Laminin-alpha1 globular domains 3 and 4 induce heterotrimeric G protein binding to alpha-syntrophin's PDZ domain and alter intracellular Ca2+ in muscle.
Alpha-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 alpha-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 betagamma-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 Gbetagamma-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, alpha- and beta-dystroglycan, or dystrophin precipitate a complex with Gbetagamma from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of Gbetagamma binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of Gsalpha and Gbetagamma occurs and active Gsalpha, measured as GTP-gamma35S bound, decreases. Because intracellular Ca2+ is elevated in Duchenne muscular dystrophy and Gsalpha is known to activate the dihydropyridine receptor Ca2+ channel, whether laminin also altered intracellular Ca2+ was investigated. Laminin-1 decreases active (GTP-gammaS-bound) Gsalpha, and the Ca2+ channel is inhibited by laminin-1. The laminin alpha1-chain globular domains 4 and 5 region, the region bound by DGC alpha-dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to alpha-dystroglycan inhibits Gbeta binding by syntrophin in C2C12 myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor.
Zhou
Yan Wen
YW
Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163, USA.
Oak
Shilpa A
SA
Senogles
Susan E
SE
Jarrett
Harry W
HW
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D049028:Dystrophin-Associated Protein Complex / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D047468:Immunoprecipitation; D007424:Intracellular Fluid / Q000737:chemistry; D007797:Laminin / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000737:chemistry / Q000502:physiology*; D020388:Muscular Dystrophy, Duchenne / Q000503:physiopathology; D011485:Protein Binding / Q000502:physiology; D020836:Protein Structure, Quaternary; D011817:Rabbits
D002135:Calcium-Binding Proteins; D049028:Dystrophin-Associated Protein Complex; D007797:Laminin; D008565:Membrane Proteins; D009124:Muscle Proteins; C091825:syntrophin alpha1; C084098:laminin A; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium