Interaction of the protein nucleobindin with Gαi2, as revealed by the yeast two‐hybrid system

Abstract

The heterotrimeric G protein, Gαi2, 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 Gαi2 by use of the yeast two-hybrid system. A human B cell cDNA library was screened by this system using full length Gαi2. 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 Gαi2 and nucleobindin was confirmed by an in vitro binding assay using recombinant proteins. Transfection of Gαi2 and nucleobindin in COS cells increased Gαi2 expression relative to cells transfected with Gαi2 and mock vector. Our results indicate that the yeast two-hybrid system provides a means to identify novel proteins that interact with Gα proteins. Nucleobindin appears to represent one of those proteins