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Am J Physiol Cell Physiol 292: C200-C208, 2007. First published August 2, 2006; doi:10.1152/ajpcell.00103.2006
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Inhibition of G_q-dependent PLC-1 activity by PKG and PKA is mediated by phosphorylation of RGS4 and GRK2

Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia

Submitted 3 March 2006 ; accepted in final form 24 July 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
In smooth muscle of the gut, G_q-coupled receptor agonists activate preferentially PLC-1 to stimulate phosphoinositide (PI) hydrolysis and inositol 1,4,5-trisphosphate (IP₃) generation and induce IP₃-dependent Ca²⁺ release. Inhibition of Ca²⁺ mobilization by cAMP- (PKA) and cGMP-dependent (PKG) protein kinases reflects inhibition of PI hydrolysis by both kinases and PKG-specific inhibitory phosphorylation of IP₃ receptor type I. The mechanism of inhibition of PLC-1-dependent PI hydrolysis has not been established. Neither G_q nor PLC-1 was directly phosphorylated by PKA or PKG in gastric smooth muscle cells. However, both kinases 1) phosphorylated regulator of G protein signaling 4 (RGS4) and induced its translocation from cytosol to plasma membrane, 2) enhanced ACh-stimulated association of RGS4 and G_q·GTP and intrinsic G_q·GTPase activity, and 3) inhibited ACh-stimulated PI hydrolysis. RGS4 phosphorylation and inhibition of PI hydrolysis were blocked by selective PKA and PKG inhibitors. Expression of RGS4(S52A), which lacks a PKA/PKG phosphorylation site, blocked the increase in GTPase activity and the decrease in PI hydrolysis induced by PKA and PKG. Blockade of PKA-dependent effects was only partial. Selective phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which contains a RGS domain, by PKA augmented ACh-stimulated GRK2:G_q·GTP association; both effects were blocked in cells expressing GRK2(S685A), which lacks a PKA phosphorylation site. Inhibition of PI hydrolysis induced by PKA was partly blocked in cells expressing GRK2(S685A) and completely blocked in cells coexpressing GRK2(S685A) and RGS4(S52A) or G_q(G188S), a G_q mutant that binds GRK2 but not RGS4. The results demonstrate that inhibition of PLC-1-dependent PI hydrolysis by PKA is mediated via stimulatory phosphorylation of RGS4 and GRK2, leading to rapid inactivation of G_q·GTP. PKG acts only via phosphorylation of RGS4.

regulators of G protein signaling; G protein-coupled receptor kinase 2; phospholipase C; cAMP-dependent protein kinase; cGMP-dependent protein kinase

Relaxation of contracted smooth muscle is mediated by cAMP- or cGMP-dependent protein kinase (PKA or PKG), which acts on several targets in the signaling cascades to induce dephosphorylation of MLC₂₀ (1, 15, 32). Relaxation of initial contraction correlates with the decrease in [Ca²⁺]_i, which could occur as the result of a decrease in IP₃ generation and/or inhibition of IP₃-induced Ca²⁺ release (16, 24). Only PKG is capable of phosphorylating IP₃ receptor type I (IP₃R-I) and inhibiting IP₃-induced Ca²⁺ release, whereas both PKA and PKG are capable of inhibiting IP₃ generation (12, 26, 27).

Several studies have examined whether inhibition of IP₃ generation reflects inhibitory phosphorylation of PLC- (2, 17, 37, 38). In various cell lines transfected with G protein subunits and PLC- isozymes, phosphorylation of Ser²⁶ and/or Ser¹¹⁰⁵ in PLC-3 by PKA or PKG inhibited G_q- or G_i-stimulated PLC-3 activity (37, 38). However, in cell lines or native cells, PLC-1 is not phosphorylated in vivo or in vitro by PKA or PKG (37). In smooth muscle cells, PLC-3 is preferentially activated by G_i, whereas PLC-1 is selectively activated by G_q (20, 22, 25, 28). The mechanism by which PLC-1 activity is inhibited by PKA or PKG could reflect a proximal step, such as inhibitory phosphorylation of G_q or stimulatory phosphorylation of a RGS protein.

The strength and duration of G·GTP signaling are regulated by a family of GTPase-activating proteins known as regulators of G protein signaling (RGS) (8). The proteins contain a RGS domain, capable of binding to activated G subunits and accelerating their intrinsic GTPase activity. Smooth muscle cells of the gut express several RGS proteins, including RGS3, RGS4, RGS6, RGS8, RGS12, and RGS16 (Huang J and Murthy KS, unpublished studies). Activation of G_q in these cells is regulated by RGS4 (10).

