Premium
Identification of a C‐terminal binding site for G‐protein βγ‐subunits in phosducin‐like protein
Author(s) -
Schröder Stefan,
Blüml Klaus,
Dees Christian,
Lohse Martin J
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(96)01483-4
Subject(s) - g protein , gtp binding protein regulators , gtpase , biochemistry , protein subunit , rhodopsin , biology , fusion protein , chemistry , signal transduction , recombinant dna , gene , retinal
Phosducin‐like protein (PhLP) has recently been identified as a ubiquitous inhibitor of G‐protein βγ‐subunit (G βγ )‐mediated signaling, with an affinity about 5‐fold lower than that of phosducin. The G βγ binding site of phosducin has been suggested to be contained in its N‐terminus. A region corresponding to this N‐terminus is lacking in PhLP, suggesting that PhLP must utilize a different mode of G βγ binding. To map the G βγ binding site in PhLP, a series of deletion mutants were constructed, expressed in E. coli as glutathione S ‐transferase (GST) fusion proteins, and the purified fusion proteins were examined for their ability to attenuate G o GTPase activity. Progressive N‐terminal truncations of PhLP caused only minor reductions in potency, whereas the complementary N‐terminal PhLP fragments turned out to be inactive. We further identified a short C‐terminal segment comprising residues 168 to 195 that inhibited G o GTPase activity similar in efficacy and potency to full‐length PhLP. This C‐terminal fragment was also capable of antagonizing a second G βγ ‐mediated function, the enhancement of rhodopsin phosphorylation by the β‐adrenergic receptor kinase. Taken together, these data indicate that PhLP interacts with G βγ via a short C‐terminal binding site which is distinct from that identified previously in phosducin.