The applicant intends to understand the role of non-receptor tyrosine kinases in heterotrimeric G protein-mediated signal transduction. He plans to use the activation of the mitogen-activated protein kinase (MAPK) pathway by G protein-coupled receptors in tyrosine kinase-deficient cells as a functional assay. The MAPK signaling cascade is a prominent cellular pathway used by many growth factors, cytokines, hormones and neurotransmitters to regulate numerous physiological responses. Although the activation mechanism of the MAPK pathway by receptor tyrosine kinases is well understood, the mechanism utilized by heterotrimeric G protein-coupled receptors to activate the MAPK pathway is less clear in mammalian cells. Several studies have demonstrated the ability of G protein-coupled receptors and G proteins to activate MAPK. Some of these activations are sensitive to tyrosine kinase inhibitors. However, none of the involved tyrosine kinases have been identified. A combination of molecular, biochemical and genetic approaches have been employed to demonstrate a tyrosine kinase-dependent signal transduction pathway from G protein to the activation of mitogen-activated protein kinase (MAPK). In Src-related tyrosine kinase Lyn-deficient cells, stimulation of MAPKK and MAPK by Gq-coupled m1 muscarinic acetylcholine receptor (mAChR) was blocked while Gi-coupled m2 mAChR-mediated stimulation was not affected. In another family, tyrosine kinase Syk-deficient cells, both m1 and m2mAChRs failed to simulate MAPKK and MAPK. To extend these observations and to search the underlining biochemical processes, the applicant will continue to perform molecular genetic analysis of the involvement of other sub-families of tyrosine kinases in G protein signaling and to learn biochemically how G proteins regulate the activities of nonreceptor tyrosine kinases.
