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. 2014 Apr 18;289(16):11206-18.
doi: 10.1074/jbc.M113.538546. Epub 2014 Mar 4.

M3 Muscarinic Receptor Interaction With Phospholipase C β3 Determines Its Signaling Efficiency

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Free PMC article

M3 Muscarinic Receptor Interaction With Phospholipase C β3 Determines Its Signaling Efficiency

Wei Kan et al. J Biol Chem. .
Free PMC article

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.

Keywords: G Protein-coupled Receptors (GPCR); G Proteins; Phosphatidylinositol Signaling; Phospholipase C; Protein-Protein Interactions; Scaffolding.

Figures

FIGURE 1.
FIGURE 1.
M3 muscarinic receptor stably interacts with PLCβ3. A, HEK293 cells were transfected with PLCβ3 and empty vector or PLCβ3 and 3xHA-M3R with and without stimulation with 100 μm carbachol for 5 min (* indicates treatment with carbachol). Cells were lysed, immunoprecipitated (IP), and immunoblotted (IB) for either PLCβ3 or HA as described under “Experimental Procedures.” Representative Western blots shown were from three or more independent experiments. B, M3R or PLCβ3 was immunoprecipitated (IP) from rat lung lysates and assayed for associated PLC activity as described under “Experimental Procedures.” The data were compiled from four independent assays, each with internal triplicates. C, YFP-PLCβ3 or YFP-PLCβ2 was expressed in CHO cells with empty vector or 3xHA-M3R, and cells were analyzed by live cell confocal microscopy as described under “Experimental Procedures.” Line scans from a to b are shown below each image. D, multiple images treated as in C were analyzed as described under “Experimental Procedures,” compiled, and plotted. E, YFP-PLCβ3, 3xHA-M3R, or YFP-PLCβ3+M3R was expressed in CHO cells. Surface M3R was immunostained in red as described under “Experimental Procedures.” Line scans are shown to the right of each image set. Error bars indicate S.E. *, p < 0.05; ***, p < 0.001; N.S., not significant. Ab, antibody.
FIGURE 2.
FIGURE 2.
Plasma membrane localization of PLCβ3 C terminus depends on M3R expression and binding. A, primary structure of PLCβ with PH domain (residues 1–147) followed by four EF hands, X and Y catalytic cores, C2 domain (residues 712–809), and CT (residues 845–1234). B, YFP-PLCβ3 fragment constructs were expressed in CHO cells. Either empty vector or 3xHA-M3R was cotransfected. Live cells were analyzed by confocal microscopy as described under “Experimental Procedures.” Line scans from a to b are shown below each image. C, multiple images treated as in B were analyzed as described under “Experimental Procedures,” compiled, and plotted. D, HEK293 cells were cotransfected with YFP-PLCβ3 CT (residues 845–1234) and 3xHA-M3R. Cells were lysed and immunoprecipitated (IP) with or without anti-HA specific antibody. Input lysate (rightmost lanes) and immunoprecipitated (left) samples were immunoblotted (IB) for either PLCβ3 or HA as described under “Experimental Procedures.” Representative Western blots shown were from two independent experiments. Error bars represent S.E. ***, p < 0.001; N.S., not significant.
FIGURE 3.
FIGURE 3.
Enrichment of PLCβ3 at plasma membrane by M3R and full binding of PLCβ3 to M3R require the PDZ ligand at the extreme C terminus of PLCβ3. A, deletion of PDZ ligand (NTQL, residues 1231–1234) in PLCβ3 resulted in loss of M3R-mediated enrichment of PLCβ3 at plasma membrane. YFP-PLCβ3 mutant constructs (ΔCT, 1–886; ΔPDZ, 1–1230) were expressed in CHO cells. Either empty vector or 3xHA-M3R was cotransfected. Live cells were analyzed by confocal microscopy as described under “Experimental Procedures.” Line scans from a to b are shown below each image. B, multiple images treated as in A were analyzed as described under “Experimental Procedures,” compiled, and plotted. Additional quantitation yielded the following results: PLCβ3ΔCT without M3R expression, 0 of 42 exhibited PM localization; PLCβ3ΔCT with M3R, 2 of 34 cells exhibited PM localization; PLCβ3ΔPDZ without M3R, 2 of 45 exhibited PM localization; PLCβ3ΔPDZ with M3R coexpression; 0 of 54 exhibited PM localization. C, PLCβ3ΔCT (1–886) or PLCβ3 full length was cotransfected in HEK293 cells with 3xHA-M3R. Cells were lysed and immunoprecipitated (IP) and immunoblotted (IB) for either PLCβ3 N terminus (NT) or HA as described under “Experimental Procedures.” Representative Western blots shown were from three independent experiments. D, untagged PLCβ3 full length, YFP-PLCβ3 full length, or YFP-PLCβ3ΔPDZ (1–1231) was cotransfected in HEK293 cells with 3xHA-M3R. Cells were lysed and immunoprecipitated (IP) and immunoblotted (IB) for either PLCβ3 N terminus (NT) or HA as in C. Representative Western blots shown were from four independent experiments. Error bars represent S.E.
FIGURE 4.
FIGURE 4.
Intracellular loops of M3 muscarinic receptor bind PLCβ3. A, fragments from the intracellular surface of M3R were expressed as GST fusion proteins demarcated as follows: M3Ri1, 92–104; M3Ri2, 165–184; M3Ri3N, 252–389; M3Ri3M, 352–469; M3Ri3C, 389–491; M3R/H8, 547–560; M3R/CT, 564–590; and M3R/H8-CT, 547–590. Each fragment was tested for binding to purified PLCβ3 as described under “Experimental Procedures.” Representative Western blots shown were from three independent experiments. B, M3Ri3 loop subfragments. C, the indicated fragments from B were tested for binding to purified PLCβ3. Representative Western blots shown were from three independent experiments. D, M3Ri3Na was tested for binding to various purified PLCβ3 proteins (FL; ΔPDZ, 1–1230; ΔCT, 1–886; each at 3 nm). Representative Western blots shown were from three independent experiments. IB, immunoblot.
FIGURE 5.
FIGURE 5.
Intracellular loops of M3 muscarinic receptor specifically enhance efficiency of Gαq-dependent activation of PLC. A, [3H]IP3 release from [3H]PIP2-labeled vesicles due to PLCβ3 activation by Gαq-GDP-AlF4 was measured as a function of M3R loop concentration as described under “Experimental Procedures.” B, IP3 release from [3H]PIP2-labeled vesicles due to PLCβ3 activation was measured in the presence of calcium only, Gβ1γ2, or Gαq-GDP-AlF4. Buffer (in which all fusion proteins were suspended) or a 300 nm concentration of GST, M3Ri2, M3Ri3Na, or M3R/H8-CT was added. C, PLCβ3 activation was measured as a function of [Gαq-Mg-GDP-AlF4]. A representative plot is shown. D, IP3 release from [3H]PIP2-labeled vesicles due to activation of PLCβ3 (filled) and PLCβ2 (empty) was measured in the presence of Gαq-GDP-AlF4. For C and D, M3Ri2, M3Ri3Na, and M3R/H8-CT were included at 200 nm. All assay results are representative of at least three independent experiments that contained internal triplicates per experimental condition. Error bars represent S.E.
FIGURE 6.
FIGURE 6.
Intracellular loops of M3 muscarinic receptor bind Gαq and Gβγ. A, similar to Fig. 4A, M3R constructs fused to GST were tested for binding to purified Gαq or Gβ1γ2 in a glutathione bead pulldown assay. Results were analyzed by Western blot. Nonspecific recognition for GST fusion proteins by Gαq antibody W082 is indicated by * as shown. Representative Western blots shown were each from three independent experiments. B, within M3Ri3Na (252–322): 1, residues 252–273; 2, residues 274–294; 3, residues 295–322; 4, residues 289–314; 5, residues 262–285; 6, residues 286–309. C, binding site mapping for PLCβ3, Gαq, and Gβγ at M3Ri3N residues 252–322. To map binding sites for PLCβ3 (top), Gαq (middle), or Gβγ (bottom), GST fusion proteins as described in B were used in a pulldown assay. Representative Western blots shown were each from two independent experiments. D, binding of PLCβ3 and different isoforms of G protein α subunits (Gαq, Gαs-long, and Gαolf) to M3Ri3Na fragments 3 and 4 was tested. Results were analyzed by Western blot. Representative Western blots are shown from three independent experiments. Fractions of original input were loaded in the leftmost lanes. Samples were also immunoblotted (IB) with anti-GST antibodies to validate loading equal amounts of protein.
FIGURE 7.
FIGURE 7.
Residues 309–314 of M3Ri3N and PDZ ligand of PLCβ3 are determinants for M3Ri3N-mediated potentiation of PLCβ3 activation. A, to identify M3Ri3Na residues that contribute to the potentiation of Gαq-dependent PLCβ3 activation, constructs M3Ri3Na, M3Ri3Na 2, M3Ri3Na 3, and M3Ri3Na 4 were tested. The phospholipase C assay and data analysis were performed as in Fig. 5. A representative plot from four independent experiments is shown. B, M3R-mediated potentiation of PLCβ3 activation was compared between PLCβ3 variants (PLCβ3 FL, 1–1234; PLCβ3ΔPDZ, 1–1230) at 30 and 60 nm M3Ri3Na. *, p < 0.05, paired Student's t test from seven independent experiments. Coomassie staining of purified PLCβ3 proteins (1.5 μg each) is shown on the right. C, PLCβ3 activation by Ca2+ only or Gαq was tested for purified PLCβ3 constructs. The data were compiled from seven independent assays. D, within M3Ri3Na 252–322, alanine-scanning mutagenesis was performed across residues 309RCHFWF314. To facilitate screening for mutations that affect binding, alanine mutant proteins were partially purified by GST-glutathione affinity chromatography. A representative Western blot from three independent experiments is shown. Coomassie staining of each loaded GST fusion protein is shown on the bottommost panel. E, each M3Ri3Na variant was tested at 30 nm. * denotes significantly different (p < 0.05) relative to i3WT-PLCβ3 FL. Data were compiled from at least four independent experiments. Coomassie staining of M3Ri3Na WT (i3wt), M3Ri3Na F312A (i3FA), and M3Ri3Na W313A (i3WA) proteins (1.5 μg each) that were purified using His6-nickel affinity is shown on the left. Error bars represent S.E. IB, immunoblot.
FIGURE 8.
FIGURE 8.
M3R interaction with PLCβ3 determines PLCβ3 signaling efficiency. M3R binding to PLCβ3 localizes PLCβ3 at the plasma membrane via the C-terminal tail of PLCβ3 and the intracellular loops and C-terminal tail of the receptor. This may result in an unfolding of the PLCβ3 enzyme to result in a more optimal interaction with G protein and substrate PIP2. Upon receptor activation, more PLCβ3 is recruited from the cytosol to the receptor. Gαq either prescaffolded to the receptor (not depicted) or recruited by collision coupling can then interact with the scaffolded PLC. PLC recruitment after activation could be due to direct interactions with activated Gαq, M3R, or both. EF, EF hands; XY, X and Y catalytic cores.

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