The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to elucidate the mechanisms of action of the phorbol esters and their endogenous analog, the lipophilic second messenger sn-1,2- diacylglycerol. Protein kinase C (PKC) is the major receptor for these compounds, and our emphasis is correspondingly directed at this family of isozymes. In a collaborative effort, we seek to combine mutational analysis with computer modeling and chemical synthesis to probe ligand - PKC interactions. Reflecting our developing insights, we are now able to make synthetic ligands approaching an affinity of 1 nM. A related issue is the significance of twin phorbol ester binding domains in typical PKCs. By mutating the individual binding domains, we find that the pattern of interaction of ligands with PKC depends both on the specific isotype and on the specific ligand. Our long term objective is to exploit such isotype differences to dissect subpathways of PKC mediated signal transduction. An on-going effort is directed at understanding the distinct biological roles of specific isoforms and the structural basis for their specificity. Using chimeric constructs, we find that both the regulatory and catalytic domains contribute to their specificity of action. The bryostatins, although activators of PKC in vitro, function as partial antagonists in intact cells. Protection of PKC delta from down regulation represents one mechanism contributing to bryostatin's unique activity. This resistance to down regulation in the presence of bryostatin resides in the catalytic domain of PKC delta. The phorbol-related diterpene resiniferatoxin acts as an ultrapotent analog of capsaicin and has permitted characterization of specific capsaicin receptors. We are now able to define distinct receptor subclasses with distinct functions. It had been believed that these receptors were found exclusively on sensory neurons. We have now demonstrated that the C-type vanilloid receptors are also found on other cell types, including mast cells, keratinocytes, and glial cells. These findings have important implications for the therapeutic development of vanilloids in the treatment of pain.
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