Polarized epithelial cells, by definition, segregate the functions of their apical and basolateral membranes. This separation of functional membrane domains allows epithelial cells to translate messages received at one membrane surface into responses at the opposite plasma membrane domain. In the gastric parietal cell, activation of second messenger systems at the basolateral membrane results in the fusion of tubulovesicles containing the H/K-ATPase with an apically oriented intracellular canalicular target membrane. In order to carry out the complex integrated processes required, polarized cells have evolved intricate mechanisms to sequester critical links in the signaling pathway within discrete subcellular domains. In the case of the Type II cAMP-dependent protein kinases (A-kinases), the kinase has been localized to both cytosolic and particulate fractions in epithelial cells and neurons. The Type II kinase associates with particulate fractions through the binding of its regulatory subunits (RII) to A- kinase anchoring proteins (AKAPs). We have indentified A-kinase anchoring proteins (AKAPs) in parietal cells which may be involved in localizing A-kinase within particular subcellular domains. In particular, we have recently recognized that the major parietal cell AKAP, AKAP78, is ezrin, the apical F-actin associated protein. These data suggest that Type II A-kinase may be anchored to the secretory canaliculus, the site of membrane fusion during parietal cell secretion. We have also cloned from parietal cells a second novel AKAP, AKAP120, which appears to be present in a number epithelial tissues. We have hypothesized that AKAPs, in particular AKAP78/erin and AKAP120, map function as sites for the assembly of critical kinase/phosphatase signaling complexes anchored to discrete areas of the parietal cell. The present proposal has four specific aims: First, we will establish definitively the association of RII with ezrin by characterizing the RII binding site and the immunocytochemical co-localization of the two proteins. Second, since ezrin is phophorylated in response to histamine, we will determine the effects of phosphorylation on ezrin/RII interactions and characterize ezrin kinase. Third, we will characterize the association of RII with the novel AKAP120 protein. Fourth, we will seek to identify parietal cell proteins which interact with ezrin and AKAP120. These investigations will provide important insights into the mechanisms by which epithelial cells in general, and parietal cells specifically, segregate their second messenger-dependent signaling systems.
