Identification of a C-terminal binding site for G-protein betagamma-subunits in phosducin-like protein.
Phosducin-like protein (PhLP) has recently been identified as a ubiquitous inhibitor of G-protein betagamma-subunit (G betagamma)-mediated signaling, with an affinity about 5-fold lower than that of phosducin. The G betagamma 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 betagamma binding. To map the G betagamma 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 G0 GTPase activity similar in efficacy and potency to full-length PhLP. This C-terminal fragment was also capable of antagonizing a second G betagamma-mediated function, the enhancement of rhodopsin phosphorylation by the beta-adrenergic receptor kinase. Taken together, these data indicate that PhLP interacts with G betagamma via a short C-terminal binding site which is distinct from that identified previously in phosducin.
Schröder
S
S
Institute of Pharmacology, University of Würzburg, Germany.
Blüml
K
K
Dees
C
C
Lohse
M J
MJ
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D002352:Carrier Proteins; D009419:Nerve Tissue Proteins; C098789:PDCL protein, human; D011993:Recombinant Fusion Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins