Interaction of heterotrimeric G protein Galphao with Purkinje cell protein-2. Evidence for a novel nucleotide exchange factor.
The heterotrimeric G protein Galphao is ubiquitously expressed throughout the central nervous system, but many of its functions remain to be defined. To search for novel proteins that interact with Galphao, a mouse brain library was screened using the yeast two-hybrid interaction system. Pcp2 (Purkinje cell protein-2) was identified as a partner for Galphao in this system. Pcp2 is expressed in cerebellar Purkinje cells and retinal bipolar neurons, two locations where Galphao is also expressed. Pcp2 was first identified as a candidate gene to explain Purkinje cell degeneration in pcd mice (Nordquist, D. T., Kozak, C. A., and Orr, H. T. (1988) J. Neurosci. 8, 4780-4789), but its function remains unknown as Pcp2 knockout mice are normal (Mohn, A. R., Feddersen, R. M., Nguyen, M. S., and Koller, B. H. (1997) Mol. Cell. Neurosci. 9, 63-76). Galphao and Pcp2 binding was confirmed in vitro using glutathione S-transferase-Pcp2 fusion proteins and in vitro translated [35S]methionine-labeled Galphao. In addition, when Galphao and Pcp2 were cotransfected into COS cells, Galphao was detected in immunoprecipitates of Pcp2. To determine whether Pcp2 could modulate Galphao function, kinetic constants kcat and koff of bovine brain Galphao were determined in the presence and absence of Pcp2. Pcp2 stimulates GDP release from Galphao more than 5-fold without affecting kcat. These findings define a novel nucleotide exchange function for Pcp2 and suggest that the interaction between Pcp2 and Galphao is important to Purkinje cell function.
Luo
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Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Denker
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BM
D000818:Animals; D001704:Biopolymers; D019556:COS Cells; D002417:Cattle; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D011689:Purkinje Cells / Q000378:metabolism*
D001704:Biopolymers; D009419:Nerve Tissue Proteins; D006153:Guanosine Diphosphate; D019204:GTP-Binding Proteins