Identification of human liver cDNA clones whose products interact with G-protein β subunit of Schizosccharomyces pombe

Abstract

Heterotrimeric GTP binding proteins (G-proteins) are composed of α, β, and γ subunits and have important roles in the cell signalling from cell surface receptors to effectors. The a subunit as well as the βγ complex are known to interact with effector molecules to deliver specific signals to downstream genes. In an attempt to identify the molecules interacting directly with G-protein β subunit (Gβ) in signal pathway, G-protein β subunit gene of Schizosaccharomyces pombe was used as bait to screen a human liver cDNA library in a yeast two-hybrid system. When the coding region DNA of the S. pombe β subunit gene, gpb1+, was cloned into the 3′-end of the GAL4 DNA binding domain sequence and used to screen a human liver cDNA library cloned at the 3′-end of the GAL4 activation domain, 42 novel clones showing β-galactosidase activity were obtained from the 9×106 transformants. The sequence analysis of these clones revealed that the four clones contain sequence homologies with a receptor tyrosine kinase, a mammalian homolog of hsp40 (DNAJ), a NMDA receptor glutamate-binding protein, and a protein serine kinase. These four clones showed specific binding to G-protein β subunit of S. pombe in vitro. The characterization of these proteins in the G-protein-mediated signal pathway will be useful in elucidating the precise mechanism of G-protein function.