FIGURE 8.
Gαi3 W258F binds GAIP and LPAR1 as efficiently as wt Gαi3. A, mutation of Trp-258 to Phe does not affect Gαi3 interaction with GAIP. GDP·AlF4−-preloaded Gαi3 W258F (lane 6, upper panel) binds GAIP to the same extent as wt Gαi3 (lane 3, upper panel). Neither of the GDP-loaded G proteins binds GAIP (lanes 3 and 6, lower panel). ∼2 μg of purified wt His-Gαi3 (lanes 1–3) or His-Gαi3 W258F (lanes 4–6) pre-loaded with GDP·AlF4− (upper panel) or GDP (lower panel) were incubated with ∼5 μg of GST (lanes 2 and 5) or GST-GAIP (lanes 3 and 6) immobilized on glutathione beads. Bound proteins were analyzed by immunoblotting (IB) for His. 10% of the input His-Gα proteins were loaded in lanes 1 and 4. Equal loading of GST proteins was confirmed by Ponceau S staining. In B: Upper panel, FLAG-tagged LPAR1 (lanes 4–6) co-immunoprecipitates with both exogenously expressed wt Gαi3 (lane 5) and Gαi3 W258F mutant (lane 6). Endogenous Gαi3 (lane 4) is not detected in FLAG-LPAR1 immunoprecipitates, and no Gαi3 is present in FLAG-immunoprecipitates from controls (lanes 1–3). 48 h after transfection cells were harvested and lysates used for immunoprecipitation (IP) with anti-FLAG IgG (∼2 μg) as described under “Experimental Procedures.” IP was followed by immunoblotting (IB) for FLAG (LPAR1) and Gαi3. Equal IgG loading was confirmed by Ponceau S staining. Lower panel, aliquots of cell lysates (∼5%) were analyzed for expression of FLAG (LPAR1), Gαi3, and α-tubulin by immunoblotting (IB) to confirm the expression of the analyzed proteins.