The dystrophin glycoprotein complex has been proposed to be involved in signal transduction. Here we have shown that laminin binding causes syntrophin to recruit Rac1 from the rabbit skeletal muscle. LamininSepharose and syntrophin-Sepharose bind a protein complex containing Rac1 from the muscle membranes. The presence of heparin, which inhibits laminin interactions, prevents recruitment of Rac1. The dystrophin glycoprotein complex recruits Rac1 via syntrophin through a Grb2⅐Sos1 complex. A syntrophin antibody also prevents recruitment of Rac1, suggesting that the signaling complex requires syntrophin. PAK1 is in turn bound by Rac1. c-Jun NH 2 -terminal kinase-p46 is phosphorylated and activated only when laminin is present, and the p54 isoform is activated when laminin is depleted or binding is inhibited with heparin. In the presence of laminin, c-Jun is activated in both skeletal muscle microsomes and in C2C12 myoblasts, and proliferation increases in C2C12 myoblasts. We postulate that this pathway signals muscle homeostasis and hypertrophy.

Previously, a signaling pathway was described [Oak, Zhou, and Jarrett (2003) J. Biol. Chem. 278, 39287-39295] that links matrix laminin binding on the outside of the sarcolemma to Grb2 binding to syntrophin on the inside surface of the sarcolemma and by way of Grb2-Sos1-Rac1-PAK1-JNK ultimately results in the phosphorylation of c-jun on Ser(65). How this signaling is initiated was investigated. Grb2-binding to syntrophin is increased by the addition of either laminin-1 or the isolated laminin alpha1 globular domain modules LG4-5, a protein referred to as E3. This identifies the LG4-5 sequences as the region of laminin responsible for signaling. Since laminin alpha1 LG4 is known to bind alpha-dystroglycan, this directly implicates alpha-dystroglycan as the laminin-signaling receptor. E3 or laminin-1 increase Grb2-binding and Rac1 activation. In the presence of E3 or laminin-1, syntrophin is phosphorylated on a tyrosine residue, and this increases and alters Grb2 binding. The alpha-dystroglycan antibody, IIH6, which blocks binding of laminins to alpha-dystroglycan, blocks both the laminin-induced Sos1/2 recruitment and syntrophin phosphorylation, showing that it is alpha-dystroglycan binding the LG4-5 region of laminin that is responsible. The C-terminal SH3 domain of Grb2 (C-SH3) binds only to nonphosphorylated syntrophin, and phosphorylation causes the Grb2 SH2 domain to bind and prevents SH3 binding. Syntrophin, tyrosine phosphate, beta-dystroglycan, and Rac1 all co-localize to the sarcolemma of rat muscle sections. A model for how this phosphorylation may initiate downstream events in laminin signaling is presented.

(DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with ␣-dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the ␤␥-subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain G␤␥-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, ␣-and ␤-dystroglycan, or dystrophin precipitate a complex with G␤␥ from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of G␤␥ binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of G s␣ and G␤␥ occurs and active G s ␣, measured as GTP-␥ 35 S bound, decreases. Because intracellular Ca 2ϩ is elevated in Duchenne muscular dystrophy and Gs␣ is known to activate the dihydropyridine receptor Ca 2ϩ channel, whether laminin also altered intracellular Ca 2ϩ was investigated. Laminin-1 decreases active (GTP-␥S-bound) G s␣, and the Ca 2ϩ channel is inhibited by laminin-1. The laminin ␣1-chain globular domains 4 and 5 region, the region bound by DGC ␣-dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to ␣-dystroglycan inhibits G␤ binding by syntrophin in C 2C12 myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor.

In our previous studies, we showed laminin binds α-dystroglycan in the dystrophin glycoprotein complex and initiates cell signaling pathways. Here, differentiated C2C12 myocytes serve as a model of skeletal muscle. C2C12 cells have a biphasic response to the laminin-α(1) laminin globular (LG) 4-5 domains (1E3) dependent on the concentration used; at low concentrations of 1E3 (<1 μg/ml), myoblast proliferation is increased while higher concentrations (>1 μg/ml) cause apoptosis in myoblasts and differentiated myotubes. This alters the activation of the transcription factors activator protein-1 (AP-1) and NF-κB via laminin-dystrophin glycoprotein complex (DGC)-src-grb2-sos1-Rac1-Pak1-c-jun N-terminal kinase (JNK)p46 and laminin-DGC-Gβγ-phosphatidylinositol 3-kinase (PI3K)-Akt pathways, respectively. A specific antibody against Ser(63) phosphorylated c-jun completely blocks or supershifts the AP-1-DNA binding resulting from laminin binding but only partially blocks or supershifts the AP-1-DNA binding resulting from 1E3. This suggests that AP-1 contains phosphorylated c-jun in the presence of hololaminin but contains a different composition in the presence of 1E3. Nuclear NF-κB was only upregulated by a low concentration of 1E3 and is then diminished by a higher concentration; it also has a biphasic response. Nuclear localization of NF-κB is affected by PI3K/Akt signaling, and DGC associated PI3K activity also shows a biphasic response to 1E3. Furthermore, our data suggest that activation of c-jun N-terminal kinase participates in the cell survival pathway and suggest that NF-κB is involved in both survival and cell death. A model is presented which incorporates these observations.