Dystrophin and its isoforms in a sympathetic ganglion of normal and dystrophic mdx mice: immunolocalization by electron microscopy and biochemical characterization.

In normal mouse superior cervical ganglion, dystrophin immunoreactivity is present in ganglionic neurons, satellite cells and Schwann cells. It is associated with several cytoplasmic organelles and specialized plasma membrane domains, including two types of structurally and functionally different intercellular junctions: synapses, where it is located at postsynaptic densities, and adherens junctions. Dystrophin immunostaining can be ascribed to the 427,000 mol. wt full-length dystrophin, as well as to the several dystrophin isoforms present in superior cervical ganglion, as revealed by western immunoblots. In mdx mouse superior cervical ganglion, which lacks the 427,000 mol. wt dystrophin, the unchanged pattern of dystrophin immunolabelling observed at several subcellular structures indicates the presence of dystrophin isoforms at these sites. Moreover, the absence of labelled adherens junctions indicates the presence of full-length dystrophin at this type of junction in the normal mouse superior cervical ganglion. The lower number of immunopositive postsynaptic densities in mdx mouse superior cervical ganglion than in normal mouse ganglion suggests the presence, in the latter, of postsynaptic densities with differently organized dystrophin cytoskeleton: some containing dystrophin isoforms alone or together with 427,000 mol. wt dystrophin, and others containing 427,000 mol. wt dystrophin alone.

Poly(ADP-ribosyl)ation is implicated in the G0-G1 transition of resting cells.

Poly(ADP-ribosyl)ation, catalysed by a family of poly(ADP-ribose) polymerases (PARPs), plays an important role in a large variety of physiological processes, including cell proliferation, but its role in cell cycle progression is not yet completely defined. As reported here, the examination of early times following serum stimulation of quiescent fibroblasts suggests that poly(ADP-ribosyl)ation is necessary for the transition from the G0 phase to the G1 phase. We show that PARP activity is involved in this step through the regulation of immediate-early response genes, such as c-Fos and c-Myc. This is supported by the finding that exogenous Myc expression substantially restores cell cycle reactivation in the absence of polymer synthesis. Furthermore, using RNA interference, we show that PARP-1 is the PARP family member playing the most prominent role in the upregulation of c-Fos and c-Myc during G0-G1 transition. We report that even in lectin-stimulated peripheral blood mononucleated cells, the inhibition of PARP activity interferes with the upregulation of immediate-early genes and delays the induction of proliferation, suggesting a general role for PARP-1 in linking growth factor signaling with cell cycle entry.

Association of the dystroglycan complex isolated from bovine brain synaptosomes with proteins involved in signal transduction.

Dystroglycan is a transmembrane heterodimeric complex of alpha and beta subunits that links the extracellular matrix to the cell cytoskeleton. It was originally identified in skeletal muscle, where it anchors dystrophin to the sarcolemma. Dystroglycan is also highly expressed in nonmuscle tissues, including brain. To investigate the molecular interactions of dystroglycan in the CNS, we fractionated a digitonin-soluble extract from bovine brain synaptosomes by laminin-affinity chromatography and characterized the protein components. The 120-kDa alpha-dystroglycan was the major 125I-laminin-labeled protein detected by overlay assay. This complex, in addition to beta-dystroglycan, was also found to contain Grb2 and focal adhesion kinase p125FAK (FAK). Anti-FAK antibodies co-immunoprecipitated Grb2 with FAK. However, no direct interaction between beta-dystroglycan and FAK was detected by co-precipitation assay. Grb2, an adaptor protein involved in signal transduction and cytoskeleton organization, has been shown to bind beta-dystroglycan. We isolated both FAK and Grb2 from synaptosomal extracts by chromatography on immobilized recombinant beta-dystroglycan. In the CNS, FAK phosphorylation has been linked to membrane depolarization and neurotransmitter receptor activation. At the synapses, the adaptor protein Grb2 may mediate FAK-beta-dystroglycan interaction, and it may play a role in transferring information between the dystroglycan complex and other signaling pathways.

Localization of the dystrophin binding site at the carboxyl terminus of beta-dystroglycan.

Alpha- and beta-dystroglycan form a heteromeric transmembrane complex linking the extracellular matrix to the cytoskeleton. In muscle beta-dystroglycan interacts with dystrophin on the inside of the cell and with alpha-dystroglycan, which binds the extracellular matrix protein laminin, on the outside. Dystroglycan is expressed not only in muscle but also in other tissues. We cloned beta-dystroglycan from rabbit brain by RT-PCR and expressed deletion mutants of the beta-dystroglycan cytoplasmic domain as GST-fusion proteins. We identified the dystrophin binding region on beta-dystroglycan by protein overlay and co-precipitation assays with skeletal muscle dystrophin and recombinant apo-dystrophin I. We demonstrate that the beta-dystroglycan carboxyl terminus interacts with dystrophin and that the binding site is restricted to the last 20 amino acids. Our data also suggest that the region adjacent to the beta-dystroglycan transmembrane domain might modulate beta-dystroglycan-dystrophin interaction.