NO synthase II in mouse skeletal muscle is associated with caveolin 3.
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The inducible-type NO synthase (NOS II; iNOS) is constitutively expressed in slow-twitch skeletal muscle fibres of guinea-pigs [Gath, Closs, Godtel-Armbrust, Schmitt, Nakane, Wessler and Forstermann (1996) FASEB J. 10, 1614-1620]. Here we studied the expression of NOS II in skeletal muscle of wild-type and NOS II-deficient mice and investigated the molecular basis for the membrane association of this NOS in muscle. A basal expression of NOS II mRNA and protein was detected in skeletal muscle from untreated wild-type mice; expression increased when mice were treated with bacterial lipopolysaccharide (LPS). No NOS II was found in any tissue of untreated or LPS-treated NOS II-deficient mice. Immunoprecipitation experiments were performed with homogenates of gastrocnemius muscle from untreated or LPS-treated wild-type mice. A NOS II-specific antibody precipitated caveolin 3 in all homogenates investigated, the effect being most pronounced in skeletal muscle from LPS-treated animals. Conversely, an antibody against caveolin 3 co-precipitated NOS II in muscle homogenates. Similarly, a weak co-precipitation of NOS II and caveolin 3 was seen in homogenates of untreated murine C2C12 myotubes; co-precipitation was markedly enhanced in cells stimulated with LPS/interferon gamma. The association of NOS II with caveolin 3 might have implications for the regulation of contraction of, and/or glucose uptake by, slow-twitch muscle fibres. (+info)
Neuregulin signaling in the heart. Dynamic targeting of erbB4 to caveolar microdomains in cardiac myocytes.
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Two of the neuregulins (NRG1 and NRG2) and their receptors (erbB2 and erbB4) are essential for normal cardiac development and can mediate hypertrophic growth and enhance survival of embryonic, postnatal, and adult rat ventricular myocytes. The expression of erbB4, the predominant NRG receptor in postnatal rat ventricular muscle, declines after midembryogenesis, and its expression is limited to cardiac myocytes. A full-length erbB4 rat cDNA isolated from neonatal ventricular muscle was found to be highly homologous to human erbB4 and contained a caveolin binding motif within the cytoplasmic kinase domain. Using the complementary techniques of detergent-free density-gradient ultracentrifugation of myocyte lysates and coimmunoprecipitation of erbB4 and caveolin-3, the caveolin isoform expressed in cardiac myocytes, erbB4 could be localized (using both approaches) to caveolar microdomains. Moreover, addition of a soluble NRG1, recombinant human glial growth factor 2, resulted in rapid (2-minute) translocation of erbB4 out of caveolar microdomain in cardiac myocytes. Thus, erbB4 is dynamically targeted to caveolar microdomains within cardiac myocytes. Its rapid translocation after NRG1 binding may contribute to receptor desensitization in the continuous presence of ligand. (+info)
Molecular characterization of caveolin association with the Golgi complex: identification of a cis-Golgi targeting domain in the caveolin molecule.
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Caveolins are integral membrane proteins which are a major component of caveolae. In addition, caveolins have been proposed to cycle between intracellular compartments and the cell surface but the exact trafficking route and targeting information in the caveolin molecule have not been defined. We show that antibodies against the caveolin scaffolding domain or against the COOH terminus of caveolin-1 show a striking specificity for the Golgi pool of caveolin and do not recognize surface caveolin by immunofluorescence. To analyze the Golgi targeting of caveolin in more detail, caveolin mutants were expressed in fibroblasts. Specific mutants lacking the NH2 terminus were targeted to the cis Golgi but were not detectable in surface caveolae. Moreover, a 32-amino acid segment of the putative COOH-terminal cytoplasmic domain of caveolin-3 was targeted specifically and exclusively to the Golgi complex and could target a soluble heterologous protein, green fluorescent protein, to this compartment. Palmitoylation-deficient COOH-terminal mutants showed negligible association with the Golgi complex. This study defines unique Golgi targeting information in the caveolin molecule and identifies the cis Golgi complex as an intermediate compartment on the caveolin cycling pathway. (+info)
Localization of the human caveolin-3 gene to the D3S18/D3S4163/D3S4539 locus (3p25), in close proximity to the human oxytocin receptor gene. Identification of the caveolin-3 gene as a candidate for deletion in 3p-syndrome.
