| Abstrakt: |
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 (αi1, αi2, αo, αs, and αq) to associate with three β subunits (β1, β2, and β5) dimerized to a γ2subunit containing an amino-terminal hexahistidine-FLAG affinity tag (γ2HF). Sf9 insect cells were used to overexpress the recombinant proteins. The hexahistidine-FLAG sequence does not hinder the function of the β1γ2HFdimer as it can be specifically eluted from an αi1-agarose column with GDP and AlF4−, and purified β1γ2HFdimer stimulates type II adenylyl cyclase. The β1γ2HFand β2γ2HFdimers immobilized on an anti-FLAG affinity column bound all five α subunits tested, whereas the β5γ2HFdimer bound only αq. The ability of other α subunits to compete with the αqsubunit for binding to the β5γ2HFdimer was tested. Addition of increasing amounts of purified, recombinant αi1to the αqin a Sf9 cell extract did not decrease the amount of αqbound to the β5γ2HFcolumn. 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 β1γ2HFor β5γ2HFdimers, only αqbound to the β5γ2HFdimer. The αq-β5γ2HFinteraction on the column was functional as GDP, and AlF4−specifically eluted αqfrom the column. These results indicate that although the β1and β2subunits interact with α subunits from the αi, αs, and αqfamilies, the structurally divergent β5subunit only interacts with αq. |
