The G protein β5 subunit interacts selectively with the G(q) α subunit

Abstract

The diversity in the heterotrimeric G protein α, β, and γ subunits may allow selective protein-protein interactions and provide specificity for signaling pathways. We examined the ability of five α subunits (α(i1), a{i2), α(o), α(s), and α(q)) to associate with three β subunits (β1, β2, and β5) dimerized to a γ2 subunit containing an amino-terminal hexahistidine-FLAG affinity tag (γ(2HF)). Sf9 insect cells were used to overexpress the recombinant proteins. The hexahistidine-FLAG sequence does not hinder the function of the β1γ(2HF) dimer as it can be specifically eluted from an α(i1)-agarose column with GDP and AlF4/-, and purified β1γ(2HF) dimer stimulates type II adenylyl cyclase. The β1γ(2HF) and β2γ(2HF) dimers immobilized on an anti-FLAG affinity column bound all five α subunits tested, whereas the βγ(2HF) dimer bound only α(q). The ability of other α subunits to compete with the α(q) subunit for binding to the β5γ(2HF) dimer was tested. Addition of increasing amounts of purified, recombinant α(i1) to the α(q) in a Sf9 cell extract did not decrease the amount of α(q) bound to the β5γ(2HF) column. When G proteins in an extract of brain membranes were activated with GDP and AlF4/- and deactivated in the presence of equal amounts of the β1γ(2HF) or β5γ(2HF) dimers, only α(q) bound to the β5γ(2HF) dimer. The α(q)- β5γ(2HF) interaction on the column was functional as GDP, and AlF4/- specifically eluted α(q) from the column. These results indicate that although the β1 and β2 subunits interact with α subunits from the α(i), α(s), and α(q) families, the structurally divergent β5 subunit only interacts with α(q).