalization Research Articles

Filamin A Regulates Caveolae Internalization and Trafficking in Endothelial Cells

Transcytosis via caveolae is critical for maintaining vascular homeostasis by regulating the tissue delivery of macromolecules, hormones, and lipids. In the present study, we test the hypothesis that interactions between F-actin cross-linking protein filamin A and caveolin-1 facilitate the internalization and trafficking of caveolae. Small interfering RNA-mediated knockdown of filamin A, but not filamin B, reduced the uptake and transcytosis of albumin by approximately 35 and 60%, respectively, without altering the actin cytoskeletal structure or cell-cell adherens junctions. Mobility of both intracellular caveolin-1-green fluorescent protein (GFP)-labeled vesicles measured by fluorescence recovery after photobleaching and membrane-associated vesicles measured by total internal reflection-fluorescence microscopy was decreased in cells with reduced filamin A expression. In addition, in melanoma cells that lack filamin A (M2 cells), the majority of caveolin-1-GFP was localized on the plasma membrane, whereas in cells in which filamin A expression was reconstituted (A7 cells and M2 cells transfected with filamin A-RFP), caveolin-1-GFP was concentrated in intracellular vesicles. Filamin A association with caveolin-1 in endothelial cells was confirmed by cofractionation of these proteins in density gradients, as well as by coimmunoprecipitation. Moreover, this interaction was enhanced by Src activation, associated with increased caveolin-1 phosphorylation, and blocked by Src inhibition. Taken together, these data suggest that filamin A association with caveolin-1 promotes caveolae-mediated transport by regulating vesicle internalization, clustering, and trafficking.

Dynamins at a glance

The superfamily of dynamins includes classical dynamins and dynamin-related proteins. Classical dynamins are proteins that share sequence similarity with the first described dynamin, which is a large GTPase with five characteristic domains. Dynamin-related proteins differ from classical dynamins in

Albumin endocytosis via megalin in astrocytes is caveola‐ and Dab‐1 dependent and is required for the synthesis of the neurotrophic factor oleic acid

The synthesis and release of the neurotrophic factor oleic acid requires internalization of albumin into the astrocyte, which is mediated by megalin. In this study, we show that the binding and internalization of albumin involve its interaction with megalin, caveolin-1, caveolin-2 and cavin, but not with clathrin in astrocytes from primary culture. Electron microscopy analyses revealed albumin-gold complexes localized in caveolae, but not in clathrin-coated vesicles. Neither chlorpromazine nor silencing clathrin expression modified albumin uptake. Silencing caveolin-1 strongly reduced the binding and internalization of albumin and the distribution of megalin in the plasma membrane. However, silencing caveolin-2 only decreased albumin internalization, suggesting that caveolin-1 is responsible for megalin recruitment to the caveolae and that caveolin-2 participates in caveolae internalization. In most tissues, the cytosolic adaptor protein disabled (Dab)-2 connects megalin to clathrin, astrocytes lack Dab-2; instead, they express Dab-1, which interacts with caveolin-1 and megalin and is required for albumin internalization. The transcytosis of albumin in astrocytes, including the passage through the endoplasmic reticulum, which is a compulsory step for oleic acid synthesis, was confirmed by electron microscopy analyses. Thus, whereas silencing clathrin did not modify the synthesis and release of oleic acid, the knock-down of caveolin-1, caveolin-2 and Dab-1 strongly reduced the synthesis and release of this neurotrophic factor. In conclusion, caveola-mediated endocytosis of albumin requires megalin and the adaptor protein Dab-1 in cultured astrocytes. Albumin endocytosis may be a key step in brain development because it stimulates the synthesis of oleic acid, which in turn promotes neuronal differentiation.

