The long term objective of the proposed research plan is to investigate the molecular mechanisms by which cell adhesion receptor integrins transduce signals across the membrane and regulate the growth and differentiation of higher eucaryotic cells. We focus on the possibility that intracellular protein tyrosine kinases interact with integrins to mediate signal transduction and cellular function of integrins. Consistent with this possibility, we have recently found that interactions of beta1 integrins with their extracellular ligands triggers tyrosine phosphorylation of an intracellular 120 kDa protein, ppl20, which may be involved in the responses of cells to attachment. In the current proposal, pp120 will be isolated and purified, and its partial peptide sequence will be obtained at the N-terminus and internally, after trypsin digestion and separation of peptides by HPLC. cDNA clones coding for ppl20 will then be isolated using degenerate oligonucleotides based on its partial peptide sequences. Sequencing of the cDNA clones will determine the primary structure of pp120 and allow us to generate antisera for ppl20 using fusion proteins and synthetic peptides. The mechanism of integrin-dependent tyrosine phosphorylation of ppl20 will be examined by determining the putative tyrosine kinases acting on pp120, the potential physical and functional interactions among pp120, its kinase and integrins, and the effects of ppl20 tyrosine phosphorylation induced by integrin binding to ligands on these interactions. Furthermore, recombinant integrins will be prepared using the baculovirus expression systems to study the in vitro interactions of integrins with ppl20 and/or the putative tyrosine kinase for ppl20. The role of ppl20 and its tyrosine phosphorylation in mediating signal transduction of integrins resulting in cell spreading will be investigated using site-directed mutagenesis and transient expression vectors. A selected group of dominant mutants will then be expressed using inducible promoters to further assess the role of pp120 in integrin-mediated cell adhesion and growth. This research will enhance our knowledge of the mechanisms by which cell surface integrin receptors exert their regulatory functions on cellular behaviors both in normal and disease states. In particular, it might establish an important link between cell surface adhesion receptors and cytoplasmic tyrosine kinases: transmembrane protein integrins as regulators for cytoplasmic tyrosine kinases.
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