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 cells and in the pineal gland. It binds to the beta gamma subunits of guanine nucleotide-binding proteins (G proteins) (G beta gamma) and may regulate G-protein function, In this study, the ability of specific regions of phosducin to bind G beta gamma was characterized. A series of deletion mutants were made in bovine phosducin, They were tested in cotransfection assays for their ability to inhibit G beta gamma-mediated 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 residue 29, which is part of a well conserved Il-amino acid sequence, severely impaired phosducin inhibitory function, Glutathione S-transferase-phosducin fusion proteins were expressed in Escherichia coli to study phosducin-G beta gamma interaction in vitro. The N-terminal 63-amino acid fragment was able to bind to G beta gamma. In contrast, the C-terminal half failed to bind to G beta gamma. The substitution mutants showed little or no binding, Furthermore, direct measurements of interaction between G beta gamma and fragments of phosducin, using surface plasmon resonance technology, confirmed the assignment of binding activity to the 63-amino acid fragment and the importance of the tryptophan residue.