M3 muscarinic receptor interaction with phospholipase C beta3 determines its signaling efficiency

Phospholipase Cbeta (PLCbeta) enzymes are activated by G protein-coupled receptors (GPCRs), through receptor-catalyzed guanine nucleotide exchange on the Galphabetagamma heterotrimer containing Gq family G proteins. Activation of phosphoinositide (PI) hydrolysis has been successfully reconstituted using purified components of phospholipid vesicles, GPCR, Gq, and PLCbeta Thus it is often described as an outcome of diffusion-limited interactions between GPCRs, Gq, and PLCbeta however, this model does not incorporate evidence for spatial organization of PLC signaling in cells. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and PLCbeta Both expressed and endogenous M3R interacts with PLCbeta in coimmunoprecipitation experiments. Expression of unstimulated M3R in CHO cells promotes plasma membrane localization of YFP-PLCbeta3. Deletion of the PLCbeta3 C-terminus or deletion of the PLCbeta3 PDZ ligand inhibits coimmunoprecipitation with M3R and M3R-dependent PLCbeta3 plasma membrane localization. Purified PLCbeta3 binds directly to glutathione-S-transferase (GST-) fused M3R intracellular loops 2 and 3 (M3Ri2 and M3Ri3), as well as M3R C-terminus (M3RCT). PLCbeta3 binding to the N-terminus of M3Ri3 was inhibited when the PDZ ligand was removed. In assays using reconstituted purified components in vitro, M3Ri2, M3Ri3, and M3RCT potentiated Galphaq-dependent, but not Gbetagamma -dependent PLCbeta3 activation. At the N-terminus of M3Ri3 (M3Ri3N), PLCbeta3 binding is necessary for potentiating PLCbeta3 activation by Galpha Disruptions of a key W313 residue in M3Ri3N and of the PDZ ligand in PLCbeta3 together significantly hinder M3Ri3-mediated potentiation. We conclude that PDZ sensitivity in M3R-mediated PLCbeta3 binding and localization is attributable to a direct M3R-PLCbeta3 interaction. We propose that the M3 muscarinic receptor maximizes the efficiency of PLCbeta3 signaling, beyond its canonical role as a guanine nucleotide exchange factor for Galpha.