Interaction of the protein nucleobindin with G alpha i2, as revealed by the yeast two-hybrid system.
The heterotrimeric G protein, G alpha i2, transduces signals from seven membrane spanning receptors to effectors such as adenylyl cyclase and ion channels. The purpose of this study was to identify these or other cellular proteins that interact with G alpha i2 by use of the yeast two-hybrid system. A human B cell cDNA library was screened by this system using full length G alpha i2. Four positive colonies were obtained. Two of the four were identified as nucleobindin, a calcium binding protein and a putative antigen to which anti-nuclear antibodies are generated in mice with a disorder that resembles systemic lupus erythematosus. Nucleobindin has a leucine zipper, EF hands, and a signal peptide sequence and is thought to localize to the nucleus as well as being secreted. The specificity of intehraction between G alpha i2 and nucleobindin was confirmed by an in vitro binding assay using recombinant proteins. Transfection of G alpha i2 and nucleobindin in COS cells increased G alpha i2 expression relative to cells transfected with G alpha i2 and mock vector. Our results indicate that the yeast two-hybrid system provides a means to identify novel proteins that interact with G alpha proteins. Nucleobindin appears to represent one of those proteins.
Mochizuki
N
N
Department of Pharmacology, University of California San Diego, La Jolla 92093-0636, USA.
Hibi
M
M
Kanai
Y
Y
Insel
P A
PA
D000818:Animals; D000911:Antibodies, Monoclonal; D001402:B-Lymphocytes / Q000502:physiology; D001483:Base Sequence; D002135:Calcium-Binding Proteins; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D015723:Gene Library; D005982:Glutathione Transferase / Q000096:biosynthesis; D006133:Growth Substances / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D006133:Growth Substances; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D001616:beta-Galactosidase; D019204:GTP-Binding Proteins