γ1- and γ2-Syntrophins, Two Novel Dystrophin-binding Proteins Localized in Neuronal Cells

Abstract

Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies. At the last 3 terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound. These are a family of cytoplasmic peripheral membrane proteins. Three genes have been described encoding one acidic (␣1) and two basic (␤1 and ␤2) proteins of ϳ57-60 kDa. Here, we describe the characterization of two novel putative members of the syntrophin family, named ␥1-and ␥2-syntrophins. The human ␥1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids. The human ␥2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues. We mapped the ␥1-syntrophin gene to human chromosome 8q11 and the ␥2-syntrophin gene to chromosome 2p25. Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins. We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections. Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system.Since the identification of dystrophin, the product of the Duchenne muscular dystrophy gene at Xp21, molecular genetics has moved quickly (1, 2). The deficiency of dystrophin in Duchenne muscular dystrophy (DMD) 1 and its first animal model, the mdx mouse, leads to a dramatic reduction in a group of previously unknown proteins identified as the dystrophinassociated protein complex.In the last few years, the dystrophin-associated protein complex proteins have been isolated; their genes have been cloned; and the following model of the complex has been hypothesized (3-5). Dystrophin is a large rod-shaped protein, primarily localized beneath the muscle cell membrane. Its actinin-like N terminus binds F-actin (6), whereas its C terminus is anchored to the transmembrane protein, ␤-dystroglycan, which is linked through ␣-dystroglycan to the extracellular merosin (laminin-2) (7). Then, this complex bridges the muscle membrane from the cytoskeleton to the extracellular matrix. In addition, dystroglycan is the receptor for agrin, a protein with a pivotal role in the clustering of acetylcholine receptors at the neuromuscular junction (8 -10) and a fundamental element of the basal lamina (11). At the muscle membrane, this complex is associated with the hydrophobic sarcospan DAP25 (dystrophinassociated protein; A5) (12) and the sarcoglycan complex, which is composed of at least four interacting transmembrane glycoproteins: ␣-sarcoglycan (DAG50 (dystrophin-associated glycoprotein), A2, adhalin) (13, 14), ␤-sarcoglycan (DAG43, A3b) (15, 16), ␥-sarcoglycan (DAG35, A4) (17), and ␦-sarcoglycan (18). Mutations in the laminin-␣2 gene are responsible for congenital muscular dystrophy (19); mutations in the ␥-, ␣-, ␤-, and ␦-sarcoglycan genes cause limb-girdle mus...

Potassium channel Kir4.1 macromolecular complex in retinal glial cells

“…Aside from a-syntrophin, the ubiquitously expressed b 1 and b 2 (Ahn et al, 1996) and the brain-specific g 1 and g 2 isoforms (Piluso et al, 2000) have all been characterized. Furthermore, recent studies showed that there can be up to four distinct syntrophin binding sites within a single DGC, which can theoretically bind any combination of syntrophin (Peters et al, 1997;Newey et al, 2000).…”

Quel avenir pour la dystrophine ?

“…Dès 1987, date de sa découverte [1], et durant environ 2 décennies la dystrophine voyait presque chaque année autour d'elle apparaître de nouveaux partenaires aussi bien ancrés dans la membrane musculaire que distribués dans le cytoplasme environnant et formant ainsi un large complexe macromoléculaire [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. On va ainsi identifier une protéine apparentée à la dystrophine comme la dystrobrévine avec 2 versions différentes (α et β) et 7 à 8 isoformes respectivement.…”

Syntrophin γ2 Regulates SCN5A Gating by a PDZ Domain-mediated Interaction

“…Sections were incubated with 10% normal donkey serum and 0.3% Triton X-100 for 1 h to block nonspecific absorption sites and then incubated overnight at 4°C with anti-syntrophin ␥2 rabbit polyclonal antibody (diluted 1:200 in 5% normal donkey serum; a kind gift from Dr. Vincenzo Nigro). The specificity of this antibody for syntrophin ␥2 has been previously shown by Piluso et al (22). After several rinses in PBS, the sections were incubated for 1 h with donkey, anti-rabbit IgG conjugated to CY3 (1:100 dilution in 2.5% normal donkey serum), rinsed in PBS, and coverslipped in glycerol-PBS containing an anti-fade reagent.…”

“…Each syntrophin is encoded by a separate gene but shares a common domain organization. Each syntrophin contains two tandem pleckstrin homology domains at the N terminus, a single PDZ domain, and a highly conserved C terminus syntrophin-unique region (22,23). The PDZ domains of syntrophins ␣, ␤1, and ␤2 but not ␥1 are known to interact with SCN5A and with SCN4A, a skeletal muscle Na ϩ channel, via the C terminus sequence motif (E(S/T)XV) (14,15,24).…”

“…Syntrophins are a multigene family of homologous proteins (18 -22). Five syntrophins, ␣, ␤1, ␤2, ␥1, and ␥2, have been characterized (22,23). Each syntrophin is encoded by a separate gene but shares a common domain organization.…”