Leiden Muscular Dystrophy pages

Caveolin-3

(last modified November 29, 2007)


Contents



Summary


Caveolae are small membrane invaginations on the surface of cells that participate in membrane trafficking, sorting, transport and signal transduction (including endocytosis and potocytosis). Caveolins are believed to play a role in the formation of the caveolae membranes, acting as scaffolding proteins organizing and concentrating caveolin-interacting proteins and lipids in caveolae microdomains (Couet, Lisanti).

Caveolin is a small molecular weight protein shown to be associated with the caveolar plasma membranes. Caveolin-3, or M-caveolin, was identified as a muscle-specific form of the caveolin family (Way, Tang). In skeletal muscle, caveolin-3 is localized to the sarcolemma, coinciding with dystrophin. Some caveolin-3 co-purifies with the dystrophin-associated glycoprotein complex and can be immunoprecipitated with dystrophin antibodies (Song, Ervasti, McNally), which suggests the existence of a discrete protein complex containing both proteins. However, Crosbie showed that CAV3 is not an integral member of the DGC, since its association is not as strong as that of other members. The number and size of caveolae are abnormal in DMD-patients. Caveolin-3 directly interacts with neuronal nitric oxide synthase (nNOS) and binding results in the loss of NOS activity (Venema...).


The caveolin-3 gene


Links to other databases:
Gene Symbol nomenclature   LocusLink    OMIM Gene Map   GDB

The human caveolin gene (Gene Symbol CAV3, alternative names M-cavelin, LGMD1C, VIP21, RMD) was cloned by Minetti et al. using a mouse Cav3 cDNA. Using FISH with a YAC containing the gene (CEPH773C11 or 887B5), CAV3 could be mapped to human chromosome 3p25 (Minetti, McNally). The CAV3-gene contains two exons (McNally) and spans 12 kb of genomic DNA. Sequence database comparisons show that the second exon is differentially spliced in the 3'UTR splitting it into two seperate exons and removing 96 bp of sequence (den Dunnen, unpublished). 

To be added; study of Biederer et al., promoter analysis, JBiolChem 2000 275: 26245-26251.

Exon Exon size (bp) Intron size (bp) 5' cDNA position Splice after Remarks
1 181 11,535 -67 0 5'UTR / 114 bp coding
2
(2a/2b)
1236 -
(96)
115   338 bp coding/ 3'UTR;
differential splicing occurs in this exon, splicing out 96 bp from the 3'UTR (position c.*7-*102 in the coding DNA Reference Sequence)

Legend:
Exon: numbering of exons and intron/exon boundaries are according to McNally with the first base of the Met-codon counted as position 1 (see coding DNA Reference Sequence). Exon size: size of exon indicated in basepairs. Intron size: size of intron indicated in kilobasepairs. 5' cDNA position: first base of the exon (according to cDNA sequence reported by Minetti / McNally). Splice after: splicing occurs in between of two coding triplets (0), after the first (1) or the second (2) base of a triplet. Remarks: 5'UTR = 5' untranslated region, 3'UTR = 3' untranslated region.

primers for DNA-amplification


The caveolin-3 mRNA


Links to other databases: UniGene: Hs.98303   RefSeq: NM_001234

On Northern-blot, caveolin-3 RNA expression was detected exclusively in cardiac and skeletal muscle and not in brain, kidney, liver, lung, pancreas and placenta (McNally, Minetti). The transcript measures about 1.5 kb. The CAV3 cDNA contains a 453 bp open reading frame.

primers for RNA-amplification


The caveolin-3 protein


Links to other databases: RefSeq: NP_001225  PFAM: PF01146

Caveolin-3, or M-caveolin, is a 151 amino acid protein in which three seperate segments can be identified; an N- region (amino acid 1-73), a central hydrophobic transmembrane domain (aa 75-106 PFAM:01146) and a C-terminal (aa 107-151) domain. This transmembrane domain is believed to form a hairpin loop structure in the cell membrane, allowing both the N- and C-terminal ends to face the cytoplasm (both extremities are known to face the cytoplasm). The N-terminal domain contains a caveolin signature sequence (aa 41-48, FEDVIAEP present in all caveolins) and a  scaffolding domain (aa 55-74) known to bind various signaling proteins. The N-terminal region is responsible for the homo-oligomerization and the interaction with caveolin-associated signalling molecules.

Currently, three different caveolins are known: caveolin-1 (or VIP21), caveolin-2 and caveolin-3 (or M-caveolin). Caveolin-3, or M-caveolin, is expressed in muscle, caveolin-1 and caveolin-2 not. Caveolins are proteins of about 20 Kd, they form high molecular mass homo-oligomers. Structurally they all have N-terminal and C-terminal hydrophilic segments and a long central transmembrane domain. They are a family of integral membrane proteins which are the principal components of caveolae membranes. Cavoleae are flask-shaped plasma membrane invaginations whose exact cellular function is not yet clear. Caveolins may act as scaffolding proteins within caveolar membranes. They interact directly with G-protein alpha subunits and can functionally regulate their activity.

