Published July 9, 2001 JCBArticle Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly Paul Young,1 Elisabeth Ehler,2 and Mathias Gautel3 1European Molecular Biology Laboratory, Structural Biology Division, 69117 Heidelberg, Germany 2Institute of Cell Biology, Hönggerberg, CH-8093 Zürich, Switzerland 3Department of Physical Biochemistry, Max-Planck Institute for Molecular Physiology, 44202 Dortmund, Germany ertebrate-striated muscle is assumed to owe its re- scurin shows a modular architecture of tandem Ig domains markable order to the molecular ruler functions of reminiscent of the elastic region of titin. The COOH-terminal V the giant modular signaling proteins, titin and nebu- region of obscurin interacts via two specific Ig-like domains lin. It was believed that these two proteins represented with the NH2-terminal Z-disk region of titin. Both proteins unique results of protein evolution in vertebrate muscle. In coassemble during myofibrillogenesis. During the progres- this paper we report the identification of a third giant protein sion of myofibrillogenesis, all obscurin epitopes become de- from vertebrate muscle, obscurin, encoded on chromosome tectable at the M band. The presence of a calmodulin-binding Downloaded from -kD and is expressed specifically in IQ motif, and a Rho guanine nucleotide exchange factor do 800ف 1q42. Obscurin is skeletal and cardiac muscle. The complete cDNA sequence main in the COOH-terminal region suggest that obscurin is of obscurin reveals a modular architecture, consisting of involved in Ca2ϩ/calmodulin, as well as G protein–coupled Ͼ67 intracellular immunoglobulin (Ig)- or fibronectin-3–like signal transduction in the sarcomere. domains with multiple splice variants. A large region of ob- on May 24, 2017 Introduction Sarcomeres, the smallest contractile units of striated muscles, The process of myofibril assembly requires both spatial are assembled from thousands of protein subunits into the and temporal coordination of protein interactions with high largest and most regular macromolecular complex known. precision (Gautel et al., 1999). To achieve this long-range Sarcomeres are assembled during the embryonic differentia- coordination, two giant modular proteins, acting as molec- tion of heart and skeletal muscle, but also on a continuous ular scaffolds or blueprints, are found in vertebrate mus- basis during the physiological turnover of muscle. New sar- cle. Titin (Wang et al., 1979), also known as connectin, comeres are also formed at a high rate in hypertrophying (Maruyama, 1976) and nebulin provide specific attachment muscle: either as a result of exercise, increased pressure and sites for other proteins and thus specify their sarcomeric po- volume load of the heart, or pathological or hormonal stim- sitions (Trinick, 1996; Trinick and Tskhovrebova, 1999). ulation. The mechanisms which cooperate to regulate mus- Recently, it was shown that the deletion of titin leads to a to- cle-specific gene transcription are only beginning to emerge tal loss of myofibril assembly despite the persisting ex- (Chien, 2000). It remains largely unclear how signaling at pression of other sarcomeric proteins (Van der Ven et al., the molecular level within the sarcomere and the control of 2000). Apart from binding sites for other sarcomeric pro- assembly are coordinated. Therefore, identifying and charac- teins, these giant proteins contain potential signaling domains: terizing key elements of sarcomeric signal transduction and a COOH-terminal Src homology 3 (SH3)* domain in neb- their roles in the control of myofibrillogenesis are essential to ulin (Labeit and Kolmerer, 1995a), and multiple phosphor- elucidate basic mechanisms of the cell biology of muscle, ylation sites and a COOH-terminal catalytic protein kinase leading to a molecular understanding of associated diseases. domain in titin implicated in myofibril assembly (Mayans et al., 1998). These domains suggest that the molecular scaf- Address correspondence to Dr. Mathias Gautel, Max-Planck Institute for fold proteins of the myofibril receive and propagate signals Molecular Physiology, Department of Physical Biochemistry, Postfach from various pathways. 