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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 obscurin IQ domain interacts with calmodulin in a Ca++-independent manner. The expressed and purified obscurin fragment Ob51–52, which contains the IQ motif, is shown in lane 1. The binding of this fragment to either calmodulin-coupled Sepharose beads (lanes 2, 4, and 6), or to control beads coupled with an unrelated protein (lanes 3, 5, and 7), was analyzed in a pulldown assay. The assay was carried out in the presence of 1 mM CaCl2 (lanes 2–5) or 1 mM EDTA (lanes 6 and 7). Bound protein was eluted from the beads with either 5 mM EDTA (lanes 2 and 3) or 6 M urea (lanes 4 to 7) and analyzed by SDS-PAGE and Coomassie blue staining. The obscurin fragment binds specifically to calmodulin both in the presence and absence of Ca++ and can be eluted under denaturing conditions (6 M urea), but not by chelating agents (EDTA). Elution by EDTA or urea is indicated by E or U, respectively.
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fig5: The obscurin IQ domain interacts with calmodulin in a Ca++-independent manner. The expressed and purified obscurin fragment Ob51–52, which contains the IQ motif, is shown in lane 1. The binding of this fragment to either calmodulin-coupled Sepharose beads (lanes 2, 4, and 6), or to control beads coupled with an unrelated protein (lanes 3, 5, and 7), was analyzed in a pulldown assay. The assay was carried out in the presence of 1 mM CaCl2 (lanes 2–5) or 1 mM EDTA (lanes 6 and 7). Bound protein was eluted from the beads with either 5 mM EDTA (lanes 2 and 3) or 6 M urea (lanes 4 to 7) and analyzed by SDS-PAGE and Coomassie blue staining. The obscurin fragment binds specifically to calmodulin both in the presence and absence of Ca++ and can be eluted under denaturing conditions (6 M urea), but not by chelating agents (EDTA). Elution by EDTA or urea is indicated by E or U, respectively.

Mentions: Ligands for the obscurin IQ domain were identified by a yeast two hybrid screen using domains 51–52 as bait. The IQ domain is immediately NH2-terminal to Ob52 and is preceded by ∼160 amino acids of nonmodular sequence. Six of seven clones from this screen were found to encode full-length calmodulin. To verify this interaction and to test for Ca2+ sensitivity of the interaction, binding of recombinant Ob51–52 was assayed on calmodulin-Sepharose beads in the presence and absence of Ca2+. The Ob51–52 fragment was found to bind to calmodulin-beads in a Ca2+-independent way (Fig. 5) . Control obscurin fragments show no calmodulin binding (not shown).


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 obscurin IQ domain interacts with calmodulin in a Ca++-independent manner. The expressed and purified obscurin fragment Ob51–52, which contains the IQ motif, is shown in lane 1. The binding of this fragment to either calmodulin-coupled Sepharose beads (lanes 2, 4, and 6), or to control beads coupled with an unrelated protein (lanes 3, 5, and 7), was analyzed in a pulldown assay. The assay was carried out in the presence of 1 mM CaCl2 (lanes 2–5) or 1 mM EDTA (lanes 6 and 7). Bound protein was eluted from the beads with either 5 mM EDTA (lanes 2 and 3) or 6 M urea (lanes 4 to 7) and analyzed by SDS-PAGE and Coomassie blue staining. The obscurin fragment binds specifically to calmodulin both in the presence and absence of Ca++ and can be eluted under denaturing conditions (6 M urea), but not by chelating agents (EDTA). Elution by EDTA or urea is indicated by E or U, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196875&req=5

fig5: The obscurin IQ domain interacts with calmodulin in a Ca++-independent manner. The expressed and purified obscurin fragment Ob51–52, which contains the IQ motif, is shown in lane 1. The binding of this fragment to either calmodulin-coupled Sepharose beads (lanes 2, 4, and 6), or to control beads coupled with an unrelated protein (lanes 3, 5, and 7), was analyzed in a pulldown assay. The assay was carried out in the presence of 1 mM CaCl2 (lanes 2–5) or 1 mM EDTA (lanes 6 and 7). Bound protein was eluted from the beads with either 5 mM EDTA (lanes 2 and 3) or 6 M urea (lanes 4 to 7) and analyzed by SDS-PAGE and Coomassie blue staining. The obscurin fragment binds specifically to calmodulin both in the presence and absence of Ca++ and can be eluted under denaturing conditions (6 M urea), but not by chelating agents (EDTA). Elution by EDTA or urea is indicated by E or U, respectively.
Mentions: Ligands for the obscurin IQ domain were identified by a yeast two hybrid screen using domains 51–52 as bait. The IQ domain is immediately NH2-terminal to Ob52 and is preceded by ∼160 amino acids of nonmodular sequence. Six of seven clones from this screen were found to encode full-length calmodulin. To verify this interaction and to test for Ca2+ sensitivity of the interaction, binding of recombinant Ob51–52 was assayed on calmodulin-Sepharose beads in the presence and absence of Ca2+. The Ob51–52 fragment was found to bind to calmodulin-beads in a Ca2+-independent way (Fig. 5) . Control obscurin fragments show no calmodulin binding (not shown).

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