In the present study, we have shown that both PKA and PKG phosphorylated RGS4, enhancing its ability to bind G_q, and accelerated the intrinsic GTPase activity of G_q·GTP. Furthermore, PKA, but not PKG, phosphorylated G protein-coupled receptor kinase 2 (GRK2), which contains a RGS-like domain, enhancing its ability to bind G_q and accelerate the intrinsic GTPase activity of G_q·GTP. The rapid inactivation of G_q via phosphorylation of GRK2 and/or RGS4 resulted in inhibition of PLC-1 activity. The results provide firm evidence that PKA inhibits PLC-1 activity by phosphorylating both RGS4 and GRK2, thereby enhancing their ability to accelerate deactivation of G_q·GTP. In contrast, PKG inhibits PLC-1 activity by phosphorylating RGS4 only.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Gastric smooth muscle cell culture. Smooth muscle cells were isolated from the circular muscle layer of rabbit stomach by sequential enzymatic digestion in 25 mM HEPES medium as described previously (19, 24, 28, 29). Briefly, muscle strips were incubated at 31°C for 20 min in HEPES medium with type II collagenase (0.1%) and soybean trypsin inhibitor (0.1%). The partly digested strips were washed, and muscle cells were allowed to disperse spontaneously for 30 min. The cells were harvested by filtration through 500-µm Nitex and then centrifuged twice at 350 g for 10 min. Dispersed smooth muscle cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum until they attained confluence and were then passaged once for use in various studies (28, 29). Experiments were performed in both freshly dispersed muscle cells and in cultured muscle cells at confluence in first passage.

RGS4, GRK2, and G_q mutant constructs. The following mutants were constructed: 1) RGS4(S52A), which lacks a PKA/PKG phosphorylation site, 2) G_q(G188S), which binds GRK2 but not RGS protein (5–7), and 3) GRK2(S685A), which lacks a PKA phosphorylation site. These constructs were subcloned separately into the multiple cloning site (EcoRI) of the eukaryotic expression vector pEXV. Recombinant plasmid DNAs (2 µg each) were transiently transfected into cultured smooth muscle cells in first passage with the use of Lipofectamine Plus reagent for 48 h. The cells were cotransfected with 1 µg of pGreen Lantern-1 to monitor expression. Control cells were cotransfected with 2 µg of vector (pEXV) and 1 µg of pGreen Lantern-1 DNA. Transfection efficiency (75%) was monitored by the expression of green fluorescent protein using FITC filters (28, 29).

Assay of phosphoinositide hydrolysis. Phosphoinositide (PI) hydrolysis was measured as total inositol phosphate formation with the use of anion exchange chromatography as described previously (20, 22, 28). Freshly dispersed and cultured smooth muscle cells were labeled with myo-2-[³H]inositol (0.5 µCi/ml) in inositol-free medium. Muscle cells (2 x 10⁶ cells) were treated with isoproterenol or sodium nitroprusside (SNP) for 10 min and then with acetylcholine (ACh) for 60 s in 1 ml of 25 HEPES medium containing 115 mM NaCl, 5.8 mM KCl, 2.1 mM KH₂PO₄, 2 mM CaCl₂, 0.6 mM MgCl₂, and 14 mM glucose. The reaction was terminated by the addition of 940 µl of chloroform-methanol-HCl (50:100:1). After extraction with 340 µl of chloroform and 340 µl of H₂O, the aqueous phase was applied to DOWEX AG-1 columns. [³H]inositol phosphates were then eluted, and the radioactivity was determined by liquid scintillation.

