Recent data have demonstrated that caveolin, a major structural protein of caveolae, negatively regulates signaling molecules localized to caveolae. The interaction of caveolin with several caveolae-associated signaling proteins is mediated by the binding of the scaffolding region of caveolin to a hydrophobic amino acid-containing region within the regulated proteins. The presence of a similar motif within the insulin receptor kinase prompted us to investigate the caveolar localization and regulation of the insulin receptor by caveolin. We found that overexpression of caveolin-3 augmented insulinstimulated phosphorylation of insulin receptor substrate-1 in 293T cells but not the phosphorylation of insulin receptor. Peptides corresponding to the scaffolding domain of caveolin potently stimulated insulin receptor kinase activity toward insulin receptor substrate-1 or a Src-derived peptide in vitro and in a caveolin subtype-dependent fashion. Peptides from caveolin-2 exhibited no effect, whereas caveolin-1 and -3 stimulated activity 10-and 17-fold, respectively. Peptides which increased insulin receptor kinase activity did so without affecting insulin receptor auto-phosphorylation. Furthermore, the insulin receptor bound to immobilized caveolin peptides, and this binding was inhibited in the presence of free caveolin-3 peptides. Thus, we have identified a novel mechanism by which the insulin receptor is bound and activated by specific caveolin subtypes. Furthermore, these data define a new role for caveolin as an activator of signaling.

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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-␣, -⑀, and -were overexpressed in COS cells followed by subcellular fractionation using the sucrose gradient method, all the isoenzymes (␣, ⑀, and ) 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-␣ and -, but not of protein kinase C-⑀, 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.Several studies from independent laboratories have demonstrated that multiple phorbol ester-sensitive, classic protein kinase C (PKC) 1 isoenzymes are accumulated in caveolae. Enrichment of PKC-␣ was detected by immunoelectron microscopy (1) as well as by the cell fractionation technique using buoyant density gradient centrifugation (2). PKC-␤ and -␥ were also detected in caveolae as separated by the silica coating method from lung endothelial cells (3). Whether all PKC isoenzymes, including nonclassic isoenzymes, are similarly accumulated in caveolae, however, remains uncharacterized. PKC also regulates the function and formation of caveolae. Caveolin, the major structural protein of caveolae, contains a conserved consensus phosphorylation site of PKC as well as of v-Src (4). Phorbol ester treatment of the cell inhibits caveolae-mediated internalization and markedly reduces the number of caveola (1). Further, activation of PKC-␣ by phorbol esters dislocates this isoenzyme from caveolae. Thus, PKC is not only present in caveolae, but interacts functionally with caveolae.It has been suggested that caveolin by itself regulates the function of certain molecules accumulated in caveolae. Caveolin may directly interact with G protein, Src kinase, and HaRas as has been demonstrated using a short stretch of membrane proximal regions of the cytosolic amino terminus caveolin domain (or the caveolin scaffolding domain) (5-8).Further, a small peptide derived from this domain bound G protein directly and regulated its function (9). The specificity of binding caveolin to target molecules has been confirmed, using a random peptide sequence library, by identifying a common amino acid sequence motif (XXXXX or XXXXXX; is an aromatic residue), which is contained in ma...

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Add-on treatment with omalizumab improved asthma control without significant adverse events in Japanese patients with moderate-to-severe persistent asthma.

Responses of contractile properties of soleus to unloading and/or changes in high-energy phosphate contents were studied in rats. Reduction of high-energy phosphates, especially phosphocreatine, in ankle extensors was induced by feeding beta-guanidinopropionic acid (beta-GPA). The major finding in the study was that the fatigability and speed-related contractile properties responded to unloading and creatine supplementation in a similar manner. The high-energy phosphate contents tended to be elevated after 10-d supplementation of creatine and hindlimb suspension. The shift toward slow-type, mainly due to an increased one-half relaxation time, was seen in rats fed beta-GPA. Such a shift was reversed by feeding creatine or by hindlimb suspension; however, the suspension-induced shift of contractile properties toward fast-type was not prevented completely by beta-GPA feeding. Although the muscle fatigue resistance did not change by beta-GPA feeding alone, the decrease in fatigue resistance following suspension and creatine supply was less in the beta-GPA group. It is suggested that the levels of high-energy phosphates and tension production play important roles in the regulation of contractile properties of the soleus muscle.

The rapid amplification of ␤-adrenergic receptor signaling involves the sequential activation of multiple signaling molecules ranging from the receptor to adenylyl cyclase. The prevailing view of the agonist-induced interaction between signaling molecules is based on random collisions between proteins that diffuse freely in the plasma membrane. The recent identification of G protein ␣-and ␤␥-subunits in caveolae and their functional interaction with caveolin suggests that caveolae may participate in G protein-coupled signaling. We have investigated the potential interaction of ␤-adrenergic receptors with caveolin under resting conditions. ␤1-and ␤2-adrenergic receptors were recombinantly overexpressed in COS-7 cells. Caveolae were isolated using the detergent-free sucrose gradient centrifugation method. ␤1-and ␤2-adrenergic receptors were localized in the same gradient fractions as caveolin, where Gs␣-and ␤␥-subunits were detected as well. Immunofluorescence microscopy demonstrated the colocalization of ␤-adrenergic receptors with caveolin, indicating a nonrandom distribution of ␤-adrenergic receptors in the plasma membrane. Using polyhistidine-tagged recombinant proteins, ␤-adrenergic receptors were copurified with caveolin, suggesting that they were physically bound. Our results suggest that, in addition to clathrin-coated pits, caveolae may act as another plasma membrane microdomain to compartmentalize ␤-adrenergic receptors.

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