Figure 2

Effects of 2A, 3A and 5A mutations on Gα ₁₆ -mediated PLCβ activation. (A) Positions of the alanine mutations on the corresponding Gα₁₆ mutants were shown as an alignment with the Gα₁₆ sequence. Identical residues were simplified with dots. (B) Top: COS-7 cells were transiently transfected with 0.25 μg/ml cDNAs encoding the wild type or QL mutants of Gα₁₆, 2A, 3A and 5A. Transfectants were labeled with [³H]myo-inositol and assayed for IP accumulation. * IP accumulation stimulated by constitutively active mutants was significantly higher than that obtained with their wild type counterparts; ‡ Constitutive activity was significantly lower than that obtained with Gα₁₆QL; Tukey-Kramer's test, p < 0.05. Bottom: Transfected COS-7 cells were harvested and membrane proteins were extracted for immunodetection. A Gα₁₆-specific custom antiserum was used for recognition of Gα₁₆ and its mutants. Fluorographs were visualized with the ECL chemiluminescence detection kit. Untransfected COS-7 cells served as the negative control. Two separate sets of transfected cells yielded similar results. (C) COS-7 cells were transfected with increasing amounts of cDNA encoding Gα₁₆QL, 2A-QL, 3A-QL or 5A-QL. Empty vector pcDNA3 was added to balance the amount of cDNA used in the transfection for each sample. Gα₁₆-transfected cells served as the negative control (hollow square). Top: IP production increased dose-dependently with increasing expression levels of the constitutively active form of alanine mutants and Gα₁₆. Bottom: Expression level of constitutively active counterparts of Gα₁₆ and its mutants were determined by Western blotting.