Similarity to other proteins

Multiple protein sequence alignment for Caveolin-3

Human caveolin-3 is 95% identical to the mouse and rat caveolin-3 protein sequences. Human caveolin-3 shows 82% and 58% similarity with human caveolin-1 and -2 resp. A comparison between mammalian caveolins and C. elegans CAV-1 and CAV-2 reveals twelve absolutely conserved amino acid residues.

Caveolin-3 function

During caveolae formation, caveolin undergoes two stages of oligomerization. Shortly after synthesis, 300-350 kDa homo-oligomers are formed in the endoplasmic reticulum, each containing 14-16 caveolin monomers. At a later stage the homo-oligomers interact with each other and form clusters of some 25-50 nm in diameter.

In skeletal muscle, caveolin-3 is localized to the sarcolemma, coinciding with dystrophin. Some caveolin-3 co-purifies with the dystrophin-associated glyco-protein complex and is immunoprecipitated with dystrophin antibodies (Song, Ervasti, McNally), which suggests the existence of a discrete protein complex containing both proteins. However, Crosbie showed that CAV3 is not an integral member of the DGC, since its association is not as strong as that of other members. Caveolin-3 directly interacts with neuronal nitric oxide synthase (nNOS) and binding results in the loss of NOS activity (Venema...).

Caveolin-3 antibodies

Song et al. describe a caveolin-3 monoclonal antibody (cl 26) generated in mice against a synthetic peptide corresponding to amino acids 3-24 of rat CAV-3. The antibody gives clear staining in immunohistochemistry and on Western blot.


Caveolin-3 and disease: LGMD-1C, hyperCKemia, RMD


Links to other databases:   OMIM: 607801, 123320 and 606072

Sequence variations in the CAV3-gene were found by Minetti et al. upon analysis of muscle biopsy samples using caveolin-3 antibodies. The muscle biopsy samples were derived from a series of muscular dystrophy patients, 44 with a dystrophinopathy, 12 with a sarcoglycanopathy and 137 with Limb-Girdle muscular dystrophy (LGMD), including 12 dominant cases. Reduced caveolin-3 staining was found in 8 patients from two different families and could be confirmed in Western blot analysis; expression was reduced by 90-95%. Other membrane proteins involved in muscular dystrophies, like dystrophin, the sarcoglycans and merosin, were present at normal levels in these patients. The phenotype in these patients was designated LGMD type 1C.

The clinical features of the LGMD-1C patients reported (Minetti) involved calf hypertrophy and mild to moderate proximal muscle weakness. Histological and histochemical studies revealed only non-specific changed of moderate severity. Calveolin-3 staining was reduced 90-95%. In DMD-patients, the number and size of caveolae are abnormal.

Using RNA analysis (RT-PCR in combination with direct sequencing), Minetti et al. identified in two families two different heterozygous variations; a 9 bp (3 amino acid) deletion and a transition leading to an amino acid substitution, both involving an amino acid which is 100% conserved in all known caveolins (from man to worm). Given the fact that caveolin-3 forms homo-oligomers, these variations probably exert a dominant negative effect leading to a degradation of both variant and wild-type protein.

McNally analysed a set of 82 muscular dystrophy patients of unknown genetic etiology with a normal dystrophin immunostaining pattern on a muscle biopsy and/or normal in dystrophin multiplex PCR. One female had a homozygous 166G>A (Gly56Ser) missense change, another patient carried a 216C>G (Cys72Trp) variation on one allele. Since in the latter case the mother and two other siblings carried the same allele, inheritance was clearly autosomal dominant. A muscle biopsy staining of the 216C>G patient with dystrophin, caveolin-3 and alpha-, beta- and gamma-sarcoglycan appeared sligthly patchy.

Changes in the CAV3 gene may also cause other disease phenotypes. First, Carbone et al. reported a novel sporadic mutation in the CAV3 gene in two unrelated children with persistent elevated levels of serum creatine kinase (hyperCKemia) without muscle weakness; 80G>A (Arg27Gln). Immunohistochemistry and quantitative immunoblot analysis of caveolin-3 showed reduced expression of the protein in muscle fibers. Second, Betz et al. detected CAV3 mutations in hereditary rippling muscle disease (RMD), an autosomal dominant human disorder characterized by mechanically triggered contractions of skeletal muscle. Using a genome-wide linkage analysis they mapped an RMD locus to chromosome 3p25 and identified missense mutations in the positional candidate gene CAV3 in all five German families analyzed. Intriguingly, identical mutations can cause RMD/hyperCKemia or LGMD1C/RMD, i.e. a phenotype with and without muscle weakness !.

caveolin-3 sequence variations - mutations and polymorphisms


Miscellaneous




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