Caveolin-1 Scaffolding Domain Peptide Research Articles

Caveolin-1 Regulates Store-Operated Ca2+ Influx by Binding of Its Scaffolding Domain to Transient Receptor Potential Channel-1 in Endothelial Cells

Caveolin-1 associates with store-operated cation channels (SOC) in endothelial cells. We examined the role of the caveolin-1 scaffolding domain (CSD) in regulating the SOC [i.e., transient receptor potential channel-1 (TRPC1)] in human pulmonary artery endothelial cells (HPAECs). We used the cell-permeant antennapedia (AP)-conjugated CSD peptide, which competes for protein binding partners with caveolin-1, to assess the interactions of caveolin-1 with TRPC1 and its consequences on thrombin-induced Ca2+ influx. We observed that AP-CSD peptide markedly reduced thrombin-induced Ca2+ influx via SOC in HPAECs in contrast to control peptide. AP-CSD also suppressed thapsigargin-induced Ca2+ influx. Streptavidin-bead pull-down assay indicated strong binding of biotin-labeled AP-CSD peptide to TRPC1. Immunoprecipitation studies demonstrated an interaction between endogenous TRPC1 and ectopically expressed hemagglutinin-tagged CSD. Analysis of the deduced TRPC1 amino acid sequence revealed the presence of CSD binding consensus sequence in the TRPC1 C terminus. We also observed that an AP-TRPC1 peptide containing the CSD binding sequence markedly reduced the thrombin-induced Ca2+ influx. We identified the interaction between biotin-labeled AP-TRPC1 C terminus peptide and caveolin-1. Thus, these results demonstrate a crucial role of caveolin-1 scaffolding domain interaction with TRPC1 in regulating Ca2+ influx via SOC.

Caveolin‐1 scaffolding domain regulates store‐operated Ca 2+ influx by binding to TRPC1 in endothelial cells

Caveolin-1 associates with store-operated cation channels (SOC) in endothelial cells. We examined the importance of the caveolin-1 scaffolding domain (CSD) in regulating the predominant SOC (i.e., transient receptor potential channel-1 [TRPC1]) expressed in human pulmonary artery endothelial cells (HPAEC). We tested a cell permeable antennapedia (AP)-CSD peptide effects on thrombin-induced Ca2+ influx via SOC. AP-CSD peptide markedly reduced thrombin-induced Ca2+ influx via SOC in HPAEC. AP-CSD also suppressed thapsigargin-induced Ca2+ influx. Control-peptide had no effect on thrombin-induced Ca2+ influx. Streptavidin-bead pull-down assays indicated strong binding of biotin labeled AP-CSD peptide to TRPC1. Immunoprecipitation studies further demonstrated the interaction between endogenous TRPC1 and ectopically expressed HA-tagged-CSD. Analysis of the deduced TRPC1 amino acid sequence revealed the presence of CSD binding consensus sequence in the TRPC1-C-terminus. Incubation of HPAEC with AP-TRPC1 peptide containing the CSD binding sequence markedly reduced thrombin-induced Ca2+ influx. We also observed interaction between the biotin-labeled AP-TRPC1 C-terminus peptide and cav-1. These results indicate that the cav-1 scaffolding domain interacts with TRPC1 to regulate SOC Ca2+ influx and thus may regulate nitric oxide production and endothelial permeability.

Caveolin regulates microtubule polymerization in the vascular smooth muscle cells

Microtubule and caveolin have common properties in intracellular trafficking and the regulation of cellular growth. Overexpression of caveolin in vascular smooth muscle cells increased the polymer form of microtubule without changing in the total amount of tubulin, and downregulation of caveolin decreased the polymer form of microtubule. Fractionation of cellular proteins followed by immunodetection as well as immunostaining of caveolin and microtubule revealed that caveolin and a portion of microtubule were co-localized in caveolar fractions. A caveolin scaffolding domain peptide, which mimics caveolin function, did not alter the polymerization of microtubule in vitro, but dramatically inhibited the depolymerization of microtubule induced by stathmin, a microtubule destabilizing protein, which was also found in caveolar fractions. Accordingly, it is most likely that caveolin increased the polymer form of microtubule through the inhibition of a microtubule destabilizer, stathmin, suggesting a novel role of caveolin in regulating cellular network and trafficking.

