The objective of this proposal is to characterize the signal transduction pathways that mediate intestinal smooth muscle contraction in response to neurotransmitters, chiefly acetylcholine. The pathways consist of receptors, G proteins, effector enzymes, and cascades of messengers and protein kinases that mediate phosphorylation of myosin light chain (MLC) and muscle contraction. The PI's current studies have identified distinctive patterns of receptor-G protein coupling that are both receptor- and cell-specific. The G proteins are coupled to diverse effectors that generate distinct Ca2+-mobilizing messengers in intestinal circular muscle (IP3) and longitudinal muscle (arachidonic acid and cADP ribose). The PI has expanded his focus to include: (i) feedback regulation of G proteins by caveolin, downstream kinases, and GTPases; (ii) characterization of messengers that regulate Ca2+ release and sustained Ca2+ influx; and (iii) characterization of the kinase/phosphatase cascades that regulate phosphorylation and dephosphorylation of MLC. Accordingly, the specific aims which are supported by preliminary studies are: (1) to analyze G protein coupling to Gq/11 and G13 initiated by m3 and m2 receptors; characterize the mechanisms of desensitization that selectively target G proteins (caveolin-3 binding, Gil/Gi2-specific PKC phosphorylation, and deactivation by RGS proteins); and utilize the single-transmembrane NPR-C receptor expressed in intestinal muscle as a model for receptor-G protein coupling using deletion and site-directed mutagenesis; (2) to characterize G protein-dependent regulation of Ca2+ release in longitudinal muscle by cADPR, and capacitative Ca2+ influx by P450 mono-oxygenase metabolites; (3) to characterize the stimulatory pathway initiated by m3 receptors linking G13 and the monomeric G protein, RhoA, to sequential activation of PLD and PKC-epsilon and inhibition of MLC phosphatase, and the inhibitory pathway initiated by m2 receptors linking Gi3 and the monomeric G proteins, Rac and Cdc42, to activation of PAK-1 and inhibition of MLCK. These pathways operate in concert to maintain MLC phosphorylation and sustained contraction at low Ca2+ levels.
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