Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells.

The cytoplasmic domains of integrins provide attachment of these extracellular matrix receptors to the cytoskeleton and play a critical role in integrin-mediated signal transduction. In this report we describe the identification, expression, localization, and initial functional characterization of a novel form of beta 1 integrin, termed beta 1D. This isoform contains a unique alternatively spliced cytoplasmic domain of 50 amino acids, with the last 24 amino acids encoded by an additional exon. Of these 24 amino acids, 11 are conserved when compared to the beta 1A isoform, but 13 are unique (Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Biochem. Biophys. Res. Commun. 214:279-285; van der Flier, A., I. Kuikman, C. Baudoin, R, van der Neuf, and A. Sonnenberg. 1995. FEBS Lett. 369:340-344). Using an anti-peptide antibody against the beta 1D integrin subunit, we demonstrated that the beta 1D isoform is synthesized only in skeletal and cardiac muscles, while very low amounts of beta 1A were detected by immunoblot in striated muscles. Whereas beta 1A could not be detected in adult skeletal muscle fibers and cardiomyocytes by immunofluorescence, beta 1D was localized to the sarcolemma of both cell types. In skeletal muscle, beta 1D was concentrated in costameres, myotendinous, and neuromuscular junctions. In cardiac muscle this beta 1 isoform was found in costamers and intercalated discs. beta 1D was associated with alpha 7A and alpha 7B in adult skeletal muscle. In cardiomyocytes of adult heart, alpha 7B was the major partner for the beta 1D isoform. beta 1D could not be detected in proliferating C2C12 myoblasts, but it appeared immediately after myoblast fusion and its amount continued to rise during myotube growth and maturation. In contrast, expression of the beta 1A isoform was downregulated during myodifferentiation in culture and it was completely displaced by beta 1D in mature differentiated myotubes. We also analyzed some functional properties of the beta 1D integrin subunit. Expression of human beta 1D in CHO cells led to its localization at focal adhesions. Clustering of this integrin isoform on the cell surface stimulated tyrosine phosphorylation of pp125FAK (focal adhesion kinase) and caused transient activation of mitogen-activated protein (MAP) kinases. These data indicate that beta 1D and beta 1A integrin isoforms are functionally similar with regard to integrin-mediated signaling.

Molecular cloning of PARP (proline/arginine-rich protein) from human cartilage and subsequent demonstration that PARP is a fragment of the NH2-terminal domain of the collagen alpha 2(XI) chain.

We report the molecular cloning of a proline/arginine-rich protein (called PARP) from human cartilage using the polymerase chain reaction (PCR) and degenerate oligonucleotides based on the previously published amino acid sequence of bovine PARP [1]. Subsequently, a reverse transcription-polymerase chain reaction (RT-PCR) was performed with poly(A)-rich RNA from human cartilage using a sense oligonucleotide derived from PARP and an anti-sense oligonucleotide derived from the known sequence of the human collagen alpha 2(XI) chain [2]. Nucleotide sequencing of the PCR product demonstrated that PARP is a fragment of the NH2-terminal non-collagenous (NC3) domain of the collagen alpha 2(XI) chain.

Localization of talin in skeletal and cardiac muscles.

Antibodies to talin and vinculin were used for localization of these proteins in skeletal and cardiac muscles by the indirect immunofluorescence method. We have found that talin is localized in intercalated discs of cardiac muscle and in costameres of skeletal and cardiac muscles. It is suggested that in striated muscles talin and vinculin play an important role in interactions between actin filaments and membranes.

Meta-vinculin distribution in adult human tissues and cultured cells.

Meta-vinculin distribution in adult human tissue was studied by immunoblotting technique. Meta-vinculin was found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, kidney and cultured cells - macrophages, foreskin fibroblasts, peripheral blood lymphocytes and vascular endothelial cells. In the primary culture of smooth muscle cells from human aorta the meta-vinculin/vinculin ratio was reduced, and on the onset of cell division meta-vinculin could hardly be detected. Subcultured smooth muscle cells from human aorta did not contain meta-vinculin. The data show that the presence of meta-vinculin is characteristic of 'contractile' smooth muscle cells rather than of proliferating in vitro.

