| Popis: |
GAIP is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by some G protein α subunits. In the present studies, we have examined the structural basis for the ability of GAIP to discriminate among members of the Gαi family. Gαi1, Gαi3, and Gαo interacted strongly with GAIP, whereas Gαi2 interacted weakly and Gαs did not interact at all. A chimeric G protein composed of a Gαi2N terminus and a Gαi1 C terminus interacted as strongly with GAIP as native Gαi1, whereas a chimeric N-terminal Gαi1 with a Gαi2 C terminus did not interact. These results suggest that the determinants responsible for GAIP selectivity between these two Gαis reside within the C-terminal GTPase domain of the G protein. To further localize residues contributing to G protein-GAIP selectivity, a panel of 15 site-directed Gαi1 and Gαi2 mutants were assayed. Of the Gαi1 mutants tested, only that containing a mutation at aspartate 229 located at the N terminus of Switch 3 did not interact with GAIP. Furthermore, the only Gαi2 variant that interacted strongly with GAIP contained a replacement of the corresponding Gαi2 Switch 3 residue (Ala230) with aspartate. To determine whether GAIP showed functional preferences for Gα subunits that correlate with the binding data, the ability of GAIP to enhance the GTPase activity of purified α subunits was tested. GAIP catalyzed a 3–5-fold increase in the rate of GTP hydrolysis by Gαi1 and Gαi2(A230D) but no increase in the rate of Gαi2 and less than a 2-fold increase in the rate of Gαi1(D229A) under the same conditions. Thus, GAIP was able to discriminate between Gαi1 and Gαi2 in both binding and functional assays, and in both cases residue 229/230 played a critical role in selective recognition. |