Protein phosphorylation (RGS4, GRK2, PLC-1, PLC-3, and G_q). Phosphorylation of specific proteins was determined from the amount of ³²P incorporated into each protein after immunoprecipitation with specific antibody to RGS4, GRK2, PLC-1, PLC-3, or G_q. Briefly, cultured muscle cells were incubated with [³²P]orthophosphate for 24 h, whereas freshly dispersed cells were incubated with [³²P]orthophosphate for 4 h (21). Samples (1 ml) were then incubated with isoproterenol or SNP for 10 min in the presence or absence of inhibitors for PKA (myristoylated PKI) and PKG [Rp-8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (Rp-cGMPS)] (21). Cell lysates were separated by centrifugation at 13,000 g for 10 min at 4°C, precleared with 40 µl of protein A-Sepharose, and incubated with RGS4, GRK2, G_q, PLC-1, or PLC-3 antibody for 2 h at 4°C and with 40 µl of protein A-Sepharose for another 1 h. The immunoprecipitates were extracted with Laemmli sample buffer, boiled for 5 min, and separated by electrophoresis on SDS-PAGE. After transfer to polyvinylidene difluoride (PVDF) membranes, [³²P]RGS4, [³²P]GRK2, [³²P]G_q, [³²P]PLC-1, or [³²P]PLC-3 was visualized by autoradiography, and the amount of radioactivity in the band was measured using liquid scintillation. The results were expressed as counts per minute (cpm).

Immunoblot analysis of G_q-bound RGS4 and GRK2 proteins. Smooth muscle cells (3 x 10⁶ cells/ml) treated with ACh in the presence of isoproterenol or SNP were lysed by incubation for 30 min at 4°C in 10 mM Tris (pH 7.5), 50 mM NaCl, 1% Triton X-100, and 60 mM octyl glucoside, and the lysates were centrifuged at 15,000 g for 30 min. The supernatant was precleared by incubation with 40 µl of protein A-Sepharose for 4 h and then incubated overnight with the antibody to RGS4 or GRK2. Protein A-Sepharose was then added and incubated for another 2 h, and the mixture was centrifuged at 13,000 g for 5 min. The immunoprecipitates were washed four times in lysis buffer and boiled in Laemmli buffer. Samples were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibody to G_q subunit. After incubation with secondary antibody, the proteins were visualized. The intensity of the protein band on ECL film was determined using densitometry.

For translocation of RGS4 proteins, cells were treated with SNP or isoproterenol for different periods and the amount of RGS4 in cytosol and membrane fractions was quantified by Western blot analysis. Cell homogenates were prepared from freshly dispersed gastric smooth muscle cells in Tris·HCl (pH 7.5) medium containing 5 mM MgCl₂, 2 mM EDTA, 250 mM sucrose, 1 mM DTT, 1 mM PMSF, 20 µg/ml of leupeptin, and 20 µg/ml aprotinin. The suspension was centrifuged at 100,000 g for 30 min at 4°C, and the supernatant was collected as the cytosolic fraction. Pellets were suspended in Tris·HCl buffer medium containing 1% Triton X-100 and 1% sodium cholate. The extract was centrifuged at 1,000 g for 15 min, and the supernatant was collected as membrane fraction. Cytosolic and membrane proteins were resolved by SDS-PAGE, electrophoretically transferred onto PVDF membrane, and probed with RGS4 antibody. The bands were identified using enhanced chemiluminescence.

Assay for GTPase activity. GTPase activity was measured in smooth muscle membranes as described previously (30). Smooth muscle cells were incubated with ACh in the presence or absence of isoproterenol or SNP for various time periods. An equal amount of the membrane protein was incubated with 0.4 µCi of [³²P]GTP for 30 min at 31°C. The reaction was terminated by the addition of 750 µl of 5% activated charcoal containing 20 mM phosphoric acid, and after centrifugation at 6,000 g for 15 min, the amount of radioactivity in the supernatant (500 µl) was counted by liquid scintillation. Nonspecific release of ³²P in the absence of membrane protein was subtracted from the values. The results were expressed as counts per minute.

[³H]scopolamine binding to smooth muscle cells. Binding of [³H]scopolamine to dispersed muscle cells was done as described previously (23). Muscle cells were suspended in HEPES medium containing 1% bovine serum albumin. Triplicate 0.5-ml (10⁶ cell/ml) aliquots were incubated for 15 min with 1 nM [³H]scopolamine alone or in the presence of 10 µM ACh. Bound and free radioligands were separated by rapid filtration under reduced pressure through 5-µm polycarbonate Nucleophore filters. Nonspecific binding was calculated as the amount of radioactivity in the presence of 10 µM ACh. [³H]scopolamine binding was measured in the control cells and in cells pretreated for 10 min with isoproterenol or SNP.

Measurement of relaxation in dispersed smooth muscle cells. Inhibition of ACh-induced contraction (i.e., relaxation) by SNP or isoproterenol was expressed as the decrease in maximal cell contraction induced by 0.1 µM ACh (24–26). The mean cell length of 50 muscle cells treated with ACh alone or in the presence of SNP (1 µM) or isoproterenol (1 µM) was measured using scanning micrometry and was compared with the length of untreated muscle cells (mean control cell length 102 ± 3 µM).

Materials. Myo-[³H]inositol and [³²P]orthophosphate were obtained from NEN Life Sciences Products (Boston, MA). Polyclonal antibodies to G_q, PLC-1, PLC-3, RGS4, and GRK2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Western blotting and chromatography material were obtained from Bio-Rad Laboratories (Hercules, CA). Collagenase and soybean trypsin inhibitor were obtained from Worthington Biochemical (Freehold, NJ). All other reagents were obtained from Sigma.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Inhibition of ACh-stimulated PI hydrolysis by PKA and PKG. Two relaxant agents were used to determine the ability of PKA and PKG to inhibit ACh-stimulated PI hydrolysis: SNP, which stimulates cGMP and selectively activates PKG at concentrations <1 µM, and isoproterenol, which stimulates cAMP and selectively activates PKA at concentrations <1 µM (21, 24). These agents were used in the presence or absence of selective inhibitors of PKA (myristoylated PKI_14–22 amide) and PKG (Rp-cGMPS) to evaluate the involvement each kinase in inhibition of PI hydrolysis.

As shown previously, ACh acts via G_q-coupled m3 receptors to activate PLC-1 and via G_i3-coupled m2 receptors to activate PLC-3 (28). In the presence of the m2 receptor antagonist methoctramine, ACh caused a concentration-dependent increase in PI hydrolysis in freshly dispersed smooth muscle cells (Fig. 1). Pretreatment of the cells with isoproterenol or SNP inhibited ACh-stimulated PI hydrolysis: the inhibition by isoproterenol was selectively blocked by myristoylated PKI, whereas the inhibition by SNP was selectively blocked by Rp-cGMPS (Fig. 1). Inhibition of maximally stimulated PI hydrolysis by isoproterenol (EC₅₀ 25 nM) and SNP (EC₅₀ 60 nM) was concentration dependent. These results suggested that selective activation of PKA by isoproterenol and PKG by SNP inhibited PI hydrolysis. Inhibition of maximally stimulated PI hydrolysis by isoproterenol and SNP was time dependent: inhibition was significant within 2 min (61 ± 5% with SNP and 68 ± 5% with isoproterenol) and reached a peak within 5 min (78 ± 6% with SNP and 82 ± 4% with isoproterenol) that was sustained for 10 min.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Inhibition of ACh-stimulated phosphoinositide (PI) hydrolysis in dispersed smooth muscle cells by cAMP- (PKA) and cGMP-dependent (PKG) protein kinases. PI hydrolysis in response to ACh was measured in muscle cells labeled with myo-[3H]inositol. Freshly dispersed muscle cells were pretreated with isoproterenol (Isop; 1 µM) or sodium nitroprusside (SNP; 1 µM) for 10 min in the presence and absence of myristoylated PKI14–22 amide (Myr-PKI; 1 µM) or Rp-8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (Rp-cGMPS; 1 µM). PI hydrolysis is expressed as [3H]inositol phosphate formation in counts per minute (cpm) per mg protein above basal levels (485 ± 72 cpm/mg protein). Values are means ± SE of 4 experiments. **P < 0.01, significant inhibition of ACh-stimulated PI hydrolysis.

Phosphorylation of RGS4 by PKA and PKG and stimulation of RGS4:G_q·GTP association. Both isoproterenol and SNP phosphorylated RGS4 in freshly dispersed smooth muscle cells. Phosphorylation by isoproterenol was inhibited by myristoylated PKI but was not affected by Rp-cGMPS, whereas phosphorylation by SNP was inhibited by Rp-cGMPS but was not affected by myristoylated PKI (Fig. 2).