500 247, 44202 Dortmund, Germany. Tel.: (49) 231-133-2354. Fax: (49) 231-133-2399. E-mail: [email protected] *Abbreviations used in this paper: DH, dbl homology; E, embryo day Paul Young’s present address is Department of Neurobiology, Duke Uni- (gestation days); Fn, fibronectin; GEF, guanine nucleotide exchange fac- versity Medical Center, Durham, NC 27710. tor; PH, pleckstrin homology; PI3, phosphatidyl inositol 3; PiP, phos- Key words: obscurin; Rho GTPases; GEF proteins; myofibril; heart muscle phatidylinositol-phosphates; SH3, Src homology 3. The Rockefeller University Press, 0021-9525/2001/07/123/14 $5.00 The Journal of Cell Biology, Volume 154, Number 1, July 9, 2001 123–136 http://www.jcb.org/cgi/doi/10.1083/jcb.200102110 123 Published July 9, 2001 124 The Journal of Cell Biology | Volume 154, 2001 Nematodes contain two large muscle proteins, encoded Results kb ORF-20ف by the unc-22 and unc-89 genes in Caenorhabditis elegans. An obscurin cDNA sequence contains an The unc-22 product is twitchin, which is localized along We have identified obscurin as a novel titin interacting pro- the myosin filament and shows homology to titin (Benian tein in a yeast two hybrid screen (see below). Searches of the et al., 1989). Unc-89 has been implicated in the assembly EMBL and GenBank databases with this sequence retrieved of the sarcomeric M band (Benian et al., 1996); a mamma- no identical cDNA sequence, suggesting the identification lian homologue of unc-89 has not been identified to date. of a novel protein. We have named this protein obscurin, af- Titin, twitchin, and unc-89 are all at least partly associated ter the adjective obscure, defined in the New Oxford Dictio- with the myosin filament. These proteins share a similar nary as meaning: (a) difficult to see or make out, (b) not well molecular architecture, being largely composed of 100-resi- known, or (c) not easily understood. All three meanings are due domains of the intracellular Ig superfamily, and also appropriate to obscurin, which has not been identified pre- contain domains involved in signal transduction (Benian et viously and has proven difficult to characterize because of its al., 1989, 1996; Labeit et al., 1992; Heierhorst et al., complexity, large size, and relatively low abundance. 1994). Titin and twitchin contain a myosin light chain ki- Starting with the 750 bp of obscurin cDNA sequence from nase–like protein kinase domain which has been implicated positive yeast two hybrid clones, a lambda phage cardiac in the control of myofibril formation in titin (Mayans et al., cDNA library was screened in order to extend this sequence 1998), whereas unc-89 contains a G protein–activating in both directions. Multiple rounds of screening yielded a GDP/GTP exchange factor domain (GEF domain; Benian panel of 18 cDNA clones which can be assembled into a 20.4 et al., 1996). kb contig (Fig. 1 A). This cDNA encodes an ORF of 19,860 Among the giant proteins, the complex modular architecture bp. The ORF is preceded by an in-frame stop codon 27-bp of titin is probably the one best understood at the functional upstream of a putative start methionine. The sequence pre- level, and may therefore serve as a paradigm for the analysis of ceding this methionine codon fits to the Kozak sequence other large modular proteins. Apart from Ig-like domains, titin Downloaded from found around the start methionine of eukaryotic genes contains unique sequences which are involved in signal trans- (Kozak, 1989). The first domain starts nine amino acids after duction or interactions with other sarcomeric proteins (Gautel the putative start methionine. At the 3Ј end of the ORF there et al., 1999). Specific titin domains interact with myomesin, is an in-frame stop codon followed by 500 bp of predicted 3Ј myosin, myosin-binding protein C, ␣-actinin, and telethonin untranslated sequence which contains stop codons in all along the distance from M-line to Z-disk (Gautel et al., 1999; reading frames. No consensus polyadenylation signal was Gregorio et al., 1999; Trinick and Tskhovrebova, 1999; Sanger identified, although the 3Ј untranslated region shows perfect et al., 2000). These interactions specify the sarcomeric localiza- on May 24, 2017 homology to several entries from the EST database which tion of these other protein components and define the length were cloned using oligo dT primers. The ORF is predicted to of myosin filaments or the thickness of the Z-disk (Gautel et encode a 720-kD protein of 6,620 amino acids. One shorter al., 1999). In the I-band, titin is composed of Ig domains ar- splice variant was sequenced (termed obscurin-a1). Attempts ranged in tandem and a unique sequence, the so-called PEVK to further characterize alternative splice variants by reverse region (Labeit and Kolmerer, 1995b). These two secondary transcriptase PCR failed because of the low abundance of the structure elements act as serial springs and are responsible for obscurin message and the extreme homology over most of the passive elasticity of muscle (Gautel and Goulding, 1996; the tandem Ig-regions (see below). Linke et al., 1996), with the tandem Ig stretch being largely re- sponsible for extension at low forces and resisting stretch at higher forces (Trinick, 1996). Obscurin is a modular protein built from adhesion