THE N-TERMINUS OF PHOSDUCIN IS INVOLVED IN BINDING OF BETA-GAMMA-SUBUNITS OF G-PROTEIN

Phosducin is a soluble phosphoprotein found in retinal photoreceptor c ells and in the pineal gland. It binds to the beta gamma subunits of g uanine nucleotide-binding proteins (G proteins) (G beta gamma) and may regulate G-protein function, In this study, the ability of specific r egions of phosducin to bind G beta gamma was characterized. A series o f deletion mutants were made in bovine phosducin, They were tested in cotransfection assays for their ability to inhibit G beta gamma-mediat ed phospholipase C beta(2) isoform activation, Overexpression of the N -terminal half of phosducin showed inhibition, whereas overexpression of the C-terminal half did not. The first 63 amino acid residues were required for inhibition. A tryptophan-to-valine substitution at residu e 29, which is part of a well conserved Il-amino acid sequence, severe ly impaired phosducin inhibitory function, Glutathione S-transferase-p hosducin fusion proteins were expressed in Escherichia coli to study p hosducin-G beta gamma interaction in vitro. The N-terminal 63-amino ac id fragment was able to bind to G beta gamma. In contrast, the C-termi nal half failed to bind to G beta gamma. The substitution mutants show ed little or no binding, Furthermore, direct measurements of interacti on between G beta gamma and fragments of phosducin, using surface plas mon resonance technology, confirmed the assignment of binding activity to the 63-amino acid fragment and the importance of the tryptophan re sidue.