The Role of Dynamin Family Members in Membrane Fission

The dynamin family of proteins are required for numerous membrane fission and remodeling events throughout the eukaryotic cell. Dynamin is involved in the final stages of fission during clathrin‐mediated endocytosis, caveolae internalization, and vesiculation from the Golgi and recycling endosome, while Drp1 (dynamin‐related protein 1) is necessary for mitochondrial division. During membrane fission, dynamin or Drp1 is believed to wrap around the constricted site to facilitate vesiculation or organelle division. In support of this model purified dynamin has been shown to self‐assemble into spirals (50 nm diameter) and readily form dynamin‐lipid tubes, which constrict and fragment upon addition of GTP. The three‐dimensional structures of dynamin in the constricted and non‐constricted states were solved by cryo‐electron microscopy methods. Placement of the GTPase and Pleckstrin Homology crystal structures into the cryo‐EM densities revealed a twisting motion that suggests a corkscrew model for dynamin constriction. We are currently examining the structure and function of the Drp1 homologue in yeast, Dnm1, to determine if a common mechanism of action exists among dynamin family members. In collaboration with Dr. Jodi Nunnari (UC Davis), we have shown that Dnm1 assembles into large spirals with a diameter of ~110 nm, which is a similar diameter to the observed mitochondrial constriction sites seen in vivo. Dnm1 also assembles onto lipid and forms Dnm1 decorated tubes that constrict significantly upon GTP addition. A preliminary 3D map of Dnm1‐lipid tubes reveals a slightly different architecture to dynamin. For example, the Dnm1 map lacks a strong inner radial density near the lipid bilayer, which correlates well with the absence of a pleckstrin homology domain in Dnm1. Overall, these results suggest that although dynamin family members share common mechanochemical properties, the structure of each member vary to fit their unique function.

Dynamin and caveolae in cardiac ischemic preconditioning

Caveolae are specialized membranes enriched in lipids and proteins that regulate numerous signaling processes. Caveolae participate in clathrin‐independent endocytosis that is regulated by dynamin‐2, large GTPase proteins that induce membrane fission. We have previously shown that ischemic preconditioning (IPC) induces protection in cardiac myocytes via modulated formation of caveolae; however, the dynamics of caveolae formation and internalization by IPC is unknown. We hypothesize that IPC leads to the localization of dynamin to caveolae in cardiac myocytes. Adult male mice underwent 5 minutes of cardiac ischemia followed by 15 minutes of reperfusion or 20 minutes of sham surgery. For cell fractionation and SDS‐PAGE analysis, hearts were excised and homogenized in sodium carbonate buffer. The hearts that were used for immunofluorescence experiments were perfused and prepared for cryo‐sectioning. Western blot analysis of whole cell lysates confirmed equal dynamin expression in both samples. Following cell fractionation, there were higher levels of dynamin in caveolae rich fractions of IPC samples than in sham samples. Immunofluorescence images showed decreased cytosolic dynamin and increased amounts of dynamin along the plasma membrane, with caveolin‐3 co‐localization in IPC cardiac myocytes relative to sham cardiac myocytes. Our data suggests that IPC causes cytosolic dynamin to migrate to caveolae in cadiac myocytes, such migration may be key to modulation of caveolar mobilization from the plasma membrane that can affect cardiac protection.

Nitration of p190RhoGAP secondary to caveolae‐mediated endocytosis increases endothelial junctional permeability

Depletion of caveolin‐1, which increases endothelial nitric oxide synthase (eNOS) activity, may influence the function of endothelial adherens junctions (AJs), thereby increases the junctional permeability. We showed that stimulation of caveolae‐mediated endocytosis increased the activity of eNOS (Miniatis et al., Circ Res. 2006; 99:870‐7). We now show that activation caveolae‐mediated endocytosis augments the increase in endothelial permeability in response to activation of protease activated receptor‐1 (PAR‐1) suggesting a relationship between signals emanating from caveolae and disruption of the junctional barrier. Following endocytosis, eNOS was translocated to AJs, resulting in augmented NO production after PAR‐1 activation. Thus, we addressed the possibility that protein nitration of p190RhoGAP (which constitutively associated with the AJ protein p120‐catenin) occurring secondary to the NO production p190RhoGAP nitration might interfere with the GAP activity, and hence RhoA activation. We observed nitration of p190RhoGAP that led to inhibition of its GAP activity resulting in sustained activation of RhoA. We conclude that signals initiated by caveolae internalization regulate junctional endothelial permeability by activating eNOS and through NO‐mediated inhibition of p190RhoGAP activity.