View larger version (14K): [in this window] [in a new window]   Fig. 2. Phosphorylation of regulator of G protein signaling 4 (RGS4) by PKA and PKG. Gastric smooth muscle cells labeled with 32P were incubated with isoproterenol (1 µM) or SNP (1 µM) for 10 min in the presence and absence of Myr-PKI (1 µM) or Rp-cGMPS (1 µM). Immunoprecipitates derived from 500 µg of protein using RGS4 antibody were separated on SDS-PAGE. Bands corresponding to [32P]RGS4 were identified using autoradiography. Radioactivity in the bands is expressed as cpm. Immunoblot analysis showed equal amounts of loaded protein. Values are means ± SE of 4 experiments. **P < 0.01, significant inhibition of isoproterenol- or SNP-induced RGS4 phosphorylation.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Translocation of RGS4 to plasma membrane upon phosphorylation by PKA and PKG. Dispersed smooth muscle cells were incubated for various time periods with isoproterenol (1 µM) or SNP (1 µM) in the presence and absence of Myr-PKI (1 µM) or Rp-cGMPS (1 µM). Immunoblot analysis using RGS4 antibody was performed on cytosolic and particulate fractions prepared from muscle cell homogenates. An equal amount of protein was loaded in each well (data not shown). The results shown are from a single experiment representative of at least 3 experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Stimulation of RGS4 association with Gq·GTP by PKA and PKG. Dispersed smooth muscle cells were treated with ACh (0.1 µM) for 1 min in the presence and absence of isoproterenol (1 µM) or SNP (1 µM). Immunoprecipitates derived from 500 µg of protein using RGS4 antibody were separated on SDS-PAGE and immunoblotted using Gq antibody. Values are means ± SE of 4 experiments. **P < 0.01, significant increase in ACh-stimulated Gq·GTP:RGS4 association by SNP and isoproterenol.

Augmentation of ACh-stimulated G·GTPase activity by PKA and PKG.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Stimulation of G·GTPase activity by PKA and PKG. Dispersed smooth muscle cells were treated with ACh for various time periods in the presence and absence of isoproterenol or SNP. G·GTPase activity was determined in membrane fractions labeled with [32P]GTP as described in MATERIALS AND METHODS, and the results are expressed as cpm. Values are means ± SE of 4 experiments. **P < 0.01, significant increase in ACh-stimulated G·GTPase activity by isoproterenol or SNP.

Effect of RGS4 and G_q mutants on ACh-stimulated G_q:RGS4 association and GTPase and PLC-1 activities.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Blockade of isoproterenol- or SNP-induced increase in Gq·GTPase activity and decrease in PI hydrolysis in cells expressing phosphorylation-deficient RGS4 mutant. A: cultured smooth muscle cells expressing wild-type RGS4 (vector alone) or RGS4(S52A), which lacks a PKA/PKG phosphorylation site, were treated with isoproterenol (1 µM) or SNP (1 µM) for 10 min and then with ACh (0.1 µM) for 2 min. Gq·GTPase activity was determined in membrane fractions labeled with [32P]GTP as described in MATERIALS AND METHODS, and the results are expressed as cpm. Values are means ± SE of 4 experiments. The increase in ACh-stimulated Gq·GTPase activity induced by isoproterenol or SNP was significantly blocked (**P < 0.01) in cells expressing RGS4(S52A). B: cultured smooth muscle cells expressing wild-type RGS4 (vector alone) or RGS4(S52A) were treated with isoproterenol (1 µM) or SNP (1 µM) for 10 min and then with ACh (0.1 µM) for 1 min. PI hydrolysis was measured in cells labeled with myo-[3H]inositol and is expressed as total [3H]inositol phosphate in cpm/mg protein. Values are means ± SE of 4 experiments. The inhibition of ACh-stimulated PI hydrolysis by isoproterenol or SNP was significantly blocked (**P < 0.01) in cells expressing RGS4(S52A).

View larger version (18K): [in this window] [in a new window]   Fig. 7. Blockade of isoproterenol- or SNP-induced increase RGS4 association with Gq·GTP in cells expressing RGS(S52A). Smooth muscle cells expressing wild-type RGS4 (vector alone) or RGS4(S52A) were treated with ACh (0.1 µM) for 1 min in the presence and absence of isoproterenol (1 µM) or SNP (1 µM). Immunoprecipitates derived from 500 µg of protein using RGS4 antibody were separated on SDS-PAGE and immunoblotted using Gq antibody. Immunoblot analysis showed equal amounts of loaded protein. Values are means ± SE of 4 experiments. **P < 0.01, significant increase in ACh-stimulated Gq·GTP:RGS4 by isoproterenol or SNP in cells expressing wild-type RGS4 but not RGS4(S52A).

View larger version (14K): [in this window] [in a new window]   Fig. 8. Blockade of isoproterenol- or SNP-induced inhibition of PI hydrolysis in cells expressing Gq mutant. Cultured smooth muscle cells expressing wild-type Gq (vector alone) or Gq(G188S), which binds GRK2 but not RGS4, were treated with ACh (0.1 µM) for 1 min in the presence and absence of isoproterenol (1 µM) or SNP (1 µM). PI hydrolysis was measured in cells labeled with myo-[3H]inositol and is expressed as total [3H]inositol phosphate in cpm/mg protein above basal levels (512 ± 76 to 548 ± 87 cpm/mg protein). Values are means ± SE of 4 experiments. **P < 0.01, significant inhibition of ACh-stimulated PI hydrolysis by isoproterenol and SNP in cells expressing wild type Gq. ##P < 0.01, significant inhibition of ACh-stimulated PI hydrolysis by isoproterenol only in cells expressing Gq(G188S).

Isoproterenol, but not SNP, induced GRK2 phosphorylation and increased GRK2:G_q·GTP association in the presence of ACh to the same extent in freshly dispersed and cultured smooth muscle cells. Both GRK2 phosphorylation and GRK2:G_q·GTP association were blocked in cultured muscle cells overexpressing GRK2(S685A) (Fig. 9). The mutated Ser⁶⁸⁵ site is one of three potential (RXS⁶⁸⁵) sites for phosphorylation by PKA in the COOH terminus of GRK2. Treatment of the cells with isoproterenol or ACh alone did not increase GRK2:G_q·GTP association, implying that GRK2 was selectively bound to activated G_q·GTP and the association was dependent on GRK2 phosphorylation by PKA (Fig. 9).

View larger version (15K): [in this window] [in a new window]   Fig. 9. Selective phosphorylation of GRK2 by PKA and blockade of isoproterenol-induced GRK2 phosphorylation and GRK2 association with Gq·GTP in cells expressing GRK2(S685A). A: dispersed smooth muscle cells labeled with 32P were incubated with isoproterenol (1 µM) or SNP (1 µM) for 10 min. Immunoprecipitates derived from 500 µg of protein using GRK2 antibody were separated on SDS-PAGE. Bands corresponding to [32P]GRK2 were identified using autoradiography. Radioactivity in the bands is expressed as cpm. Immunoblot analysis showed equal amounts of loaded protein. Values are means ± SE of 4 experiments. ** P < 0.01, significant increase in GRK2 phosphorylation. B: cultured muscle cells expressing wild-type GRK2 and GRK2(S685A) were incubated with isoproterenol for 10 min, and GRK2 phosphorylation was determined in GRK2 immunoprecipitates as described in A. C: cultured smooth muscle cells expressing wild-type GRK2 (vector alone) or GRK2(S685A) were treated with ACh (0.1 µM) for 1 min in the presence and absence of isoproterenol (1 µM). Immunoprecipitates derived from 500 µg of protein using GRK2 antibody were separated on SDS-PAGE and immunoblotted using Gq antibody. Values are means ± SE of 4 experiments. **P < 0.01, significant increase in ACh-stimulated Gq·GTP:GRK2 association by isoproterenol in cells expressing wild-type GRK2 but not GRK2(S685A).

View larger version (16K): [in this window] [in a new window]   Fig. 10. Inhibition of isoproterenol-induced increase in Gq·GTPase activity in cells expressing GRK2(S685A). Cultured smooth muscle cells expressing wild-type GRK2 (vector alone) or GRK2(S685A), which lacks a PKA phosphorylation site, were treated with ACh (0.1 µM) for 2 min in the presence and absence of isoproterenol (1 µM) or SNP (1 µM). Gq·GTPase activity was determined in membrane fractions labeled with [32P]GTP as described in METHODS, and the results are expressed as cpm above basal levels (1,465 ± 186 to 1,584 ± 184 cpm). Values are means ± SE of 4 experiments. The increase in ACh-stimulated Gq·GTPase activity induced by isoproterenol only was significantly blocked (**P < 0.01) in cells expressing GRK2(S685A).

View larger version (13K): [in this window] [in a new window]   Fig. 11. Selective blockade of isoproterenol-induced inhibition of PI hydrolysis in cells expressing GRK2(S685A). Cultured smooth muscle cells expressing wild-type GRK2 (vector alone) or GRK2(S685A), which lacks a PKA phosphorylation site, were treated with ACh (0.1 µM) for 1 min in the presence and absence of isoproterenol (1 µM) or SNP (1 µM). PI hydrolysis was measured in cells labeled with myo-[3H]inositol and is expressed as total [3H]inositol phosphate in cpm/mg protein above basal levels (461 ± 51 to 524 ± 68 cpm/mg protein). Values are means ± SE of 4 experiments. The inhibition of ACh-stimulated PI hydrolysis by isoproterenol only was significantly blocked (**P < 0.01) in cells expressing GRK2(S685A).

View larger version (17K): [in this window] [in a new window]   Fig. 12. Blockade of isoproterenol-induced inhibition of PI hydrolysis in cells coexpressing GRK2(S685A) with RGS4 or Gq mutants. Cultured smooth muscle cells coexpressing GRK2(S685A) with RGS4(S52A) or Gq(G188S) were treated with ACh (0.1 µM) for 1 min in the presence and absence of isoproterenol (1 µM) or SNP (1 µM). PI hydrolysis was measured in muscle cells labeled with myo-[3H]inositol and is expressed as [3H]inositol phosphate formation in cpm/mg protein above basal levels (494 ± 68 to 553 ± 82 cpm/mg protein). Values are means ± SE of 4 experiments. **P < 0.01, significant inhibition of ACh-stimulated PI hydrolysis.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Inhibition of agonist-induced Ca²⁺ mobilization in smooth muscle by PKA and PKG reflects the net effect of three processes: a proximal process involving inhibition of IP₃ generation by both kinases and two distal, PKG-specific processes involving phosphorylation of IP₃R-I, leading to inhibition of IP₃-dependent Ca²⁺ release and phosphorylation of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2), leading to Ca²⁺ sequestration in sarcoplasmic stores (1, 12, 15, 16, 24, 26, 27, 35) . The mechanism for inhibition of IP₃ generation by PKA in particular is not known but could involve phosphorylation of the receptor, G protein, a G protein regulator (i.e., RGS protein), or a PLC- isozyme.

In smooth muscle of the gut, G_q-coupled receptor agonists preferentially activate PLC-1 to stimulate PI hydrolysis and IP₃ generation (18, 28). As shown previously (37, 38) and as confirmed in the present study, PLC-1, unlike PLC-3, is not a substrate for inhibitory phosphorylation by PKA or PKG. Involvement of a RGS protein is suggested by studies in which PKG inhibited PI hydrolysis in aortic smooth muscle cells expressing wild-type but not dominant negative RGS2 (9, 36). PKG phosphorylated RGS2 and inhibited PLC-1 activity in aortic muscle cells from control but not RGS^–/– mice (9, 36). In the present study we have shown that PKA phosphorylates RGS4 and GRK2, which together regulate G_q activity in smooth muscle cells of the gut, whereas PKG selectively phosphorylates RGS4. The stimulatory phosphorylation of RGS4 and GRK2 accelerates deactivation of G_q, resulting in inhibition of PLC-1 activity.

RGS proteins bind to switch regions in the activated G subunit and increase its intrinsic GTPase activity (8). The ability of RGS4 and GRK2 to bind G_q·GTP and enhance GTPase activity was augmented upon phosphorylation of both RGS4 and GRK2 by PKA or of RGS4 only by PKG. The rapid inactivation of G_q·GTP precluded further activation of PLC-1. Several lines of evidence support this notion. First, PKG and PKA induced phosphorylation of RGS4, causing its translocation to the plasma membrane and increasing its association with activated G_q, enhanced ACh-stimulated GTPase activity, and inhibited ACh-stimulated PI hydrolysis. All these effects were dependent on phosphorylation of RGS4 in the case of PKG but only partially dependent on RGS4 in the case of PKA. Expression of phosphorylation-deficient RGS4(S52A) abolished RGS4:G_q association and blocked completely (for PKG) or partly (for PKA) the increase in GTPase activity and the inhibition of PI hydrolysis. Unlike PKG, PKA induced phosphorylation of GRK2, leading to an increase in GRK2:G_q association that contributed to the increase in GTPase activity and the decrease in PI hydrolysis; coexpression of phosphorylation-deficient RGS4(S52A) and GRK2(S685A) completely blocked the inhibition of PI hydrolysis induced by PKA.

