Interaction of neuronal nitric-oxide synthase with alpha1-syntrophin in rat brain.
(65/8990)
Neuronal nitric-oxide synthase (nNOS) has a PSD-95/Dlg/ZO-1 (PDZ) domain that can interact with multiple proteins. nNOS has been known to interact with PSD-95 and a related protein, PSD-93, in brain and with alpha1-syntrophin in skeletal muscle in mammals. In this study, we have purified an nNOS-interacting protein from bovine brain using an affinity column made of Sepharose conjugated with glutathione S-transferase-rat nNOS fusion protein and identified it as alpha1-syntrophin by microsequencing. Immunostaining of primary cultures of rat embryonic brain neuronal cells with antibodies against these proteins showed that nNOS and alpha1-syntrophin were colocalized in neuronal cell bodies and neurites. Immunohistochemical analysis indicated that the nNOS- and alpha1-syntrophin-like immunoreactive substances were highly expressed in the rat hypothalamic suprachiasmatic nucleus (SCN) and paraventricular nucleus. In the SCN, nNOS- and alpha1-syntrophin-like immunoreactive substances were colocalized in the same neurons as detected by confocal microscopy. These results indicate that nNOS in brain interacts with alpha1-syntrophin in specific neurons of the SCN and paraventricular nucleus and that this interaction might play a physiological role in functions of these neurons. (+info)
Rib truncations and fusions in the Sp2H mouse reveal a role for Pax3 in specification of the ventro-lateral and posterior parts of the somite.
(66/8990)
The splotch (Pax3) mouse mutant serves as a model for developmental defects of several types, including defective migration of dermomyotomal cells to form the limb musculature. Here, we describe abnormalities of the ribs, neural arches, and acromion in Sp2H homozygous embryos, indicating a widespread dependence of lateral somite development on Pax3 function. Moreover, the intercostal and body wall muscles, derivatives of the ventrolateral myotome, are also abnormal in Sp2H homozygotes. Pax3 is expressed in the dermomyotome, but not in either the sclerotome or the myotome, raising the possibility that Pax3-dependent inductive influences from the dermomyotome are necessary for early specification of lateral sclerotome and myotome. Support for this idea comes from analysis of gene expression markers of lateral sclerotome (tenascin-C and scleraxis) and myotome (myogenin, MyoD, and Myf5). All exhibit ventrally truncated domains of expression in Sp2H homozygotes, potentially accounting for the rib and intercostal muscle truncations. In contrast, the medial sclerotomal marker Pax1 is expressed normally in mutant embryos, arguing that Pax3 is not required for development of the medial sclerotome. Most of the somitic markers show ectopic expression in anteroposterior and mediolateral dimensions, suggesting a loss of definition of somite boundaries in splotch and explaining the rib and muscle fusions. An exception is Myf5, which is not ectopically expressed in Sp2H homozygotes, consistent with the previous suggestion that Pax3 and Myf5 function in different pathways of skeletal myogenesis. PDGFalpha and its receptor are candidates for mediating signalling between myotome and sclerotome. We find that both genes are misexpressed in Sp2H embryos, suggesting that PDGFalpha/PDGFRalpha may function downstream of Pax3, accounting for the close similarities between the splotch and Patch mutant phenotypes. Our findings point to additional regulatory functions for the Pax3 transcription factor, apart from those already demonstrated for development of the neural tube, neural crest, and dermomyotome. (+info)
bves: A novel gene expressed during coronary blood vessel development.
(67/8990)
We have used a subtractive method to clone novel messages enriched in the heart. Here we show that one such message, bves (blood vessel/epicardial substance) is a novel protein that is highly conserved between chicken and mouse. The bves message is detected at high levels in early chick hearts. Using anti-Bves antibodies, we show expression in cells of the proepicardial organ, migrating epicardium, epicardial-derived mesenchyme, and smooth muscle of the developing intracardiac arterial system, including the coronary arteries. Our data suggest that Bves is an early marker of developing vascular smooth muscle cells. In addition, the expression pattern of Bves protein reveals the patterning of intracardiac vascular smooth muscle and possible insights into the cellular regulation of smooth muscle differentiation during vasculogenesis. (+info)
A PDZ-containing scaffold related to the dystrophin complex at the basolateral membrane of epithelial cells.
