Characterization of a Gene Coamplified with Ki-ras in Y1 Murine Adrenal Carcinoma Cells That Codes for a Putative Membrane Protein

Scott, Alan F.; Elizaga, Andrew; Morrell, James C.; Bergen, Andrew W.; Penno, Margaret B.

doi:10.1006/geno.1994.1157

Cited by 8 publications

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“…This analysis revealed a homology between peptide I and Kirsten ras-associated gene (KRAG) from human and mouse (accession numbers X89105 and U02487, respectively). Murine KRAG was first identified based on coamplification of its transcript with Kirsten ras in Y1 adrenal carcinoma cells (17). Similarly, the human KRAG gene was also identified through analysis of transcripts co-amplified with KRAS2 in lung and ovarian carcinoma cells (13).…”

Section: Resultsmentioning

confidence: 99%

Sarcospan, the 25-kDa Transmembrane Component of the Dystrophin-Glycoprotein Complex

Crosbie

Heighway

Venzke

et al. 1997

Journal of Biological Chemistry

178

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The dystrophin-glycoprotein complex is a multisubunit protein complex that spans the sarcolemma and forms a link between the subsarcolemmal cytoskeleton and the extracellular matrix. Primary mutations in the genes encoding the proteins of this complex are associated with several forms of muscular dystrophy. Here we report the cloning and characterization of sarcospan, a unique 25-kDa member of this complex. Topology algorithms predict that sarcospan contains four transmembrane spanning helices with both N-and C-terminal domains located intracellularly. Phylogenetic analysis reveals that sarcospan's arrangement in the membrane as well as its primary sequence are similar to that of the tetraspan superfamily of proteins. Sarcospan co-localizes and co-purifies with the dystrophin-glycoprotein complex, demonstrating that it is an integral component of the complex. We also show that sarcospan expression is dramatically reduced in muscle from patients with Duchenne muscular dystrophy. This suggests that localization of sarcospan to the membrane is dependent on proper dystrophin expression. The gene encoding sarcospan maps to human chromosome 12p11.2, which falls within the genetic locus for congenital fibrosis of the extraocular muscle, an autosomal dominant muscular dystrophy.In skeletal muscle fibers, the dystrophin-glycoprotein complex (DGC) 1 (1-5) is located at the sarcolemma and is composed of both peripheral and integral membrane proteins. Collectively, these proteins provide a physical connection between the extracellular matrix and the intracellular cytoskeleton of muscle cells. Disruption of this linkage eventually progresses to muscle cell necrosis, as evidenced by the dystrophic muscle phenotypes that result from defects in several of the DGC components (for review, see Refs. 6 and 7). Although the DGC is known to be essential for normal muscle function, the precise role of this multi-protein complex remains to be determined.Purification of the DGC has led to the identification of many of its constituent polypeptides, which range in size from 25 kDa to over 400 kDa and include glycosylated as well as non-glycosylated proteins (1-4). Characterization of these proteins has increased our understanding of how the DGC is oriented in the sarcolemma and has provided clues to its function. In addition to dystrophin, the DGC consists of ␣/␤-dystroglycan, the sarcoglycans (␣, ␤, ␥, and ␦ subunits), and the syntrophins. While most of the "dystrophin-associated proteins" (DAPs) have been identified, one of them, a 25-kDa protein (also called A5) (1,(3)(4)(5), has remained an enigma.In the present work, we have determined the amino acid sequence of two peptides derived from 25DAP and have isolated the corresponding human cDNA. Previous analysis of the 25DAP indicated that it is an integral membrane protein, since it reacts strongly with a probe for protein hydrophobicity (4). We now report that 25DAP is a novel component of the DGC and is predicted to span the sarcolemma four times. This is unusual for integral membrane...

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Section: Resultsmentioning

confidence: 99%

Sarcospan, the 25-kDa Transmembrane Component of the Dystrophin-Glycoprotein Complex

Crosbie

Heighway

Venzke

et al. 1997

Journal of Biological Chemistry

178

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“…In fact, TID binding to the 25-kDa DAP was greater than its binding to the SGs or β-DG, which possess a single transmembrane span, providing strong evidence that the 25-kDa DAP contained multiple membrane-spanning regions and was unlikely to be a protein degradation product, as had been speculated based on its weak staining with Coomassie-Brilliant blue [5,7]. Identification of the 25-kDa DAP was accomplished by in-gel digestion and sequencing of two amino acid peptides, leading to isolation of the corresponding human cDNA [7] that was previously identified in part as Kirsten ras associated gene (krag), a gene that is co-amplified with Ki-ras in the Y1 murine adrenal carcinoma cell line [8,9]. The gene was renamed to sarcospan (SSPN) for its multiple sarcolemma spanning helices predicted from hydropathy analysis [7].…”

Section: Reviewmentioning

confidence: 99%

“…The sarcospan gene is localized to human chromosome 12p11.2 and is encoded by three small exons that are separated by very large introns [8,9]. A novel exon 4 was recently identified to encode for an alternative C-terminal region in humans.…”

