Protein-protein interactions have emerged as an important mechanism providing for specificity in cellular signal transduction. Two splice variants of type I cGMP-dependent protein kinase (PKG I␣ and I␤) differ only in their N-terminal ϳ100 amino acids, which mediate binding to different target proteins. PKG I␤, but not I␣, binds to the general transcriptional regulator TFII-I and the inositol 1,4,5-trisphosphate receptor-associated PKG substrate IRAG. Using a combination of site-directed

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... esis and in vitro binding assays, we identified a group of acidic amino acids in the N-terminal leucine zipper dimerization domain of PKG I␤ required for its binding to both TFII-I and IRAG. Small clusters of basic amino acids in possible ␣-helical regions in TFII-I and IRAG were found to mediate their interaction with PKG I␤. Mutation of two negatively charged residues in the PKG I␤ leucine zipper (D26K/E31R) to positively charged residues, found in corresponding positions in PKG I␣, completely abrogated binding to TFII-I and IRAG without disrupting PKG dimerization. Mutation of specific basic residues in TFII-I or IRAG abolished binding of the full-length proteins to PKG I␤ in intact cells. Based on these results, we propose a model for specific PKG I␤ interaction with target proteins. . 3 The abbreviations used are: PKA, cAMP-dependent protein kinase; AKAP, cAMP-dependent protein kinase anchoring protein; GST, glutathione S-transferase; IRAG, inositol 1,4,5-trisphosphate receptor-associated PKG substrate; MBS, myosin binding subunit; PKG, cGMP-dependent protein kinase.