Cholesterol depletion induces anoikis‐like apoptosis via FAK down‐regulation and caveolae internalization

Caveolae (lipid rafts), microdomains of the plasma membrane, are known to contain various signalling molecules and consequently are involved in the regulation of many biological functions. To investigate the role of the caveolae in cell survival and adhesion, we disrupted the caveolae by depletion of cholesterol, a major lipid component of the caveolae, with methyl-beta cyclodextrin (MbetaCD) treatment of A431 cells. We found that cholesterol depletion induced an anoikis-like cell death involving actin reorganization, resulting in a decrease in cell spreading and an increase in cell detachment, which was reversed by cholesterol addition. Disruption of caveolae led to the down-regulation of FAK, Src activation, tyrosine phosphorylation of caveolin-1 and mobilization of caveolae markers, GM1 and caveolin-1, from the cell surface to the cytoplasm, which were also recovered by cholesterol addition. The expression of dominant-active FAK was able to delay caveolae internalization and apoptosis and attenuated Akt inactivation by MbetaCD, whereas dominant-negative FAK expression resulted in enhanced apoptosis. Moreover, FAK down-regulation by si-RNA resulted in Akt inactivation and thus increased cell death by MbetaCD treatment. Our results suggest that the cholesterol content and/or surface levels of the caveolae affect the activity of FAK, which in turn regulates caveolae internalization and cell survival.

Regulation of transendothelial permeability by Src Kinase

Transcellular transport of albumin from the endothelial lumen to the abluminal perivascular interstitium via caveolae is a primary determinant of basal endothelial permeability. Albumin binding to specific caveolae-associated proteins induces the internalization of caveolae from the endothelial plasma membrane. Albumin-containing caveolae detach from the plasma membrane and traffic to the opposite membrane where they release albumin into the extravascular space. The events initiating transcytosis have been shown to be tightly regulated by Src family kinases, and thus Src signaling is thought to be a critical “switch” regulating caveolae-mediated transcellular transport of the plasma protein albumin. Recently, accumulating evidence indicates the importance of caveolae-mediated albumin transport in endothelial hyperpermeability in response to inflammatory stimuli. In this review, we focus on the current understanding of Src signaling in regulating basal permeability and inflammation-evoked increase in transcellular albumin permeability of the pulmonary endothelium.

Respiratory syncytial virus glycoproteins uptake occurs through clathrin-mediated endocytosis in a human epithelial cell line

Cell-surface viral proteins most frequently enter the cell through clathrin or caveolae endocytosis. Respiratory syncytial virus antigen internalization by immune cells is via caveolin, however, uptake of paramyxovirus cell membrane proteins by non-immune cells is done through clathrin-coated pits. In this work, the uptake of respiratory syncytial virus cell surface glycoproteins by non-immune human epithelial cells was investigated through indirect immunofluorescence with polyclonal anti-RSV antibody and confocal lasser-scanner microscopy. Clathrin and caveolae internalization pathways were monitored through specific inhibitors monodansylcadaverine (MDC) and methyl-beta-cyclodextrin (MBCD), respectively. Internalization of RSV antigens was inhibited by MDC but not by MBCD, implying that clathrin-mediated endocytosis is the major uptake route of RSV antigens by an epithelial human cell line.

Dynamin regulates UT‐A1 urea transporter membrane expression

Dynamin plays a direct role in endocytosis, synaptic vesicle recycling, and caveolae internalization. The urea transporter UT‐A1, located in the kidney inner medullary collecting duct (IMCD), is important for urine concentrating ability. In this study, we found that both UT‐A1 and dynamin are present in caveolae isolated from HEK293 cells by OptiPrep gradient ultracentrifugation. The direct relationship of UT‐A1 and dynamin was identified by co‐immunoprecipitation. Dynamin was precipitated by UT‐A1 antibody when both UT‐A1 and dynamin were transfected into 293 cells. However, phosphorylation does not enhance the binding of UT‐A1 and dynamin. UT‐A1 has both cytosolic amino and carboxyl termini and a large intracellular loop. We therefore tested of which of the three domains dynamin bound. The three intracellular fragments of UT‐A1 were prepared and labeled by 35S methionine by in vitro translation with rabbit reticulocyte lysate. Pull down study showed dynamin specifically binds to the intracellular loop of UT‐A1, but not the N‐ and C‐termini. Cell biotinylation experiments of UT‐A1 demonstrated that co‐expression of dynamin and UT‐A1 in 293 cells resulted in a decrease of UT‐A1 cell surface expression. Conversely, cells expressing dynamin mutants deficient in GTP binding (K44A) revealed a significant increase of UT‐A1 protein cell surface accumulation. Our data suggest that a dynamin‐dependent pathway might be an important mechanism for the regulation of urea transport activity in vivo.

Intracellular trafficking of raft/caveolae domains: Insights from integrin signaling

Cells have a complex system for delivering and compartmentalizing proteins and lipids in order to achieve spatio-temporal coordination of signaling. Rafts/caveolae are plasma membrane microdomains that regulate signaling pathways and processes such as cell migration, polarization and proliferation. Regulation of raft/caveolae trafficking involves multiple steps regulated by different proteins to ensure coordination of signaling cascades. The best studied raft-mediated endocytic route is controlled by caveolins. Recent data suggest integrin-mediated cell adhesion is a key regulator of caveolar endocytosis. In this review we examine the regulation of caveolar trafficking and the interplay between integrins, cell adhesion and caveolae internalization.

Intersectin-1s Regulates the Mitochondrial Apoptotic Pathway in Endothelial Cells

Intersectins (ITSNs) are multidomain adaptor proteins implicated in endocytosis, regulation of actin polymerization, and Ras/MAPK signaling. We have previously shown that ITSN-1s is required for caveolae fission and internalization in endothelial cells (ECs). In the present study, using small interfering RNA to knock down ITSN-1s protein expression, we demonstrate a novel role of ITSN-1s as a key antiapoptotic protein. Knockdown of ITSN-1s in ECs activated the mitochondrial pathway of apoptosis as determined by genomic DNA fragmentation, extensive mitochondrial fission, activation of the proapoptotic proteins BAK and BAX, and cytochrome c efflux from mitochondria. ITSN-1 knockdown acts as a proapoptotic signal that causes mitochondrial outer membrane permeabilization, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. These effects were secondary to decreased activation of Erk1/2 and its direct activator MEK. Bcl-X(L) overexpression prevented BAX activation and the apoptotic ECs death induced by suppression of ITSN-1s. Our findings demonstrate a novel role of ITSN-1s as a negative regulator of the mitochondrial pathway-dependent apoptosis secondary to activation of the Erk1/2 survival signaling pathway.

Internalization of caveolae and their relationship with endosomes in cultured human and mouse endothelial cells

Treatment of cells with pervanadate or vanadate induces the phosphorylation of caveolin-1 and its internalization from the cell surface, but the intracellular fate of caveolae has not been fully elucidated. In the present study, we examined the fate of endocytosed caveolae in human umbilical vein endothelial cells and mouse endothelial KOP2.16 cells. The localization of internalized caveolae and their relationship with the endosomes were examined by immunofluorescence microscopy as well as by immunoprecipitation and chasing of biotinylated transferrin. In untreated cells, caveolin-1 was mostly confined to the cell surface. When cells were treated with either pervanadate for 30 min or vanadate for 3 h, many caveolin-1-labeled vesicles were formed inside the cells, some of which were colocalized with Rab5 or Rab4. The internalized caveolin-1 was colocalized with the endocytosed transferrin in the Rab5-, Rab4- or early endosome antigen-1-labeled compartment where caveolin-1 was phosphorylated. It then moved to the Rabl 1-associated compartment. Immunogold electron microscopy revealed that internalized caveolin-1 colocalized with Rab5 or Rab4 in vesicles larger than caveolae. These results suggest that the internalized caveolae interact with early endosomes.