Caveolae Targeting and Regulation of Large Conductance Ca2+-activated K+ Channels in Vascular Endothelial Cells

The vascular endothelium is richly endowed with caveolae, which are specialized membrane microdomains that facilitate the integration of specific cellular signal transduction processes. We found that the large conductance Ca(2+)-activated K+ (BK) channels are associated with caveolin-1 in bovine aortic endothelial cells (BAECs). OptiPrep gradient cell fractionation demonstrated that BK channels were concentrated in the caveolae-rich fraction in BAECs. Immunofluorescence imaging showed co-localization of caveolin-1 and BK channels in the BAEC membrane. Immunoprecipitation and glutathione S-transferase pull-down assay results indicated that caveolin-1 and BK channels are physically associated. However, whole cell patch clamp recordings could not detect BK (iberiotoxin-sensitive) currents in cultured BAECs under baseline conditions, even though the presence of BK mRNA and protein expression was confirmed by reverse transcription-PCR and Western blots. Cholesterol depletion redistributed the BK channels to non-caveolar fractions of BAECs, resulting in BK channel activation (7.3 +/- 1.6 pA/picofarad (pF), n = 5). BK currents were also activated by isoproterenol (ISO, 1 microM, 6.9 +/- 2.4 pA/pF, n = 6). Inclusion of a caveolin-1 scaffolding domain peptide (10 microM) in the pipette solution completely abrogated the effects of ISO on BK channel activation, whereas inclusion of the scrambled control peptide (10 microM) did not inhibit the ISO effects. We have also found that caveolin-1 knockdown by small interference RNA activated BK currents (5.3 +/- 1.4 pA/pF, n = 6). We conclude that: 1) BK channels are targeted to caveolae microdomains in vascular endothelial cells; 2) caveolin-1 interacts with BK channels and exerts a negative regulatory effect on channel functions; and 3) BK channels are inactive under control conditions but can be activated by cholesterol depletion, knockdown of caveolin-1 expression, or ISO stimulation. These novel findings may have important implications for the role of BK channels in the regulation of endothelial function.

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.

Identification of caveolin-1-interacting sites in neuronal nitric-oxide synthase. Molecular mechanism for inhibition of NO formation.

Caveolin is known to down-regulate both neuronal (nNOS) and endothelial nitric-oxide synthase (eNOS). In the present study, direct interactions of recombinant caveolin-1 with both the oxygenase and reductase domains of nNOS were demonstrated using in vitro binding assays. To elucidate the mechanism of nNOS regulation by caveolin, we examined the effects of a caveolin-1 scaffolding domain peptide (CaV1p1; residues (82-101)) on the catalytic activities of wild-type and mutant nNOSs. CaV1p1 inhibited NO formation activity and NADPH oxidation of wild-type nNOS in a dose-dependent manner with an IC(50) value of 1.8 microM. Mutations of Phe(584) and Trp(587) within a caveolin binding consensus motif of the oxygenase domain did not result in the loss of CaV1p1 inhibition, indicating that an alternate region of nNOS mediates inhibition by caveolin. The addition of CaV1p1 also inhibited more than 90% of the cytochrome c reductase activity in the isolated reductase domain with or without the calmodulin (CaM) binding site, whereas CaV1p1 inhibited ferricyanide reductase activity by only 50%. These results suggest that there are significant differences in the mechanism of inhibition by caveolin for nNOS as compared with those previously reported for eNOS. Further analysis of the interaction through the use of several reductase domain deletion mutants revealed that the FMN domain was essential for successful interaction between caveolin-1 and nNOS reductase. We also examined the effects of CaV1p1 on an autoinhibitory domain deletion mutant (Delta40) and a C-terminal truncation mutant (DeltaC33), both of which are able to form NO in the absence of CaM, unlike the wild-type enzyme. Interestingly, CaV1p1 inhibited CaM-dependent, but not CaM-independent, NO formation activities of both Delta40 and DeltaC33, suggesting that CaV1p1 inhibits interdomain electron transfer induced by CaM from the reductase domain to the oxygenase domain.