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Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cell types (cardiac and skeletal). Recently, we identified an autosomal dominant form of limb girdle muscular dystrophy in humans that is due to mutations within exon 2 of the caveolin-3 gene (3p25). However, the detailed location of the human caveolin-3 gene and its position with regard to neighboring genes remains unknown. Here, we have isolated three independent BAC clones containing the human caveolin-3 gene. Using a PCR-based approach, we determined that these clones contain both exons 1 and 2 of the human caveolin-3 gene. In addition, we performed microsatellite marker analysis of these BAC clones, using a panel of 13 markers that are known to map within the 3p25 region. Our results indicate that these BAC clones contain the following three markers: D3S18, SHGC-1079 (also known as D3S4163) and D3S4539. Interestingly, D3S18 is a marker for two known human diseases, von Hippel-Lindau disease and 3p-syndrome. As D3S4163 and D3S4539 are known to map in the vicinity of the 3' end of the human oxytocin receptor gene, we determined if these caveolin-3 positive BACs also contain the oxytocin receptor gene. We show that (i) these BACs contain all four exons of the oxytocin receptor gene and (ii) that the genes encoding caveolin-3 and the oxytocin receptor are located approximately 7-10 kb apart and in the opposite orientation. As 3p-syndrome is characterized by cardiac septal defects and caveolin-3 is expressed primarily in the heart and skeletal muscle, caveolin-3 is a candidate gene that may be deleted in 3p-syndrome. (+info)
Caveolin-3 upregulation activates beta-secretase-mediated cleavage of the amyloid precursor protein in Alzheimer's disease.
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Here, we investigate the involvement of caveolins in the pathophysiology of Alzheimer's disease (AD). We show dramatic upregulation of caveolin-3 immunoreactivity in astroglial cells surrounding senile plaques in brain tissue sections from authentic AD patients and an established transgenic mouse model of AD. In addition, we find that caveolin-3 physically interacts and biochemically colocalizes with amyloid precursor protein (APP) both in vivo and in vitro. Interestingly, recombinant overexpression of caveolin-3 in cultured cells stimulated beta-secretase-mediated processing of APP. Immunoreactivities of APP and presenilins were concomitantly increased in caveolin-3-positive astrocytes. Because the presenilins also form a physical complex with caveolin-3, caveolin-3 may provide a common platform for APP and the presenilins to associate in astrocytes. In AD, augmented expression of caveolin-3 and presenilins in reactive astrocytes may alter APP processing, leading to the overproduction of its toxic amyloid metabolites. (+info)
Phenotypic behavior of caveolin-3 mutations that cause autosomal dominant limb girdle muscular dystrophy (LGMD-1C). Retention of LGMD-1C caveolin-3 mutants within the golgi complex.
