Figure 5.

Conformational efficacy of N-terminal and C-terminal Gα₁₂ mutants uncoupled from RhoGEFs. (A) Trypsin protection of selected Gα₁₂ mutants. HEK293 cell lysates expressing the indicated variants of myc-Gα₁₂^QL, or unmodified myc-Gα₁₂^QL (12^QL), or the G²²⁸A variant of myc-Gα₁₂ (12^G228A) were subjected to trypsin protection assays as described in Methods. Samples were incubated 20 min at 30°C in the presence (+) or absence (−) of TPCK-treated trypsin, and were analyzed by SDS-PAGE and immunoblotting using J169 antibody (1:700 dilution). Small horizontal arrows indicate position of the trypsin-protected fragment in selected lanes. Data presented are representative of two or more independent experiments per sample. (B) Specificity of uncoupling in selected Gα₁₂ variants. For each cassette mutant of myc-Gα₁₂^QL (indicated at top), interaction with each Gα₁₂ target (indicated at left) was quantified as a pulldown:load ratio as described in Methods, and was calculated as a percent of the identical ratio determined for myc-Gα₁₂^QL within the same experiment. Values are indicated as follows: (++) = >60%, (+) = 20 to 60%, (−) = 0 to 20%. Interacting proteins are GST fusions of the following: RH domain of LARG (LARG), C-terminal 107 amino acids of heat shock protein-90 alpha (Hsp90), protein phosphatase-5 (PP5), scaffolding Aα subunit of protein phosphatase-2A (PP2A), C-terminal 98 amino acids of E-cadherin (E-cad). Values presented indicate the mean of two or more trials per interaction sample.