Skeletal muscle is an ideal system with which to study adhesion molecules and the membrane-cytoskeletal linkages in which they participate as they play a central role in muscle development, structure and physiology, and pathology. Once muscle precursors have migrated to their targets, the program of terminal differentiation commences, which is regulated by the extracellular matrix. An elaborate contractile apparatus is synthesized and organized, which contains several cell surface associations including the myotendinous and costomeric junctions. Muscle cells are innervated at neuromuscular junctions. It is now clear that dystrophin, the muscular dystrophy gene product, has homologies to cytoskeletal proteins and is associated with adhesion molecules like integrin. The integrin family of receptors for extracellular matrix molecules are implicated in all of the above phenomena by virtue of their localization in junctional regions, their functions as dual receptors for extracellular matrix and cytoskeletal molecules, and as mediators of signal transductions. The hypothesis that guides our current research is that the integrins play a central role in organizing the surface, the extracellular matrix, and the contractile apparatus of skeletal muscle and in addition mediate signals from the extracellular matrix triggering its differentiation. Our general aims for the project period are to identify and characterize the amino acid sequences on integrin cytoplasmic and extracellular domains that determine the organization of junctional regions and determine their role in adhesion. This will be done using a recently constructed library of single-amino acid substitutions in the Beta1- cytoplasmic domain and synthetic peptides corresponding to active and mutant sequences. A similar library will be constructed for alpha subunits. Analogous, but different, methods are proposed to find extracellular matrix binding sequences in the extracellular domain. The second major aim is to identify and purify novel integrin associated cytoplasmic proteins. Previous specificity problems will be addressed using peptide sequences derived from mutant and wild type cytoplasmic domain sequences. Recently we have identified two novel integrin associated molecules. Both are cytoskeletal and one is a complex of 5 proteins. They will be characterized further for binding specificities and localization on muscle. The third objective is to elucidate the role of integrins in organizing and stabilizing junctional regions. This will be done using molecular genetic techniques to identify functional domains, alter regulation of expression, and reduce or eliminate the expression of specific integrins.
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