FIGURE 1.

Characterization of Gα_i/13 mutants. A, a primary sequence alignment of murine Gα₁₂, Gα₁₃, and Gα_q was generated using the program T-Coffee. Residues in Gα₁₃ analyzed by site-directed mutagenesis in this study are underlined. Secondary structure was assigned based on the crystal structure of the Gα_13/i1-p115 RH domain complex (Protein Data Bank entry 1SHZ) (17). B, the T274E and N278A mutations in Gα₁₃ impair Rho activation in cells. HeLa cells were transiently transfected with empty vector or the indicated Gα₁₃ QL construct. The luciferase activity of cell lysates was determined as described under “Experimental Procedures.” Total cell lysate was immunoblotted for either Gα₁₃ or GAPDH. Data are presented as the mean ± S.E. (error bars) of triplicate determinations from a single experiment, representative of three independent experiments with similar results. Data were analyzed by one-way analysis of variance followed by Dunnett's post-test. Statistically significant difference from Gα₁₃ QL is shown as follows. ns, not significant; **, p < 0.01. C, Gα_i/13 mutants can undergo activation-dependent conformational changes. Gα_i/13 (wild type or mutant) was subjected to limited trypsin digestion in the presence or absence of AlF₄⁻. After proteolysis, proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The primary protected species is indicated with an arrow. The positions of molecular mass standards, in kDa, are shown on the left.