Caveolin-1 regulates contractility in differentiated vascular smooth muscle.

Caveolin is a principal component of caveolar membranes. In the present study, we utilized a decoy peptide approach to define the degree of involvement of caveolin in PKC-dependent regulation of contractility of differentiated vascular smooth muscle. The primary isoform of caveolin in ferret aorta vascular smooth muscle is caveolin-1. Chemical loading of contractile vascular smooth muscle tissue with a synthetic caveolin-1 scaffolding domain peptide inhibited PKC-dependent increases in contractility induced by a phorbol ester or an alpha agonist. Peptide loading also resulted in a significant inhibition of phorbol ester-induced adducin Ser662 phosphorylation, an intracellular monitor of PKC kinase activity, ERK1/2 activation, and Ser789 phosphorylation of the actin binding protein caldesmon. alpha-Agonist-induced ERK1-1/2 activation was also inhibited by the caveolin-1 peptide. Scrambled peptide-loaded tissues or sham-loaded tissues were unaffected with respect to both contractility and signaling. Depolarization-induced activation of contraction was not affected by caveolin peptide loading. Similar results with respect to contractility and ERK1/2 activation during exposure to the phorbol ester or the alpha-agonist were obtained with the cholesterol-depleting agent methyl-beta-cyclodextrin. These results are consistent with a role for caveolin-1 in the coordination of signaling leading to the regulation of contractility of smooth muscle.

Insulin-mimetic signaling by the sulfonylurea glimepiride and phosphoinositolglycans involves distinct mechanisms for redistribution of lipid raft components.

The insulin signal transduction cascade provides a number of sites downstream of the insulin receptor (IR) for cross-talk from other signaling pathways. Tyrosine phosphorylation of the IR substrates IRS-1/2 and metabolic insulin-mimetic activity in insulin-responsive cells can be provoked by soluble phosphoinositolglycans (PIG), which trigger redistribution from detergent-insoluble glycolipid-enriched raft domains (DIGs) to other areas of the plasma membrane and thereby activation of nonreceptor tyrosine kinases (NRTK) [Müller, G., Jung, C., Wied, S., Welte, S., Jordan, H., and Frick, W. (2001) Mol. Cell. Biol. 21, 4553-4567]. Here we describe that stimulation of glucose transport in isolated rat adipocytes by a different stimulus, the sulfonylurea glimepiride, is also based on IRS-1/2 tyrosine phosphorylation and downstream insulin-mimetic signaling involving activation of the NRTK, pp59(Lyn), and pp125(Fak), as well as tyrosine phosphoryation of the DIGs component caveolin. As is the case for PIG 41, glimepiride causes the concentration-dependent dissociation of pp59(Lyn) from caveolin and release of this NRTK and the glycosyl-phosphatidylinositol-anchored (GPI) proteins, Gce1 and 5'-nucleotidase, from total and anti-caveolin-immunoisolated DIGs. This results in their movement to detergent-insoluble raft domains of higher buoyant density (non-DIGs areas). IRS-1/2 tyrosine phosphorylation and glucose transport activation by both glimepiride and PIG are blocked by introduction into adipocytes of the caveolin scaffolding domain peptide which mimicks the negative effect of caveolin on pp59(Lyn) activity. Tyrosine phosphorylation of the NRTK, IRS-1/2, and caveolin as well as release of the NRTK and GPI proteins from DIGs and their redistribution into non-DIGs areas in response to PIG is also inhibited by treatment of intact adipocytes with either trypsin plus salt or N-ethylmaleimide (NEM). In contrast, the putative trypsin/salt/NEM-sensitive cell surface component (CIR) is not required for glimepiride-induced glucose transport, IRS-1/2 tyrosine phosphorylation, and redistribution of GPI proteins and NRTK. The data suggest that CIR is involved in concentrating signaling molecules at DIGs vs detergent-insoluble non-DIGs areas. These inhibitory interactions are relieved in response to putative physiological (PIG) or pharmacological (sulfonylurea) stimuli via different molecular mechanisms (dependent on or independent of CIR, respectively) thereby inducing IR-independent positive cross-talk to metabolic insulin signaling.

