Laminin-α1globular domains 3 and 4 induce heterotrimeric G protein binding to α-syntrophin's PDZ domain and alter intracellular Ca2+in muscle

Abstract

α-Syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with α-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 βγ-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 Gβγ-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, α- and β-dystroglycan, or dystrophin precipitate a complex with Gβγ from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of Gβγ binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of Gsα and Gβγ occurs and active Gsα, measured as GTP-γ35S bound, decreases. Because intracellular Ca2+is elevated in Duchenne muscular dystrophy and Gsα is known to activate the dihydropyridine receptor Ca2+channel, whether laminin also altered intracellular Ca2+was investigated. Laminin-1 decreases active (GTP-γS-bound) Gsα, and the Ca2+channel is inhibited by laminin-1. The laminin α1-chain globular domains 4 and 5 region, the region bound by DGC α-dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to α-dystroglycan inhibits Gβ binding by syntrophin in C2C12myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor.