Abstract
Phospholipase Cβ (PLCβ) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on Gαβγ heterotrimers containing Gq family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and PLCβ3. Both expressed and endogenous M3R interacted with PLCβ in coimmunoprecipitation experiments. Stimulation of M3R with carbachol significantly increased this association. Expression of M3R in CHO cells promoted plasma membrane localization of YFP-PLCβ3. Deletion of the PLCβ3 C terminus or deletion of the PLCβ3 PDZ ligand inhibited coimmunoprecipitation with M3R and M3R-dependent PLCβ3 plasma membrane localization. Purified PLCβ3 bound directly to glutathione S-transferase (GST)-fused M3R intracellular loops 2 and 3 (M3Ri2 and M3Ri3) as well as M3R C terminus (M3R/H8-CT). PLCβ3 binding to M3Ri3 was inhibited when the PDZ ligand was removed. In assays using reconstituted purified components in vitro, M3Ri2, M3Ri3, and M3R/H8-CT potentiated Gαq-dependent but not Gβγ-dependent PLCβ3 activation. Disruption of key residues in M3Ri3N and of the PDZ ligand in PLCβ3 inhibited M3Ri3-mediated potentiation. We propose that the M3 muscarinic receptor maximizes the efficiency of PLCβ3 signaling beyond its canonical role as a guanine nucleotide exchange factor for Gα.
Highlights
Scaffolding of signaling proteins to G protein-coupled receptors (GPCRs) may increase signaling efficiency and spatial fidelity
The M3 Muscarinic Receptor Binds to Phospholipase C3— To determine whether PLC3 could form a complex with M3R, an N-terminal HA epitope-tagged M3R was coexpressed in HEK293 cells with PLC3, extracted, and immunoprecipitated with an anti-HA antibody
Treatment with carbachol led to a 2–3-fold (2.83 Ϯ 0.65-fold from five experiments, p ϭ 0.009) increase in PLC3 association with HA-M3R (Fig. 1A)
Summary
Scaffolding of signaling proteins to GPCRs may increase signaling efficiency and spatial fidelity. Examples of PLC-interacting PDZ scaffolds are NHERF1, NHERF2, PDZK1, and Shank that organize specific signaling complexes with parathyroid hormone receptor PTH1R [24], lysophosphatidic acid receptor LPA2R [25], somatostatin receptor [26], and. There is increasing recognition that PDZ-dependent organization is required for receptor-dependent activation of PLC [25, 28]; in a classical collision-coupling model, one would not expect physical scaffolding to be required for GPCR-dependent effector activation. The direct binding interaction between M3R and PLC3 may represent a regulatory mechanism for PLC signal output beyond receptor-stimulated nucleotide exchange on G␣q
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