Identification and characterization of a novel A-kinase-anchoring protein (AKAP120) from rabbit gastric parietal cells

The type-II cAMP-dependent protein kinase (A-Kinase) partitions primarily into the particulate fraction in gastric parietal cells. Localization of this kinase to particular subcellular domains is mediated through the binding of the regulatory subunit (R(II)) dimer to A-Kinase-anchoring proteins (AKAPs). Using a [P-32]R(II) overlay assay, we have screened a rabbit gastric parietal cell cDNA library and have isolated a single R(II)-binding protein clone. Sequence analysis revealed an open reading frame coding for 1022 amino acids (AKAP120). Recombinant fragments of the full-length clone were prepared and the R(II)-binding region mapped to an area between amino acids 489 and 549. This area contained a putative alpha-helical R(II)-binding region between amino acids 503 and 516. Incubation of [P-32]R(II) with a synthetic peptide of AKAP120-(489-522) completely inhibited the binding of [P-32]R(II) to the recombinant AKAP120 fragments that demonstrated R(II) binding. In vitro R(II)-binding affinity studies indicated a high-affinity interaction between AKAP120 and R(II) with a K-app between 50 and 120 nM for the three recombinant fragments that bound [P-32]R(II). RNase-protection analysis revealed that AKAP120 is a widely distributed protein, with the highest levels of mRNA observed in gastric fundus. The presence of this novel high-affinity AKAP in gastric parietal cells suggests that it may regulate R(II) subcellular sequestration in this cell type.