Section: Reviewmentioning

confidence: 99%

Sarcospan: a small protein with large potential for Duchenne muscular dystrophy

Marshall

Crosbie-Watson

2013

Skeletal Muscle

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Purification of the proteins associated with dystrophin, the gene product responsible for Duchenne muscular dystrophy, led to the discovery of the dystrophin-glycoprotein complex. Sarcospan, a 25-kDa transmembrane protein, was the last component to be identified and its function in skeletal muscle has been elusive. This review will focus on progress over the last decade revealing that sarcospan is an important regulator of muscle cell adhesion, strength, and regeneration. Investigations using several transgenic mouse models demonstrate that overexpression of sarcospan in the mouse model for Duchenne muscular dystrophy ameliorates pathology and restores muscle cell binding to laminin. Sarcospan improves cell surface expression of the dystrophin- and utrophin-glycoprotein complexes as well as α7β1 integrin, which are the three major laminin-binding complexes in muscle. Utrophin and α7β1 integrin compensate for the loss of dystrophin and the finding that sarcospan increases their abundance at the extra-synaptic sarcolemma supports the use of sarcospan as a therapeutic target. Newly discovered phenotypes in sarcospan-deficient mice, including a reduction in specific force output and increased drop in force in the diaphragm muscle, result from decreased utrophin and dystrophin expression and further reveal sarcospan’s role in determining abundance of these complexes. Dystrophin protein levels and the specific force output of the diaphragm muscle are further reduced upon genetic removal of α7 integrin (Itga7) in SSPN-deficient mice, demonstrating that interactions between integrin and sarcospan are critical for maintenance of the dystrophin-glycoprotein complex and force production of the diaphragm muscle. Sarcospan is a major regulator of Akt signaling pathways and sarcospan-deficiency significantly impairs muscle regeneration, a process that is dependent on Akt activation. Intriguingly, sarcospan regulates glycosylation of a specific subpopulation of α-dystroglycan, the laminin-binding receptor associated with dystrophin and utrophin, localized to the neuromuscular junction. Understanding the basic mechanisms responsible for assembly and trafficking of the dystrophin- and utrophin-glycoprotein complexes to the cell surface is lacking and recent studies suggest that sarcospan plays a role in these essential processes.

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“…It was identified as a component of the rabbit skeletal muscle DGC by peptide sequence analysis of the 25-kDa DGC component (14). Primary sequence analysis predicts that sarcospan contains four transmembrane domains with cytoplasmic NH 2 and COOH termini (14,29,61). This makes sarcospan unique among other integral membrane components of the DGC, which contain single-transmembrane domains.…”

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confidence: 99%

Sarcospan-Deficient Mice Maintain Normal Muscle Function

Lebakken

Venzke

Hrstka

et al. 2000

Molecular and Cellular Biology

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Sarcospan is an integral membrane component of the dystrophin-glycoprotein complex (DGC) found at the sarcolemma of striated and smooth muscle. The DGC plays important roles in muscle function and viability as evidenced by defects in components of the DGC, which cause muscular dystrophy. Sarcospan is unique among the components of the complex in that it contains four transmembrane domains with intracellular Nand C-terminal domains and is a member of the tetraspan superfamily of proteins. Sarcospan is tightly linked to the sarcoglycans, and together these proteins form a subcomplex within the DGC. Stable expression of sarcospan at the sarcolemma is dependent upon expression of the sarcoglycans. Here we describe the generation and analysis of mice carrying a null mutation in the Sspn gene. Surprisingly, the Sspn-deficient muscle maintains expression of other components of the DGC at the sarcolemma, and no gross histological abnormalities of muscle from the mice are observed. The Sspn-deficient muscle maintains sarcolemmal integrity as determined by serum creatine kinase and Evans blue uptake assays, and the Sspn-deficient muscle maintains normal force and power generation capabilities. These data suggest either that sarcospan is not required for normal DGC function or that the Sspn-deficient muscle is compensating for the absence of sarcospan, perhaps by utilizing another protein to carry out its function.The dystrophin-glycoprotein complex (DGC) is a multisubunit protein complex composed of integral membrane, peripheral membrane, and cytoplasmic proteins expressed at the sarcolemma of striated muscle fibers (see references 7, 41, 54, and 62 for reviews). Isolation and cloning of proteins within this complex have provided key insights into the function of the DGC and its role in normal muscle physiology. The skeletal muscle DGC is composed of dystrophin (9, 31); the syntrophins (22); ␣-and ␤-dystroglycan (33); ␣-, ␤-, ␥-, and ␦-sarcoglycan (3, 36, 42, 48, 50, 51, 55, 56); and sarcospan (14). Recently, the DGC within smooth muscle fibers has been characterized (64). This complex differs from the striated muscle DGC in at least two respects. First, ε-sarcoglycan replaces ␣-sarcoglycan within the sarcoglycan subcomplex, and second, differences occur in the glycosylation pattern of ␣-dystroglycan in the smooth muscle DGC.One likely function of the DGC is to provide a structural link between the extracellular matrix and the actin cytoskeleton, thereby maintaining the stability of the sarcolemma under contractile forces (10, 21). This link occurs through dystrophin, which binds to filamentous actin (31, 58, 59), and dystroglycan-with its ␤-subunit binding to dystrophin (37) and its ␣-subunit interacting with the extracellular matrix component laminin-2 (24). The DGC likely plays other roles in normal muscle physiology by interacting with cell signaling molecules or other proteins at the sarcolemma. The skeletal muscle DGC has been the most fully characterized in this respect, and several laboratories have demonstrated i...

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Characterization of a Gene Coamplified with Ki-ras in Y1 Murine Adrenal Carcinoma Cells That Codes for a Putative Membrane Protein

Cited by 8 publications

References 0 publications

Sarcospan, the 25-kDa Transmembrane Component of the Dystrophin-Glycoprotein Complex

Sarcospan, the 25-kDa Transmembrane Component of the Dystrophin-Glycoprotein Complex

Sarcospan: a small protein with large potential for Duchenne muscular dystrophy

Sarcospan-Deficient Mice Maintain Normal Muscle Function

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