The analysis of protein-protein interaction with special reference to PRIP-1

Analysis of protein-protein interaction is one of the powerful methods for elucidating the new functions of functionally unknown proteins. Using this approach, we isolated two proteins interacting with PRIP-1, which was isolated as a new Ins (1,4,5) P₃ binding protein from brain. One was protein phosphatase 1 catalytic subunit (PP1c) and the other was GABARAP (GABA_A-receptor-associated protein). The region of PRIP-1 responsible for their interaction was the site preceding to the pleckstrin homology domain of PRIP-1 for PP1c and the EF-hand motifs of PRIP-1 for GABARAP, which were determined by β-galactosidase assay of yeast two-hybrid system. The association between PRIP-1 and PP1c was confirmed in vitro by a pull-down assay, a far-western assay, an immunoprecipitation analysis and a surface plasmon resonance analysis. The interaction of PRIP-1 with PP1c resulted in inhibition of the catalytic activity of PP1c in a PRIP-1 concentration-dependent manner. The association between PRIP-1 and GABARAP was also confirmed by a pull-down assay, and we found that PRIP-1 competitively inhibited the binding of the γ2 subunit of the GABA_A receptor to GABARAP in vitro. Our electrophysiological and behavioral analysis of PRIP-1 knockout mice revealed that PRIP-1 is essential for the function of GABA_A receptors, especially in response to the agents acting on the γ2 subunit.