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Comparative Study
. 2008 Aug;74(2):338-47.
doi: 10.1124/mol.107.044750. Epub 2008 Apr 3.

M3 muscarinic acetylcholine receptor-mediated signaling is regulated by distinct mechanisms

Jiansong Luo  1 , John M Busillo, Jeffrey L Benovic
Affiliations
Comparative Study

M3 muscarinic acetylcholine receptor-mediated signaling is regulated by distinct mechanisms

Jiansong Luo et al. Mol Pharmacol. 2008 Aug.

Abstract

We have used RNA interference previously to demonstrate that G protein-coupled receptor kinase 2 (GRK2) regulates endogenously expressed H1 histamine receptor in human embryonic kidney 293 cells. In this report, we investigate the regulation of endogenously expressed M(3) muscarinic acetylcholine receptor (M(3) mAChR). We show that knockdown of GRK2, GRK3, or GRK6, but not GRK5, significantly increased carbachol-mediated calcium mobilization. Stable expression of wild-type GRK2 or a kinase-dead mutant (GRK2-K220R) reduced calcium mobilization after receptor activation, whereas GRK2 mutants defective in Galpha(q) binding (GRK2-D110A, GRK2-R106A, and GRK2-R106A/K220R) had no effect on calcium signaling, suggesting that GRK2 primarily regulates G(q) after M(3) mAChR activation. The knockdown of arrestin-2 or arrestin-3 also significantly increased carbachol-mediated calcium mobilization. Knockdown of GRK2 and the arrestins also significantly enhanced carbachol-mediated activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), whereas prolonged ERK1/2 activation was only observed with GRK2 or arrestin-3 knockdown. We also investigated the role of casein kinase-1alpha (CK1alpha) and found that knockdown of CK1alpha increased calcium mobilization but not ERK activation. In summary, our data suggest that multiple proteins dynamically regulate M(3) mAChR-mediated calcium signaling, whereas GRK2 and arrestin-3 play the primary role in regulating ERK activation.

