Evidence for a second, high affinity Gβγ binding site on Gα_i1 (GDP) subunits

It is well known that Gα_i1(GDP) binds strongly to Gβγ subunits to form the Gα _i1(GDP)-Gβγ heterotrimer, and that activation to Gα_i1(GTP) results in conformational changes that reduces its affinity for Gβγ subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Gα _i1(GDP) can bind a second Gβγ subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Gα_i1 and Gβγ subunits. Also, we find that phospholipase Cβ2, an effector of Gβγ, does not compete with the second binding site implying that effectors can be bound to the Gα _i1(GDP)-(Gβγ)2 complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site onGα_i1 competes with binding of the second Gβγ subunit. Injection of this peptide into cultured cells expressing eYFP-Gα _i1(GDP) and eCFP-Gβγ reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.