Regulation of M₃ muscarinic acetylcholine receptor-mediated PLD stimulation by G₁₂-type G proteins

Objective: To determine the type of G protein mediating M₃mAChR-PLD coupling in comparison to M₃ mAChR-PLC coupling. Methods: we transiently transfected HEK-293 cells with empty expression vectors and expression plasmids for wild-type,constitutively active or dominant-negative mutants of Gα₁₂,Gα₁₃,Gα_q,respectively.After 48 h,PLD and PLC activities were determined by measuring accumulation of PtdEtOH and IP_x in the absence and presence of 1 mM carbachol,a typical agonist of M₃ mAChR.Expression of the individual Gα subunits were examined by immunobloting with their specific antibodies. Results: In HEK293 cells overexpressing wild-type either Gα₁₂ or Gα₁₃proteins,the PLD response to carbachol was increased by 2-fold compared to control cells,leaving basal PLD activity unaffected,although overexpression of wild-type either Gα₁₂ or Gα₁₃ did not modify M₃ mAChR signaling to PLC.Intriguingly,the Gα_q proteins potentiated greatly the PLC response to M₃ mAChR.Furthermore,the similar（more dramatic） effect on PLD and PLC coupling to M₃ mAChR compared to their wild-type proteins,was induced by constitutatively active mutants,Q229L Gα₁₂,Q226L Gα₁₃ and R183C Gα_q.In contrast,overexpression of dominant-negative mutants G228A Gα₁₂ and G225A Gα₁₃ inhibited the PLD stimulation by M₃ mAChR to a similar extent,around 50%,but no effect on PLD basal activity. Conclusion: These data suggested that the M₃mAChR expressed in HEK-293 cells signals to PLD via G₁₂ but not G_q-type G proteins.