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Am J Physiol Gastrointest Liver Physiol 292: G867-G874, 2007. First published December 14, 2006; doi:10.1152/ajpgi.00326.2006
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HORMONES AND SIGNALING

Cross-regulation of VPAC2 receptor desensitization by M3 receptors via PKC-mediated phosphorylation of RKIP and inhibition of GRK2

Jiean Huang, Sunila Mahavadi, Wimolpak Sriwai, John R. Grider, and Karnam S. Murthy

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

Submitted 21 July 2006 ; accepted in final form 4 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In gastrointestinal smooth muscle cells, VPAC2 receptor desensitization is exclusively mediated by G protein-coupled receptor kinase 2 (GRK2). The present study examined the mechanisms by which acetylcholine (ACh) acting via M3 receptors regulates GRK2-mediated VPAC2 receptor desensitization in gastric smooth muscle cells. Vasoactive intestinal peptide induced VPAC2 receptor phosphorylation, internalization, and desensitization in both freshly dispersed and cultured smooth muscle cells. Costimulation with ACh in the presence of M2 receptor antagonist (i.e., activation of M3 receptors) inhibited VPAC2 receptor phosphorylation, internalization, and desensitization. Inhibition was blocked by the selective protein kinase C (PKC) inhibitor bisindolylmaleimide, suggesting that the inhibition was mediated by PKC, derived from M3 receptor activation. Similar results were obtained by direct activation of PKC with phorbol myristate acetate. In the presence of the M2 receptor antagonist, ACh induced phosphorylation of Raf kinase inhibitory protein (RKIP), increased RKIP-GRK2 association, decreased RKIP-Raf-1 association, and stimulated ERK1/2 activity, suggesting that, upon phosphorylation by PKC, RKIP dissociates from its known target Raf to associate with, and block the activity of, GRK2. In muscle cells expressing RKIP(S153A), which lacks the PKC phosphorylation site, RKIP phosphorylation was blocked and the inhibitory effect of ACh on VPAC2 receptor phosphorylation, internalization, and desensitization and the stimulatory effect on ERK1/2 activation were abolished. This study identified a novel mechanism of cross-regulation of Gs-coupled receptor phosphorylation and internalization by Gq-coupled receptors. The mechanism involved phosphorylation of RKIP by PKC, switching RKIP from association with Raf-1 to association with, and inhibition of, GRK2.

internalization; signaling; smooth muscle

IN THE GASTROINTESTINAL TRACT, the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating peptide (PACAP) are colocalized in a subset of myenteric neurons that innervate the smooth muscle, and their release is functionally linked to smooth muscle relaxation (1, 13). The biological actions of VIP and PACAP are mediated by a family of G protein-coupled receptors, which are designated as VPAC1, VPAC2, and PAC1 receptors (9, 26). These receptors belong to class II of the G protein-coupled receptors known as the secretin receptor family that include receptors for secretin, gastric inhibitory peptide, glucagon, glucagon-like peptide 1, calcitonin, parathyroid hormone, growth hormone-releasing factor, and corticotrophin-releasing hormone (9, 23, 24, 26, 27). PAC1 receptors exhibit high affinity for PACAP and low affinity for VIP, whereas VPAC1 and VPAC2 receptors exhibit equal affinity for VIP and PACAP (9).

Smooth muscle cells of the gut express VPAC2 receptors, but not VPAC1 and PAC1 receptors (16, 25). We have recently shown that VPAC2 receptors are desensitized and internalized by a mechanism distinct from those utilized by VPAC1 or secretin receptors (15, 23, 24, 27, 31). VPAC2 receptors are exclusively phosphorylated by G protein-coupled receptor kinase 2 (GRK2), resulting in internalization of receptors and desensitization of VIP-induced adenylyl cyclase activity and cAMP formation. GRK2 consists of an NH2-terminal domain, a central catalytic domain, and a membrane-targeting COOH-terminal domain, which contains a pleckstrin homology domain for interaction with the Gbeta{gamma} subunits (7). This interaction facilitates targeting of GRK2 to the membrane and enhances its catalytic activity toward the receptor. GRK2 activity and subcellular localization are regulated by different mechanisms including binding to Gbeta{gamma}, activated receptor, phospholipids, Ca2+-binding protein calmodulin, caveolin, and anchoring proteins (2, 5, 8, 12, 22). In addition, phosphorylation by various kinases such as extracellular signal-regulated kinases (ERK1/2), c-Src, and cAMP-dependent kinase (PKA) regulates GRK2 activity (5, 22). Phosphorylation by PKA or c-Src increased GRK2 activity, whereas phosphorylation by ERK1/2 decreased GRK2 activity.