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Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cell types (cardiac and skeletal). Autosomal dominant limb girdle muscular dystrophy (LGMD-1C) in humans is due to mutations within the caveolin-3 gene: (i) a 9-base pair microdeletion that removes three amino acids within the caveolin scaffolding domain (DeltaTFT) or (ii) a missense mutation within the membrane spanning domain (P --> L). The molecular mechanisms by which these two mutations cause muscular dystrophy remain unknown. Here, we investigate the phenotypic behavior of these caveolin-3 mutations using heterologous expression. Wild type caveolin-3 or caveolin-3 mutants were transiently expressed in NIH 3T3 cells. LGMD-1C mutants of caveolin-3 (DeltaTFT or P --> L) were primarily retained at the level of a perinuclear compartment that we identified as the Golgi complex in double-labeling experiments, while wild type caveolin-3 was efficiently targeted to the plasma membrane. In accordance with these observations, caveolin-3 mutants formed oligomers of a much larger size than wild type caveolin-3 and were excluded from caveolae-enriched membrane fractions as seen by sucrose density gradient centrifugation. In addition, these caveolin-3 mutants were expressed at significantly lower levels and had a dramatically shortened half-life of approximately 45-60 min. However, caveolin-3 mutants were palmitoylated to the same extent as wild type caveolin-3, indicating that targeting to the plasma membrane is not required for palmitoylation of caveolin-3. In conclusion, we show that LGMD-1C mutations lead to formation of unstable high molecular mass aggregates of caveolin-3 that are retained within the Golgi complex and are not targeted to the plasma membrane. Consistent with its autosomal dominant form of genetic transmission, we demonstrate that LGMD-1C mutants of caveolin-3 behave in a dominant-negative fashion, causing the retention of wild type caveolin-3 at the level of the Golgi. These data provide a molecular explanation for why caveolin-3 levels are down-regulated in patients with this form of limb girdle muscular dystrophy (LGMD-1C). (+info)
The limb-girdle muscular dystrophies-multiple genes, multiple mechanisms.
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In the field of muscular dystrophy, advances in understanding the molecular basis of the various disorders in this group have been rapidly translated into readily applicable diagnostic tests, allowing the provision of more accurate prognostic and genetic counselling. The limb-girdle muscular dystrophies (LGMD) have recently undergone a major reclassification according to their genetic basis. Currently 13 different types can be recognized. Amongst this group, increasing diversity of the mechanisms involved in producing a muscular dystrophy phenotype is emerging. Recent insights into the involvement of the dystrophin glycoprotein complex in muscular dystrophy suggests that its members may play distinct or even multiple roles in the maintenance of muscle fibre integrity. In other forms of LGMD, proteins have been implicated which may be important in intracellular signalling, vesicle trafficking or the control of transcription. As these various mechanisms are more fully elucidated, further insights will be gained into the pathophysiology of muscular dystrophy. At a practical level, despite the marked heterogeneity of this group real progress can at last be made in determining a precise diagnosis. (+info)
Codistribution of NOS and caveolin throughout peripheral vasculature and skeletal muscle of hamsters.
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In isolated cell systems, nitric oxide synthase (NOS) activity is regulated by caveolin (CAV), a resident caveolae coat protein. Because little is known of this interaction in vivo, we tested whether NOS and caveolin are distributed together in the intact organism. Using immunohistochemistry, we investigated the localization of constitutive neuronal (nNOS) and endothelial (eNOS) enzyme isoforms along with caveolin-1 (CAV-1) and caveolin-3 (CAV-3) throughout the systemic vasculature and peripheral tissues of the hamster. The carotid artery, abdominal aorta, vena cava, femoral artery and vein, feed artery and collecting vein of the cheek pouch retractor muscle, capillaries and muscle fibers of retractor and cremaster muscles, and arterioles and venules of the cheek pouch were studied. In endothelial cells, eNOS and CAV-1 were present throughout the vasculature, whereas nNOS and CAV-3 were absent except in capillaries, which reacted for nNOS. In smooth muscle cells, nNOS and CAV-1 were also expressed systemically, whereas eNOS was absent; CAV-3 was present in the arterial but not the venous vasculature. Both nNOS and CAV-3 were located at the sarcolemma of skeletal muscle fibers, which were devoid of eNOS and CAV-1. These immunolabeling patterns suggest functional interactions between eNOS and CAV-1 throughout the endothelium, regional differences in the modulation of nNOS by caveolin isoforms in vascular smooth muscle, and modulation of nNOS by CAV-3 in skeletal muscle. (+info)