DISTRIBUTION OF A-KINASE ANCHORING PROTEINS IN PARIETAL-CELLS

Recent investigations have suggested that subcellular compartmentaliza tion of second messenger responsive enzyme systems may be responsible for specific patterns of cellular activation. The type II cAMP-depende nt kinase (A-kinase) is localized to particular subcellular domains th rough the binding of the regulatory subunit (R(II)) dimer to A-kinase anchoring proteins (AKAPs). Using a [(32)p]R(II) overlay assay, we hav e investigated the presence of AKAPs throughout the gastrointestinal t ract, with specific emphasis focused on the gastric parietal cell. All gastrointestinal tissues contained at least one detectable AKAP (60 k Da), with five AKAPs (50-140 kDa) in fundic and antral mucosa. Isolate d gastric glands contained four AKAPs. Two AKAPs (50 and 78 kDa) were detected in purified parietal cells, with the 78 kDa AKAP (AKAP78) spe cific to parietal cell enriched populations. R(II)-binding to all of t hese AKAPs was abolished by preincubation of [P-32]R(II) with a synthe tic peptide representing the R(II)-binding region of the AKAP, HT-31. AKAP78 was distributed throughout all membrane fractions of subfractio nated parietal cells, with the largest amount of R(II)-binding detecte d in the light membrane fraction. Identification of A-kinase regulator y subunits by photoaffinity labeling with 8-azido-[P-32]cAMP demonstra ted that R(II) segregated into the same parietal cell subfractions as AKAP78. A majority (similar to 60%) of AKAP78 was detected in the Trit on X-100-insoluble fraction, suggesting that this protein resides in a cytoskeletal domain. AKAP78 may be involved in localizing the type II A-kinase to specific intracellular locations in the parietal cell.