Expression of a G_q(G188S), which binds GRK2 but not RGS4 (5, 6), shed further light on the roles of RGS4 and GRK2 in mediating inhibition of PI hydrolysis by PKA and PKG. In the absence of RGS4 binding, ACh-stimulated PI hydrolysis was not inhibited by PKG, and was only partly inhibited by PKA; the partial inhibition by PKA reflected the effect of GRK2, since coexpression of G_q(G188S) and GRK2(S685A) completely blocked the inhibition induced by PKA. The pattern provided further evidence for the participation of RGS4 and GRK2 in inhibition of PI hydrolysis by PKA. Although earlier studies in cell lines had shown that GRK2 bound to activated forms of G_q, G₁₁, and G₁₄ and inhibited G-mediated PI hydrolysis (4), the present study is the first to demonstrate that phosphorylation of GRK2 by PKA augments GRK2 binding to G_q·GTP and enhances the intrinsic GTPase activity of G_q, resulting in inhibition of PI hydrolysis; furthermore, the effect was specific to PKA.

It is worth noting that ACh stimulated PI hydrolysis and GTPase activity and induced association of G_q with RGS4 or GRK2 to the same extent in freshly dispersed and cultured gastric smooth muscle cells in first passage. Furthermore, isoproterenol and SNP inhibited PI hydrolysis, increased ACh-stimulated GTPase activity and association of G_q with GRK2 and/or RGS4 to the same extent in both cell preparations. These similarities provide further confirmation of the functional state of cultured smooth muscle cells.

Relaxation of smooth muscle was mediated by PKA and PKG, and they act on several targets to mediate inhibition of initial and sustained contraction, resulting in MLC₂₀ dephosphorylation and relaxation (18). Relaxation of initial contraction correlated with the decrease in cytosolic Ca²⁺, which could be achieved by inhibiting IP₃ generation and/or by decreasing Ca²⁺ release from or stimulating Ca²⁺ uptake into the sarcoplasmic stores. Our previous studies (24, 26, 27) have shown that both PKA and PKG inhibit agonist-stimulated Ca²⁺ release and that only PKG phosphorylates IP₃ receptors and inhibits Ca²⁺ release. The present studies identify a mechanism for inhibition of IP₃ generation by both PKA and PKG via phosphorylation of RGS4 and GRK2 and rapid inactivation of G_q. The importance of this mechanism is in its location at the inception of the signaling cascade that leads to Ca²⁺ release and MLC₂₀ phosphorylation during initial contraction. Inhibition of IP₃ formation and Ca²⁺ release by PKA and PKG is relevant only to the initial contraction mediated by Ca²⁺/calmodulin-dependent activation of MLC kinase and MLC₂₀ phosphorylation (18, 28). Sustained contraction is largely Ca²⁺ independent and reflects inhibition of MLC phosphatase via RhoA-dependent pathways involving phosphorylation of regulatory proteins MYPT1 and CPI-17 that mediate inhibition of MLC phosphatase (28). Relaxation of sustained contraction is mediated by both PKA and PKG via phosphorylation of RhoA at serine 188 and inhibition of RhoA activity (29).

In summary, we have identified a mechanism by which PKA and PKG inhibit agonist-induced PI hydrolysis in smooth muscle cells. Both PKA and PKG phosphorylate RGS4, stimulating its binding to G_q·GTP and enhancing its ability to accelerate the intrinsic GTPase activity of G_q. PKA, but not PKG, also phosphorylates GRK2, leading to a similar enhancement in GTPase activity. The rapid inactivation of G_q·GTP prevents further activation of PLC-1 and should lead to inhibition of IP₃ generation, IP₃-dependent Ca²⁺ release, and initial muscle contraction (Fig. 13). This appears to be the sole mechanism for relaxation mediated by PKA. Relaxation mediated by PKG involves inhibition of PI hydrolysis and IP₃-dependent Ca²⁺ release, as well as inhibitory phosphorylation of IP₃ receptors.

View larger version (26K): [in this window] [in a new window]   Fig. 13. Mechanism for inhibition of PI hydrolysis by PKA and PKG. Both PKA and PKG phosphorylate RGS4 (p-RGS4) and stimulate the intrinsic GTPase activity of Gq (Gq·GTP), leading to inhibition of PI hydrolysis (PLC- activity), inositol 1,4,5-trisphosphate (IP3) generation, IP3-dependent Ca2+ release, and muscle contraction. In addition, PKA, but not PKG, phosphorylates GRK2 (p-GRK2), leading to a similar enhancement in Gq·GTPase activity and inhibition of PI hydrolysis. PIP2, phosphatidylinositol 4,5-bisphosphate.

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	FOOTNOTES

 

Address for reprint requests and other correspondence: K. S. Murthy, PO Box 980551, Medical College of Virginia Campus, Virginia Commonwealth Univ., Richmond, VA 23298 (e-mail: skarnam{at}hsc.vcu.edu)

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