Inhibition of PKCα and rhoA Translocation in Differentiated Smooth Muscle by a Caveolin Scaffolding Domain Peptide

Receptor-coupled contraction of smooth muscle involves recruitment to the plasma membrane of downstream effector molecules PKCα and rhoA but the mechanism of this signal integration is unclear. Caveolins, the principal structural proteins of caveolar plasma membrane invaginations, have been implicated in the organization and regulation of many signal transducing molecules. Thus, using laser scanning confocal immunofluorescent microscopy, we tested the hypothesis that caveolin is involved in smooth muscle signaling by investigating caveolin isoform expression and localization, together with the effect of a peptide inhibitor of caveolin function, in intact differentiated smooth muscle cells. All three main caveolin isoforms were identified in uterine, stomach, and ileal smooth muscles and assumed a predominantly plasma membranous localization in myometrial cells. Cytoplasmic introduction of a peptide corresponding to the caveolin-1 scaffolding domain—an essential region for caveolin interaction with signaling molecules—significantly inhibited agonist-induced translocation of both PKCα and rhoA. Translocation was unimpaired by a scrambled peptide and was unaltered in sham-treated cells. The membranous localization of caveolins, and direct inhibition of receptor-coupled PKCα and rhoA translocation by the caveolin-1 scaffolding domain, supports the concept that caveolins can regulate the integration of extracellular contractile stimuli and downstream intracellular effectors in smooth muscle.

Phospholipase D2: functional interaction with caveolin in low-density membrane microdomains

Low-density detergent-insoluble membrane domains contain caveolin-1 and are enriched in a phospholipase D activity that is not PLD1. Here we show that caveolin-rich fractions, prepared from HaCaT human keratinocytes by either detergent-based or detergent-free methods, contain PLD2. Caveolar membrane PLD activity is stimulated 2-fold by low concentrations (10–30 μM) of the caveolin-1 and caveolin-2 scaffolding domain peptides, whereas it is inhibited at higher concentrations of the peptides. Immunoisolated HA-tagged PLD1 and PLD2 are not stimulated by the peptides, although both enzymes retain sensitivity to their inhibitory effect. Down-regulation of caveolin-1 expression by treatment of the cells with acetyl-leucyl-leucyl-norleucinal decreased caveolar PLD activity by 50%. Similarly, expression of an active form of the sterol regulatory element-binding protein (SREBP1–490) down-regulated caveolin-1 expression by 50% and decreased caveolar PLD activity by 60%. These data identify the PLD activity in caveolin-rich membranes as PLD2 and provide in vivo evidence suggesting that caveolin-1 regulates PLD2 activity.

Caveolin Interaction with Protein Kinase C: ISOENZYME-DEPENDENT REGULATION OF KINASE ACTIVITY BY THE CAVEOLIN SCAFFOLDING DOMAIN PEPTIDE

Caveolar localization of protein kinase C and the regulation of caveolar function by protein kinase C are well known. This study was undertaken to examine whether caveolin subtypes interact with various protein kinase C isoenzymes using the caveolin scaffolding domain peptide. When protein kinase C-alpha, -epsilon, and -zeta were overexpressed in COS cells followed by subcellular fractionation using the sucrose gradient method, all the isoenzymes (alpha, epsilon, and zeta) were detected in the same fraction as caveolin. The scaffolding domain peptide of caveolin-1 and -3, but not -2, inhibited the kinase activity and autophosphorylation of protein kinase C-alpha and -zeta, but not of protein kinase C-epsilon, overexpressed in insect cells. Truncation mutation studies of the caveolin-1 and -3 peptides demonstrated that a minimum of 16 or 14 amino acid residues of the peptide were required for the inhibition or direct binding of protein kinase C. Thus, the caveolin peptide physically interacted with protein kinase C and regulated its function. Further, this regulation occurred in a protein kinase C isoenzyme-dependent manner. Our results may provide a new mechanism regarding the regulation of protein kinase C isoenzyme activity and the molecular interaction of protein kinase C with its putative binding proteins.

Caveolin versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase.

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases. The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997) J. Biol. Chem. 272, 15583-15586). Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.