Activation of muscarinic m3 receptors causes focal adhesion formation and tyrosine phosphorylation of the associated proteins paxillin and focal adhesion kinase (FAK). Tyrosine phosphorylation was found to depend on integrin engagement by the extracellular matrix (ECM). Muscarinic rector subtypes m1, m3 and m5, are coupled to phosphoinositide (PI) turnover and activation of protein kinase C (PKC), effects weakly associated with m2 and m4 receptors. Nonetheless, both m1 and m2 receptors strongly activate intermediates in key intracellular signaling pathways, including mitogen-activated protein kinase (MAPK). Cellular responses to growth factors are profoundly influenced by interactions with the ECM, and it is proposed that signaling responses to PI-coupled muscarinic receptors may be similarly dependent on the activity of cell-surface integrins. The goals of this proposal are fourfold: first, to identify intermediates in the signaling pathway regulating integrin activation in response to muscarinic m3 receptor stimulation (Aim 1); second, to identify the specific muscarinic receptor subtypes that regulate integrin activation (Aim 2); third, to determine how different ECM molecules modulate muscarinic receptor signaling in neuronal and non-neuronal cells (Aims 3 and 5); and fourth, to pursue a recent, surprising observation that integrins act as negative regulators of receptor-activated MAPKs (Aim 4). The signaling events that will be assessed include protein tyrosine phosphorylation, activation of MAPK, cytoskeletal reorganization, and focal adhesion formation. Signaling pathways will be delineated using pharmacological inhibitors, and cDNA constructs encoding wild-type and dominant negative mutant proteins. Protein phosphorylation will be measured using antibodies specific for phosphotyrosine, or for activated MAPK. Focal adhesion formation will be assessed by immunofluorescence and confocal microscopy. Human embryonic kidney cell lines stably expressing individual muscarinic receptor subtypes, and SH-SY5Y neuroblastoma cells expressing endogenous m3 receptors, will be used as models. G protein-coupled receptors, integrins, and MAPK modulate growth, differentiation, and survival in a variety of cells types. In brain, muscarinic receptors and integrins are regulators of long-term potentiation and memory formation. The effects of both receptor classes are relayed through changes in signaling pathways, and via remodeling of cytoskeletal structures. Elucidating the interactions between muscarinic receptors and integrins, using both neuronal and non-neuronal cells models, may be expected to yield novel insights into the ways in which information is integrated at the cellular level; information that may be applicable both to the modulation of neuronal function and plasticity, as well as to the regulation of cell growth in general.