A New Paradigm

See related article, pages 870–877 Caveolae, cholesterol rich microdomain platforms localized mostly at the cytoplasmic membrane of endothelial cells as well as other cell types, are importantly involved in cell signaling and enzymatic activity.1–6 Caveolae also contain cationic amino acid transporter (CAT) which provide L-Arginine to the eNOS-Ca2+-Calmodulin complex toward the production of nitric oxide (NO).1–6 Caveolin-1, a major caveolae-localized protein, represses markedly eNOS-dependent NO production by interfering with NADPH-dependent electron flux.3 Alteration in caveolin abundance or its subcellular location leads to the control of NO production.5,6 Thus, it is clear, that the more the caveolin the less the NO, and inversely.3 The conventional paradigm supports the concept that eNOS–caveolin-1 interaction promotes inhibition of NO production, whereas increased intracellular Ca2+ activates Ca2+-Calmodulin which in turn disrupts the eNOS–caveolin-1 interaction and thus liberates the enzyme. This in turn promotes NO production.1–6 A new paradigm is now suggested by Miniatis and colleagues7, who elegantly demonstrated that NO production in pulmonary endothelial cells is significantly mediated by caveolae internalization and is independent of the increase of intracellular Ca2+. …

Novel Mechanism of Endothelial Nitric Oxide Synthase Activation Mediated by Caveolae Internalization in Endothelial Cells

Caveolin-1, the caveolae scaffolding protein, binds to and negatively regulates eNOS activity. As caveolin-1 also regulates caveolae-mediated endocytosis after activation of the 60-kDa albumin-binding glycoprotein gp60 in endothelial cells, we addressed the possibility that endothelial NO synthase (eNOS)-dependent NO production was functionally coupled to caveolae internalization. We observed that gp60-induced activation of endocytosis increased NO production within 2 minutes and up to 20 minutes. NOS inhibitor N(G)-nitro-L-arginine (L-NNA) prevented the NO production. To determine the role of caveolae internalization in the mechanism of NO production, we expressed dominant-negative dynamin-2 mutant (K44A) or treated cells with methyl-beta-cyclodextrin. Both interventions inhibited caveolae-mediated endocytosis and NO generation induced by gp60. We determined the role of signaling via Src kinase in the observed coupling of endocytosis to eNOS activation. Src activation induced the phosphorylation of caveolin-1, Akt and eNOS, and promoted dissociation of eNOS from caveolin-1. Inhibitors of Src kinase and Akt also prevented NO production. In isolated perfused mouse lungs, gp60 activation induced NO-dependent vasodilation, whereas the response was attenuated in eNOS(-/-) or caveolin-1(-/-) lungs. Together, these results demonstrate a critical role of caveolae-mediated endocytosis in regulating eNOS activation in endothelial cells and thereby the NO-dependent vasomotor tone.

Involvement of the Rho/Rac family member RhoG in caveolar endocytosis

We show here that the GTPase RhoG is involved in caveolar trafficking. Wild-type RhoG moves sequentially to the plasma membrane, intracellular vesicles, and the Golgi apparatus along markers of this endocytic pathway. Such translocation is associated with changes in RhoG GDP/GTP levels and is highly dependent on lipid raft integrity and on the function of the GTPase dynamin2. In addition, the constitutively active RhoG(Q61L) mutant is preferentially located in endocytic vesicles that can be decorated with markers of the caveola-derived endocytic pathway. RhoG(Q61L), but not the analogous Rac1 mutant protein, affects caveola internalization and the subsequent delivery of endocytic vesicles to the Golgi apparatus. The expression of RhoG/Rac1 chimeric proteins and RhoG(Q61L) effector mutants in cells induces alterations in the internalization of caveolae and severe changes in vesicle structure, respectively. However, the knockdown of endogenous rhoG transcripts using small interfering RNAs does not affect significantly the trafficking of caveola-derived vesicles, suggesting that RhoG function is dispensable for this endocytic process or, alternatively, that its function is compensated by other molecules. Taken together, these observations assign a novel function to RhoG and suggest that caveolar trafficking, as previously shown for other endocytic routes, is modulated by GTPases of the Ras superfamily.

