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 11-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.
Xu
J
J
Division of Biology, California Institute of Technology, Pasadena 91125.
Wu
D
D
Slepak
V Z
VZ
Simon
M I
MI
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D000939:Epitopes / Q000032:analysis / Q000737:chemistry; D005136:Eye Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D020555:GTP-Binding Protein Regulators; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis; D007668:Kidney; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D014364:Tryptophan; D010738:Type C Phospholipases / Q000378:metabolism; D014633:Valine
D000939:Epitopes; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C065631:phosducin; D014364:Tryptophan; D005982:Glutathione Transferase; D011494:Protein Kinases; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D014633:Valine