Binding of Laminin α1-Chain LG4−5 Domain to α-Dystroglycan Causes Tyrosine Phosphorylation of Syntrophin to Initiate Rac1 Signaling

Abstract

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.

Laminin-Induced Activation of Rac1 and JNKp46 Is Initiated by Src Family Kinases and Mimics the Effects of Skeletal Muscle Contraction

“…Another important aspect of this and our previous studies (3,9,10) is that this signaling pathway is functioning throughout muscle development, in myoblast, myotubes, and skeletal muscle. Myoblast express much lower amounts of DGC proteins (9,23) and the DGC is much less well characterized in myoblast than it is in skeletal muscle and may be somewhat different.…”

“…c-Src phosphorylates syntrophin somewhere in its N-terminal half (present in Syntrophin A) and skeletal muscle microsomes contain this same or a similar activity. This region of syntrophin has four tyrosine residues, including two we had previously speculated could provide a site of phosphorylation (9). The precise residue(s) phosphorylated is a topic of current investigation.…”

“…Furthermore, syntrophin binds Grb2 and Sos1 to cause Rac1 activation and syntrophin phosphorylation on tyrosine results in the Grb2 binding by its SH2 domain (3,9). Fig 2A shows that even 2.5 µM PP2 was sufficient to inhibit the downstream activation of Rac1 in skeletal muscle microsomes whereas PP3 was ineffective, even at a higher concentration.…”

“…The cells were then immediately suspended in DMEM containing 10% FBS and removed from the plate. This treatment has been previously shown to also deplete the cells of laminin but not DGC components (9) Preparation of contracted muscle Sprague-Dawley rats (200-300 g) were used. Animals were housed in light-and temperaturecontrolled quarters where they received food and water ad libitum.…”

“…Recently, E3 has been shown to substitute for laminin and cause tyrosine phosphorylation of syntrophin and alter grb2-binding to initiate signaling (9). E3-or laminin-binding also results in heterotrimeric G-protein binding to the DGC (10).…”