TY - JOUR
T1 - The N terminus of phosducin is involved in binding of βγ subunits of G protein
AU - Xu, Jun
AU - Wu, Dianqing
AU - Slepak, Vladlen Z.
AU - Simon, Melvin I.
PY - 1995/3/14
Y1 - 1995/3/14
N2 - Phosducin is a soluble phosphoprotein found in retinal photoreceptor cells and in the pineal gland. It binds to the βγ, subunits of guanine nucleotide-binding proteins (G proteins) (Gβγ) and may regulate G-protein function. In this study, the ability of specific regions of phosducin to bind Gβγ was characterized. A series of deletion mutants were made in bovine phosducin. They were tested in cotransfection assays for their ability to inhibit Gβγ-mediated phospholipase C β2 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-Gβγ interaction in vitro. The N-terminal 63-amino acid fragment was able to bind to Gβγ. In contrast, the C-terminal half failed to bind to Gβγ. The substitution mutants showed little or no binding. Furthermore, direct measurements of interaction between Gβγ 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.
AB - Phosducin is a soluble phosphoprotein found in retinal photoreceptor cells and in the pineal gland. It binds to the βγ, subunits of guanine nucleotide-binding proteins (G proteins) (Gβγ) and may regulate G-protein function. In this study, the ability of specific regions of phosducin to bind Gβγ was characterized. A series of deletion mutants were made in bovine phosducin. They were tested in cotransfection assays for their ability to inhibit Gβγ-mediated phospholipase C β2 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-Gβγ interaction in vitro. The N-terminal 63-amino acid fragment was able to bind to Gβγ. In contrast, the C-terminal half failed to bind to Gβγ. The substitution mutants showed little or no binding. Furthermore, direct measurements of interaction between Gβγ 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.
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U2 - 10.1073/pnas.92.6.2086
DO - 10.1073/pnas.92.6.2086
M3 - Article
C2 - 7534410
AN - SCOPUS:0028968341
VL - 92
SP - 2086
EP - 2090
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 6
ER -