[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 (GABAA-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 beta-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 gamma 2 subunit of the GABAA 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 GABAA receptors, especially in response to the agents acting on the gamma 2 subunit.
Kanematsu
Takashi
T
Laboratory of Molecular Cellular Biochemistry, Faculty of Dental Science, and Station for Collaborative Research, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Hirata
Masato
M
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D020134:Catalytic Domain; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D054645:Protein Phosphatase 1; D011963:Receptors, GABA-A / Q000378:metabolism; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C116741:GABARAP protein, human; D008869:Microtubule-Associated Proteins; D011963:Receptors, GABA-A; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1