[Comparative study of the binding of human blood plasma fibronectin with clinical strains of staphylococci]. / Sravnitel'noe izuchenie sviazyvaniia fibromnektina plazmy krovi cheloveka s klinicheskimi shtammami stafilokokkov.

The interaction of 62 S. aureus clinical strains and, respectively, 20 and 17 isolated S. epidermidis and S. saprophyticus strains with human blood plasma fibronectin (FN) has been studied. The specific interaction of FN with bacteria has been evaluated simultaneously by the binding of 125I with FN (method 1), the FN-mediated agglutination of staphylococci (method 2) and the character of colonies formed in 0.15% agar medium containing FN (method 3). The data obtained in this investigation indicated that all S. aureus strains under study react with FN to a different extent. When evaluating the binding of FN with bacteria, the most pronounced correlation was observed between methods 1 and 3. None of the methods used in this investigation has revealed interaction between FN and S. epidermidis and S. saprophyticus strains under study. The authors suggest that a preliminary inference on the capacity of the isolated clinical strains of staphylococci for reaction with FN may be made from the character of colonies formed in 0.15% agar medium containing FN.

Novel isoform of beta 1 integrin expressed in skeletal and cardiac muscle.

We describe a novel isoform of the beta 1 integrin subunit called beta 1D, which contains a unique cytoplasmic domain, and is expressed specifically in skeletal and cardiac muscle. The beta 1D isoform arises from splicing into the final transcript of an additional exon located between exons 6 and 7. The nucleotide sequence of beta 1D predicts a cytoplasmic domain of 50 amino acids in which the last 21 amino acids of the beta 1A integrin isoform are replaced by a related sequence of 24 amino acids. A beta 1D-specific anti-peptide polyclonal antibody was prepared and immunoprecipitation of tissue extracts with subsequent immunoblotting showed expression of beta 1D isoform only in striated muscle cells.

Alternative mRNA processing occurs in the variable region of the pro-alpha 1(XI) and pro-alpha 2(XI) collagen chains.

An analysis was performed of differential splicing of primary transcripts in the noncollagenous variable region located in the amino terminus of the pro-alpha 1(XI) and pro-alpha 2(XI) collagen chains. The results for the pro-alpha 2(XI) chain showed that human cartilage or fibroblasts in culture contain transcripts in which a single highly acidic exon encoding for 21 amino acids is present or absent. For the chicken pro-alpha 1(XI) chain a more complex pattern of alternative splicing was detected with six possible variants. Of special interest was the alternative use of two exons (called IIA and IIB) in which IIA encodes for 39 amino acids and is highly acidic (estimated pI = 3.2), whereas IIB encodes for 49 amino acids and is highly basic (estimated pI = 10.6). A similar alternative use of exon IIA or exon IIB was also observed for human chondrocytes. Northern blotting with probes specific for IIA or IIB showed that both exons are present in transcripts from cartilage but exon IIB is preferentially utilized in transcripts from tendon. Present results suggest that both the pro-alpha 1(XI) and pro-alpha 2(XI) chains of type XI collagen undergo limited processing in vivo and that the noncollagenous variable region is initially retained on the surface of the fibrils. Differential splicing in the variable region may potentially influence the interaction of collagen fibrils with other molecules of the extracellular matrix and more specifically with sulfated glycosaminoglycan chains or with hyaluronan. Such interactions may play a key role in establishing both the organization of the collagen fibrils within the extracellular matrix and in limiting the diameter of collagen fibrils.

Organization of the human gene encoding the cytoskeletal protein vinculin and the sequence of the vinculin promoter.

