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Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Young P, Ehler E, Gautel M - J. Cell Biol. (2001)

Bottom Line: It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle.Both proteins coassemble during myofibrillogenesis.The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Structural Biology Division, 69117 Heidelberg, Germany.

ABSTRACT
Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

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The cDNA and genomic sequence of obscurin. (A) Positions of isolated obscurin cDNAs. A total of 20,435 bp of continuous cDNA sequence can be assembled from the depicted overlapping cDNAs. This cDNA sequence contains an ORF encoding 6,620 amino acids. (B) Domain pattern of obscurin. The deduced amino acid sequence predicts a protein of 721,685 kD. Within this sequence, known protein domains were identified by database searches and visual inspection. Obscurin is a polar molecule with signal transduction domains in the COOH-terminal region and repetitive tandem Ig domains NH2-terminal to the signaling region. The Ig and Fn3 modules are numbered consecutively from the NH2 terminus. Ig, immunoglobulin-like; IQ, IQ calmodulin binding motif. One shorter alternatively spliced variant was sequenced and the extra Ig domain from this splice variant is denoted a1. The positions of the epitopes for antibodies used in this study are indicated above the domain structure. (C) Partial structure of the obscurin gene. A >150-kb obscurin genomic sequence was compiled by analyzing the draft human genome sequence. Exons are indicated as vertical black lines with the position of the exons encoding selected domains indicated above. Putative exons encoding 10 Ig domains which are not present in the cDNA sequence are marked by an asterisk. Long vertical grey lines indicate gaps of undefined length in the sequence.
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fig1: The cDNA and genomic sequence of obscurin. (A) Positions of isolated obscurin cDNAs. A total of 20,435 bp of continuous cDNA sequence can be assembled from the depicted overlapping cDNAs. This cDNA sequence contains an ORF encoding 6,620 amino acids. (B) Domain pattern of obscurin. The deduced amino acid sequence predicts a protein of 721,685 kD. Within this sequence, known protein domains were identified by database searches and visual inspection. Obscurin is a polar molecule with signal transduction domains in the COOH-terminal region and repetitive tandem Ig domains NH2-terminal to the signaling region. The Ig and Fn3 modules are numbered consecutively from the NH2 terminus. Ig, immunoglobulin-like; IQ, IQ calmodulin binding motif. One shorter alternatively spliced variant was sequenced and the extra Ig domain from this splice variant is denoted a1. The positions of the epitopes for antibodies used in this study are indicated above the domain structure. (C) Partial structure of the obscurin gene. A >150-kb obscurin genomic sequence was compiled by analyzing the draft human genome sequence. Exons are indicated as vertical black lines with the position of the exons encoding selected domains indicated above. Putative exons encoding 10 Ig domains which are not present in the cDNA sequence are marked by an asterisk. Long vertical grey lines indicate gaps of undefined length in the sequence.

Mentions: Starting with the 750 bp of obscurin cDNA sequence from positive yeast two hybrid clones, a lambda phage cardiac cDNA library was screened in order to extend this sequence in both directions. Multiple rounds of screening yielded a panel of 18 cDNA clones which can be assembled into a 20.4 kb contig (Fig. 1 A). This cDNA encodes an ORF of 19,860 bp. The ORF is preceded by an in-frame stop codon 27-bp upstream of a putative start methionine. The sequence preceding this methionine codon fits to the Kozak sequence found around the start methionine of eukaryotic genes (Kozak, 1989). The first domain starts nine amino acids after the putative start methionine. At the 3′ end of the ORF there is an in-frame stop codon followed by 500 bp of predicted 3′ untranslated sequence which contains stop codons in all reading frames. No consensus polyadenylation signal was identified, although the 3′ untranslated region shows perfect homology to several entries from the EST database which were cloned using oligo dT primers. The ORF is predicted to encode a 720-kD protein of 6,620 amino acids. One shorter splice variant was sequenced (termed obscurin-a1). Attempts to further characterize alternative splice variants by reverse transcriptase PCR failed because of the low abundance of the obscurin message and the extreme homology over most of the tandem Ig-regions (see below).


Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Young P, Ehler E, Gautel M - J. Cell Biol. (2001)

The cDNA and genomic sequence of obscurin. (A) Positions of isolated obscurin cDNAs. A total of 20,435 bp of continuous cDNA sequence can be assembled from the depicted overlapping cDNAs. This cDNA sequence contains an ORF encoding 6,620 amino acids. (B) Domain pattern of obscurin. The deduced amino acid sequence predicts a protein of 721,685 kD. Within this sequence, known protein domains were identified by database searches and visual inspection. Obscurin is a polar molecule with signal transduction domains in the COOH-terminal region and repetitive tandem Ig domains NH2-terminal to the signaling region. The Ig and Fn3 modules are numbered consecutively from the NH2 terminus. Ig, immunoglobulin-like; IQ, IQ calmodulin binding motif. One shorter alternatively spliced variant was sequenced and the extra Ig domain from this splice variant is denoted a1. The positions of the epitopes for antibodies used in this study are indicated above the domain structure. (C) Partial structure of the obscurin gene. A >150-kb obscurin genomic sequence was compiled by analyzing the draft human genome sequence. Exons are indicated as vertical black lines with the position of the exons encoding selected domains indicated above. Putative exons encoding 10 Ig domains which are not present in the cDNA sequence are marked by an asterisk. Long vertical grey lines indicate gaps of undefined length in the sequence.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196875&req=5

fig1: The cDNA and genomic sequence of obscurin. (A) Positions of isolated obscurin cDNAs. A total of 20,435 bp of continuous cDNA sequence can be assembled from the depicted overlapping cDNAs. This cDNA sequence contains an ORF encoding 6,620 amino acids. (B) Domain pattern of obscurin. The deduced amino acid sequence predicts a protein of 721,685 kD. Within this sequence, known protein domains were identified by database searches and visual inspection. Obscurin is a polar molecule with signal transduction domains in the COOH-terminal region and repetitive tandem Ig domains NH2-terminal to the signaling region. The Ig and Fn3 modules are numbered consecutively from the NH2 terminus. Ig, immunoglobulin-like; IQ, IQ calmodulin binding motif. One shorter alternatively spliced variant was sequenced and the extra Ig domain from this splice variant is denoted a1. The positions of the epitopes for antibodies used in this study are indicated above the domain structure. (C) Partial structure of the obscurin gene. A >150-kb obscurin genomic sequence was compiled by analyzing the draft human genome sequence. Exons are indicated as vertical black lines with the position of the exons encoding selected domains indicated above. Putative exons encoding 10 Ig domains which are not present in the cDNA sequence are marked by an asterisk. Long vertical grey lines indicate gaps of undefined length in the sequence.
Mentions: Starting with the 750 bp of obscurin cDNA sequence from positive yeast two hybrid clones, a lambda phage cardiac cDNA library was screened in order to extend this sequence in both directions. Multiple rounds of screening yielded a panel of 18 cDNA clones which can be assembled into a 20.4 kb contig (Fig. 1 A). This cDNA encodes an ORF of 19,860 bp. The ORF is preceded by an in-frame stop codon 27-bp upstream of a putative start methionine. The sequence preceding this methionine codon fits to the Kozak sequence found around the start methionine of eukaryotic genes (Kozak, 1989). The first domain starts nine amino acids after the putative start methionine. At the 3′ end of the ORF there is an in-frame stop codon followed by 500 bp of predicted 3′ untranslated sequence which contains stop codons in all reading frames. No consensus polyadenylation signal was identified, although the 3′ untranslated region shows perfect homology to several entries from the EST database which were cloned using oligo dT primers. The ORF is predicted to encode a 720-kD protein of 6,620 amino acids. One shorter splice variant was sequenced (termed obscurin-a1). Attempts to further characterize alternative splice variants by reverse transcriptase PCR failed because of the low abundance of the obscurin message and the extreme homology over most of the tandem Ig-regions (see below).

Bottom Line: It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle.Both proteins coassemble during myofibrillogenesis.The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Structural Biology Division, 69117 Heidelberg, Germany.

ABSTRACT
Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

Show MeSH
Related in: MedlinePlus