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Figures

Figure 1.
Figure 1.
Characterization of the Muscarinic Acetylcholine Receptor Subtype Endogenously Expressed in HEK-293 Cells. A) HEK-293 cells loaded with the ratiometric calcium indicator Fura2/AM were incubated with 100 nM pirenzepine (orange), 1 μM p-FHHsiD (green), vehicle (red), or not pretreated (black) and stimulated with 100 μM carbachol. Changes in calcium mobilization were assayed by monitoring the change in Fura-2AM fluorescence. Shown is a representative tracing from three independent experiments. B) Following a 6 hr serum starve, HEK-293 cells were incubated with 100 nM pirenzepine, 1 μM p-FHHsiD, vehicle, or not pretreated and stimulated with 100 μM carbachol for the indicated times. Cells from a 6-well plate were harvested and equal amounts of total cellular lysate were separated by SDS-PAGE and probed for phospho-ERK1/2 as described in Materials and Methods. Shown is a representative immunoblot of three independent experiments. C) Cells were treated with Bis I (2.5 μM), Bis V (2.5 μM) or rottlerin (5 μM) for 30 min prior to stimulation with carbachol (100 μM) for 5 min or PMA (100 nM) for 15 min.
Figure 2.
Figure 2.
Knockdown of Endogenous GRK Isoforms in HEK-293 Cells. A) HEK-293 cells were transfected twice within a 24 hr interval with GRK-specific or non-specific control siRNA. 72 hr after the second transfection, cells were harvested and equal amounts of total cellular lysate was separated by 10% SDS-PAGE, transferred to nitrocellulose and incubated with the indicated antibodies. Blots were stripped and re-probed for α-tubulin to control for loading. Shown is a representative immunoblot. B) Mean relative level of GRK expression following siRNA quantified by densitometry from five separate experiments.
Figure 3.
Figure 3.
GRK-Mediated Regulation of Calcium Mobilization Following M3 mAchR Activation. A) Effect on calcium mobilization. 72 hr after the second siRNA transfection, HEK-293 cells were loaded with Fura2/AM and stimulated with 10 μM carbachol. B) Mean (+/− SEM) increase in the peak calcium transient following stimulation with 10 μM carbachol from five individual experiments (***p<0.001 using two-tailed T test). C) Representative immunoblot showing relative levels of GRK2 stably expressed in HEK-293 cells. D) Calcium mobilization in HEK-293 cells stably expressing bovine GRK2. Mean (+/− SEM) increase in peak calcium mobilization in cells expressing vector (pcDNA3), wild type, Gq-binding deficient (R106A; D110A), kinase-dead (K220R), or the Gq-binding deficient/kinase dead (R106A/K220R) bovine GRK2 (*p<0.05 for GRK2-K220R, ***p<0.001 for wild type GRK2).
Figure 4.
Figure 4.
Effect of Arrestin Knockdown on Calcium Mobilization Following M3 mAchR Activation. A) Cells were transfected with SMARTpool siRNA and harvested 72 hr later. Blots were incubated with a monoclonal antibody for arrestin-2 that cross-reacts with arrestin-3. Blots were stripped and re-probed for α-tubulin to control for loading. Shown is a representative immunoblot. B) Mean relative level of arrestin expression following siRNA quantified by densitometry from five separate experiments. C) Effect on calcium mobilization. Cells were harvested 72 hr post-transfection and processed as described previously. Shown is a representative calcium trace from five independent experiments. D) Mean (+/− SEM) increase in the peak calcium transient following stimulation with 100 μM carbachol from five individual experiments (***p<0.001 using two-tailed T test).
Figure 5.
Figure 5.
Effect of GRK and Arrestin Knockdown on M3 mAchR ERK Activation. A) Effect of GRK knockdown on ERK1/2 activation. Following a 6 hour serum starve, cells were treated with 100 μM carbachol for indicated times. Shown is a representative immunoblot from six independent experiments. B) Mean fold increase in ERK2 activation. Blots were incubated simultaneously with primary antibodies specific for phospho-ERK1/2 and total ERK2 overnight. Phospho-ERK1/2 fluorescence was normalized to total ERK2 fluorescence and data are presented as fold-increase in ERK2 activation over basal (n=6, +/− SEM; *p<0.05, **p<0.01). C) Effect of arrestin knockdown on ERK1/2 activation. Following a 6 hour serum starve, cells were treated with 100 μM carbachol for indicated times. Shown is a representative immunoblot from eight independent experiments. D) Mean fold increase in ERK2 activation. Blots were incubated simultaneously with primary antibodies specific for phospho-ERK1/2 and total ERK2 overnight. Phospho-ERK1/2 fluorescence was normalized to total ERK2 fluorescence and data are presented as fold-increase in ERK2 activation over basal (n=8, +/− SEM; **p<0.01).
Figure 6.
Figure 6.
Effect of CK1α Knockdown on M3 mAchR Signaling. A) 72 hr after the second siRNA transfection, cells were harvested and equal amounts of total cellular lysate were separated by SDS-PAGE and immunoblotted for CK1α using a specific antibody. Blots were stripped and re-probed for α-tubulin to control for loading, Shown is a representative immunoblot. B) Effect on calcium mobilization. 72 hr after the second siRNA transfection, cells were loaded with Fura-2/AM and stimulated with 100 μM carbachol. Shown is a representative tracing from four independent experiments (control: 103 ± 10 nM, CK1α siRNA: 163 ± 15 nM, p<0.01). C) Effect on ERK1/2 activation. Following a 6 hr serum starve, cells were stimulated with 100 μM carbachol for indicated times. Shown is a representative immunoblot from eight independent experiments. D) Mean activation of ERK2. Blots were incubated simultaneously with primary antibodies specific for phospho-ERK1/2 and total ERK2 overnight. Phospho-ERK1/2 fluorescence was normalized to total ERK2 fluorescence and data are presented as fold-increase over basal (n=8, +/− SEM).
Figure 7.
Figure 7.
Regulation of the Endogenous M3 mAchR in HEK-293 Cells. A) Carbachol binding to the M3 mAchR results in activation of the Gq family of heterotrimeric G proteins leading to the dissociation of Gαq and Gβγ. Activated Gαq activates PLC-β resulting in the hydrolysis of PIP2 to form the second messengers IP3 and DAG. IP3 interacts with the IP3 receptor located at the endoplasmic reticulum, resulting in a robust but transient increase in cytosolic calcium. The formation of DAG recruits and activates the novel PKC-isoform, PKC-δ. Once activated, PKC-δ leads to the activation of a Ras-Raf-MEK-ERK1/2 cascade. B) Phosphorylation of the M3 mAchR by GRK6 and possibly CK1α recruits arrestin-2 and arrestin-3 to the receptor, preventing further G protein activation and terminating signaling. In addition, arrestins are able to recruit diacylglycerol kinases (DGK) to the membrane and terminate the PKC-dependent arm of the signaling cascade. GRK2 and GRK3, through a conserved RGS-domain, are able to interact with and sequester free Gαq and prevent activation of PLC-β. This results in the inhibition of both calcium mobilization and activation of the ERK1/2 cascade. GRK2 is also able to regulate activation of the ERK1/2 cascade by interacting with and negatively regulating the activity of MEK1.
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