The M₃ muscarinic receptor in 1321N1 cells and that heterologously expressed in COS7 cells activates phospholipase D (PLD) through an ARF-dependent PLD pathway. Contributions from other mediators such as PKC, Rho A and tyrosine kinases appear relatively minor and both ARF1 and 6 are thought to be involved, displaying co-immunoprecipitation with the M₃ receptor and in vitro binding to GST-fusion proteins of M₃ intracellular domains (Mitchell et al. 1998, 2003). Here we have used cellular signalling assays, co-immunoprecipitation and GST-fusion proteins to investigate two novel mechanisms that cause differential facilitation of M₃ signalling through the ARF-dependent PLD pathway relative to its conventional output, G_q/11-coupled phospholipase C (PLC). Transfection of the Gβγ-sequestering construct GRK2-(495-689), GRK-ct, clearly attenuated ARF-dependent PLD responses of M₃ and AT₁ receptors but not those of 5-HT_2A or P_2U receptors nor that evoked by phorbol ester, without affecting PLC responses. This suggests that ARF-dependent coupling to PLD significantly involves Gβγ in some but not other GPCRs. In co-immunoprecipitation experiments, GRK-ct reduced ARF1 association with the M₃ receptor and in GST-fusion protein experiments purified Gβγ increased ARF1 binding to both i3 and ct domains of the receptor and ARF6 binding to only i3. PLD but not PLC signalling was inhibited by Gβ constructs with mutations at specific loci within the Gβ:effector interface. In conjunction with this work we have been assessing the pathway selectivity of a positive allosteric modulator of the M₃ receptor, N-chloromethyl brucine (CMB) and a negative modulator, N-methyl strychnine (NMS) (Birdsall et al. 1999). CMB and NMS caused markedly greater increases and decreases respectively in the potency of M₃ receptor PLD responses than PLC responses. Current work is addressing the relationship between the allosteric mechanism for facilitation of ARF-dependent PLD activation and the molecular role of Gβγ.