(68/8990)
Membrane scaffolding complexes are key features of many cell types, serving as specialized links between the extracellular matrix and the actin cytoskeleton. An important scaffold in skeletal muscle is the dystrophin-associated protein complex. One of the proteins bound directly to dystrophin is syntrophin, a modular protein comprised entirely of interaction motifs, including PDZ (protein domain named for PSD-95, discs large, ZO-1) and pleckstrin homology (PH) domains. In skeletal muscle, the syntrophin PDZ domain recruits sodium channels and signaling molecules, such as neuronal nitric oxide synthase, to the dystrophin complex. In epithelia, we identified a variation of the dystrophin complex, in which syntrophin, and the dystrophin homologues, utrophin and dystrobrevin, are restricted to the basolateral membrane. We used exogenously expressed green fluorescent protein (GFP)-tagged fusion proteins to determine which domains of syntrophin are responsible for its polarized localization. GFP-tagged full-length syntrophin targeted to the basolateral membrane, but individual domains remained in the cytoplasm. In contrast, the second PH domain tandemly linked to a highly conserved, COOH-terminal region was sufficient for basolateral membrane targeting and association with utrophin. The results suggest an interaction between syntrophin and utrophin that leaves the PDZ domain of syntrophin available to recruit additional proteins to the epithelial basolateral membrane. The assembly of multiprotein signaling complexes at sites of membrane specialization may be a widespread function of dystrophin-related protein complexes. (+info)
Self-aggregation of triadin in the sarcoplasmic reticulum of rabbit skeletal muscle.
(69/8990)
The 95 kDa transmembrane glycoprotein triadin is believed to be an essential component of excitation-contraction coupling in the junctional sarcoplasmic reticulum of skeletal muscle fibers. It is debatable whether triadin mediates intraluminal interactions between calsequestrin and the ryanodine receptor exclusively or whether this junctional protein provides also a cytoplasmic linkage between the Ca2+-release channel and the dihydropyridine receptor. Here, we could show that native triadin exists as disulfide-linked homo-polymers of above 3000 kDa. Under non-reducing conditions, protein bands representing the alpha1-dihydropyridine receptor and calsequestrin did not show an immunodecorative overlap with the extremely high-molecular-mass triadin clusters. Following chemical crosslinking, the ryanodine receptor and triadin exhibited a similarly decreased electrophoretic mobility. However, immunoblotting of diagonal non-reducing/reducing two-dimensional gels clearly demonstrated a lack of overlap between the immunodecorated bands representing triadin, the alpha1-dihydropyridine receptor, the ryanodine receptor and calsequestrin. Thus, in native membranes triadin appears to form large self-aggregates primarily. Although triadin exists in a close neighborhood relationship to the Ca2+-release channel tetramers, it does not seem to be directly linked to the other main triad components implicated in the regulation of the excitation-contraction-relaxation cycle and Ca2+-homeostasis. This agrees with a proposed role of triadin in the maintenance of overall triad architecture. (+info)
Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor.
(70/8990)
Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the co-immunoprecipitation of ryanodine receptor and triadin. We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca2+ release from sarcoplasmic reticulum. We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca2+ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca2+ channel incorporated in lipid bilayers. Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18-46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca2+ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC50 of 300 microM). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca2+ channel activity of purified RyR in bilayer. We propose that this interaction plays a critical role in the control, by triadin, of the Ca2+ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle. (+info)
Stress-activated protein kinase-3 interacts with the PDZ domain of alpha1-syntrophin. A mechanism for specific substrate recognition.
(71/8990)
Mechanisms for selective targeting to unique subcellular sites play an important role in determining the substrate specificities of protein kinases. Here we show that stress-activated protein kinase-3 (SAPK3, also called ERK6 and p38gamma), a member of the mitogen-activated protein kinase family that is abundantly expressed in skeletal muscle, binds through its carboxyl-terminal sequence -KETXL to the PDZ domain of alpha1-syntrophin. SAPK3 phosphorylates alpha1-syntrophin at serine residues 193 and 201 in vitro and phosphorylation is dependent on binding to the PDZ domain of alpha1-syntrophin. In skeletal muscle SAPK3 and alpha1-syntrophin co-localize at the neuromuscular junction, and both proteins can be co-immunoprecipitated from transfected COS cell lysates. Phosphorylation of a PDZ domain-containing protein by an associated protein kinase is a novel mechanism for determining both the localization and the substrate specificity of a protein kinase. (+info)
The transcription factor nuclear factor I mediates repression of the GLUT4 promoter by insulin.
(72/8990)
Insulin represses GLUT4 expression in 3T3-L1 adipocytes through an insulin response element located at bases -706 to -676 in the 5'-flanking sequence. Nuclear proteins related to the nuclear factor I (NF1) family of transcription factors bind to this insulin response element. Mutations that disrupt binding of NF1 proteins to the insulin response element impair the insulin response in reporter gene assays. Insulin treatment of 3T3-L1 adipocytes induces a rapid change in the level of phosphorylation of NF1 proteins, providing a potential mechanism for insulin's ability to regulate gene expression through NF1. Another as yet unidentified protein, not related to NF1, also binds to the GLUT4 insulin response element and is able to mediate partial repression of the GLUT4 promoter in reporter gene assays. (+info)