The human vinculin gene contains 22 exons ranging in size from 71 base pairs (bp) to 303 bp (average 155 bp) with the exception of exon 22 which contains 144 bp of coding sequence and 1848 bp of 3'-untranslated sequence including two polyadenylation signals. There is a limited correlation between exon boundaries and functional domains within the vinculin molecule. The talin-binding domain in vinculin spans residues 1-258, and the first 6 exons encode residues 1-261. Similarly, the predicted boundaries of the central repeat domain (residues 259-589) are close to the boundaries of exons 7 and 12. Analysis of vinculin mRNAs in human uterus showed that alternative splicing of the gene is limited to exon 19, which encodes the 68 amino acids included in the muscle-specific isoform called metavinculin. We have determined 1.1 kilobases of sequence 5' of the transcription start site. The vinculin promoter lacks a TATA box, but does contain six Sp1 sites, and a CArG box at position -262 which forms the core of the serum response element found in immediate-early response genes. Expression of a vinculin promoter/CAT construct is serum-inducible in NIH3T3 cells demonstrating that the promoter does contain a functional serum response element.

An additional exon in the human vinculin gene specifically encodes meta-vinculin-specific difference peptide. Cross-species comparison reveals variable and conserved motifs in the meta-vinculin insert.

We have analyzed the structure, origin and expression of the high-molecular-mass muscle-specific variant of vinculin, called meta-vinculin. The meta-vinculin-specific inserts from the human and avian molecules have been isolated and sequenced and the sequences confirmed via cloning of the corresponding cDNA. Comparison of the human, avian and determined porcine sequences revealed cross-species identity in the C-terminal half of the insert. Human and porcine meta-vinculin were highly similar in the insert region, showing only five amino acid exchanges; avian meta-vinculin showed 22 exchanges in the same region compared to human meta-vinculin and exhibited, in addition, one extra amino acid, making 69 in all. Each insert was flanked by characteristic KWSSK motifs. Evidence for two vinculin mRNA species in human uterus smooth muscle was provided by reverse transcription combined with the polymerase chain reaction, as well as by ribonuclease-mapping analysis of cDNA/mRNA hybrids. One of the mRNA species contained an additional 204-nucleotide insert that precisely encoded the meta-vinculin-specific peptide. Sequence analysis of the appropriate portion of the human vinculin gene showed that the section coding for the meta-vinculin-specific insert is present as a discrete exon. Thus, meta-vinculin and vinculin mRNA are generated by alternative splicing.

Complete sequence of human vinculin and assignment of the gene to chromosome 10.

We have determined the complete sequence of human vinculin, a cytoskeletal protein associated with cell-cell and cell-matrix junctions. Comparison of human and chicken embryo vinculin sequences shows that both proteins contain 1066 amino acids and exhibit a high level of sequence identity (greater than 95%). The region of greatest divergence falls within three 112-amino acid repeats spanning residues 259-589. Interestingly, nematode vinculin lacks one of these central repeats. The regions of human vinculin that are N- and C-terminal to the repeats show 54% and 61% sequence identity, respectively, to nematode vinculin. Southern blots of human genomic DNA hybridized with short vinculin cDNA fragments indicate that there is a single vinculin gene. By using a panel of human-rodent somatic cell hybrids, the human vinculin gene was mapped to chromosome 10q11.2-qter.

Autosomal dominant and recessive osteochondrodysplasias associated with the COL11A2 locus.

Identifying mutations that cause specific osteochondrodysplasias will provide novel insights into the function of genes that are essential for skeletal morphogenesis. We report here that an autosomal dominant form of Stickler syndrome, characterized by mild spondyloepiphyseal dysplasia, osteoarthritis, and sensorineural hearing loss, but no eye involvement, is caused by a splice donor site mutation resulting in "in-frame" exon skipping within the COL11A2 gene, encoding the alpha 2(XI) chain of the quantitatively minor fibrillar collagen XI. We also show that an autosomal recessive disorder with similar, but more severe, characteristics is linked to the COL11A2 locus and is caused by a glycine to arginine substitution in alpha 2(XI) collagen. The results suggest that mutations in collagen XI genes are associated with a spectrum of abnormalities in human skeletal development and support the conclusion of others, based on studies of murine chondrodysplasia, that collagen XI is essential for skeletal morphogenesis.