Identification of a C-terminal binding site for G-protein βγ-subunits in phosducin-like protein

• Published in: FEBS letters 1997-01, Vol.401 (2), p.243-246
• Main Authors: Schröder, Stefan, Blüml, Klaus, Dees, Christian, Lohse, Martin J
• Format: Article
• Language: English
• Subjects: G-protein α-subunits; G-protein β-subunits; G-protein βγ-subunit; G-protein βγ-subunits; glutathione S-transferase; GST; Gβγ; Mas-7; mastoparan analogue (Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Ala-Leu-Leu-NH2); PhLP; Phosducin-like protein; phosducin-like protein (short variant); β-Adrenergic receptor kinase; β-adrenergic receptor kinase-1; βARK
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(96)01483-4

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.