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A role for focal adhesion kinase in the contraction of gastric fundus smooth muscle evoked by cholinergic neurotransmission
AdvisorPerrino, Brian A.
Biochemistry and Molecular Biology
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Integrins are trans-membrane receptors that form focal contacts and mediate the attachment of the smooth muscle cell cytoskeleton to the extracellular matrix. Focal adhesion kinase (FAK) regulates the recruitment and assembly of focal adhesion proteins. It has been established that integrin proteins and FAK play important roles in cell adhesion, tension generation, and mechanotransduction. Integrins and FAK are abundant in smooth muscles; however, the function of FAK in gastric smooth muscle cell contractile regulation remains unclear. To better understand the role of FAK in gastric fundus smooth muscle contractile regulation, we performed two major projects: (1) addressing gastric smooth muscles contractile regulation in cholinergic neurotransmission by FAK phosphorylation (Chapter 2) and (2) characterizing the change in Ca2+ sensitization proteins distribution at focal adhesion sites under electric field stimulation induced cholinergic neurotransmission and FAK phosphorylation regulation (Chapter 4). In carrying out the first project, the contractile responses of murine gastric smooth muscles were determined using standard myobath – isometric force transducer techniques and phosphorylated or total proteins determined by automated capillary electrophoresis and immunoblotting by Wes Simple Western. For the second project, we developed smooth muscle tissue in situ proximity ligation assay (PLA) for the quantitative PLA analysis of protein-protein interaction and protein phosphorylation (Chapter 3). From these studies, we revealed a novel role of FAK in gastric fundus smooth muscle contractile regulation by cholinergic stimulation. We also demonstrate quantifiable tissue level PLA that can be extended and applied to studies of protein-protein interaction, and protein phosphorylation in various tissues and signaling pathways.