Intersectin Regulates Endothelial Cell Junction Integrity

Our recent work indicated that the intersectin-2L (ITSN-2L), via its DH-PH domain with Cdc42-specific guanine nucleotide exchange factor activity, promotes actin polymerization and decrease in caveolae internalization. Expression of the DH-PH domain of ITSN-2L in cultured endothelial cells revealed a selective activation of Cdc42. Biochemical analyses of endothelial cell lysates and double fluorescent labeling of cultured endothelial cells indicate that ITSN interacts and co-localizes with members of the Cdc42-N-WASp-Arp2/3 actin polymerization pathway. Moreover, phalloidin staining of ECs expressing the DH-PH domain of ITSN-2L showed actin cytoskeletal changes such as increases in filopodia and cortical actin, while a biochemical assay for actin polymerization indicated a shift from G- to F-actin. We now show that these actin cytoskeletal rearrangements caused by ITSN-2L also have an effect on the integrity of interendothelial junctions. Morphological analysis of ECs transfected with the DH-PH domain of ITSN-2L demonstrated the appearance of gaps between cells. Moreover, functional studies in mouse lungs, upon activation of Cdc42, indicated increased vascular permeability via the interendothelial junctions. When coupled to our previous data indicating a decrease in caveolae internalization with ITSN overexpression, these current data show the involvement of ITSN in the regulation of both transcellular and paracellular transport pathways in the endothelium. In this sense, ITSN may serve to regulate endothelial permeability by controlling both pathways. Thus, the regulation of Cdc42 activity and actin polymerization by ITSN may be crucial for the loss of endothelial junctional integrity. This research is supported in part by NIH T32 HL07289.

Caveolae: Stable Membrane Domains with a Potential for Internalization

The role of caveolae in endocytosis is hotly debated. Here, we argue that most caveolae are stable microdomains at the cell surface. Only a small fraction of caveolae is constitutively internalized, leading to a quantitatively minor uptake of ligands and receptors. In addition, we suggest that a more pronounced downregulation of caveolae from the plasma membrane can occur, presumably stimulated by receptor cross-linking and clustering in caveolae. Finally, we propose that future studies dealing with internalization of caveolae should actually document such internalization and include kinetic data.

Caveolin-1 Interacts Directly with Dynamin-2

Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2DeltaPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.

Gβγ Activation of Src Induces Caveolae-mediated Endocytosis in Endothelial Cells

Caveolae-mediated endocytosis in endothelial cells is stimulated by the binding of albumin to gp60, a specific albumin-binding protein localized in caveolae. The activation of gp60 induces its cell surface clustering and association with caveolin-1, the caveolar-scaffolding protein. This interaction leads to G(i)-induced Src kinase activation, which in turn signals dynamin-2-mediated fission and directed migration of caveolae-derived vesicles from apical to basal membrane. In this study, we investigated the possible role of the Gbetagamma heterodimer in signaling G(i)-induced Src activation and subsequent caveolae-mediated endocytosis. We observed using rat lung microvascular endothelial cells that expression of the C terminus of beta-adrenergic receptor kinase (ct-betaARK), an inhibitor Gbetagamma signaling, prevented gp60-dependent Src activation as well as caveolae-mediated endocytosis and transcellular transport of albumin and uptake of cholera toxin subunit B, a specific marker of caveolae internalization. Expression of ct-betaARK also prevented Src-mediated tyrosine phosphorylation of caveolin-1 and dynamin-2 and the resultant phosphorylation-dependent association of dynamin-2 and caveolin-1. Also, the direct activation of Gbetagamma using a specific cell-permeant activating peptide (myristoylated-SIRKALNILGYPDYD) simulated the effects of gp60 in inducing Src activation, caveolin-1, and dynamin-2 phosphorylation as well as caveolae-mediated endocytosis of cholera toxin subunit B. The myristoylated-SIRKALNILGYPDYD peptide-induced responses were inhibited by the expression of ct-betaARK. Taken together, our results demonstrate that Gbetagamma activation of Src signals caveolae-mediated endocytosis and transendothelial albumin transport via transcytosis.