In contrast to the direct phosphorylation by kinases, a recent study identified a novel mechanism for inhibition of GRK2 activity involving protein kinase C (PKC) and Raf kinase inhibitory protein (RKIP) (14). RKIP binds to Raf-1 and prevents the activation of the ERK1/2 cascade (4, 14, 28, 29). However, phosphorylation of RKIP by PKC dissociates RKIP from Raf-1 and switches its binding to GRK2, resulting in inhibition of GRK2 activity and stimulation of ERK1/2 activity (14). These studies suggest that when multiple receptors are simultaneously activated the net effect of kinases such as PKA, ERK1/2, and PKC could modulate GRK2 activity. In this study, we provide evidence that phosphorylation, internalization, and desensitization of Gs-coupled VPAC2 receptors by GRK2 were inhibited by Gq-coupled M3 receptors. The mechanism involves phosphorylation of RKIP by PKC and phosphorylation-dependent switching of RKIP from its association with Raf-1 to association with GRK2 and inhibition of GRK2 activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Dispersion and culture of gastric smooth muscle cells. Smooth muscle cells were isolated from the circular muscle layer of the rabbit stomach by sequential enzymatic digestion, filtration, and centrifugation as described previously (17, 18, 20). Briefly, smooth muscle strips were incubated at 31°C for 20 min in HEPES medium [25 mM HEPES, 120 mM NaCl, 4 mM KCl, 2.6 mM KH2PO4, 0.6 mM MgCl2, 14 mM glucose, and 2.1% (vol/vol) Eagle's essential amino acid mixture (pH 7.4)] containing type II collagenase (0.1%) and soybean trypsin inhibitor (0.1%). The partly digested strips were washed with 50 ml of enzyme-free medium, and muscle cells were allowed to disperse spontaneously for 30 min. Muscle cells were harvested by filtration through 500-µm Nitex and centrifuged twice at 350 g for 10 min. Dispersed smooth muscle cells were cultured in DMEM containing 10% fetal bovine serum until they attained confluence and were then passaged once for use in various studies (11, 20, 30).

RKIP mutant constructs. PKC phosphorylation site-deficient RKIP [RKIP(S153A)] was subcloned into the multiple cloning site (EcoRI) of the eukaryotic expression vector pEXV. Smooth muscle cells were transiently transfected with recombinant plasmid DNAs (2 µg each) in the first passage by use of Lipofectamine Plus reagent for 48 h. The cells were cotransfected with 1 µg of pGreen Lantern-1 to monitor the transfection efficiency (~75%) using FITC filters. Control cells were cotransfected with 2 µg of vector (pEXV) and 1 µg of pGreen Lantern-1 DNA (11, 30).

125I-labeled VIP binding to smooth muscle cells. Binding of 125I-labeled VIP to freshly dispersed and cultured muscle cells was done as described previously (17). Cultured muscle cells detached by incubation with 0.53 mM EDTA in phosphate-buffered saline (PBS) for 30 min were collected by centrifugation at 400 g and resuspended in DMEM containing BSA (0.1%), amastatin (10 µM), and phosphoramidon (1 µM). Cell aliquots (2 x 106 cells/ml) were incubated for 60 min at 4°C with 50 pM 125I-labeled VIP followed by separation of bound and free radioligands by rapid filtration through 5-µm polycarbonate Nucleophore filters. Nonspecific binding was calculated as the amount of radioactivity in the presence of 10 µM VIP. 125I-labeled VIP binding was measured in the control cells and in cells pretreated for 30 min with 1 µM VIP in the absence or presence of ACh (0.1 µM) plus M2 receptor antagonist methoctramine (0.1 µM) to induce VPAC2 receptor internalization.

Biotin labeling of surface receptors. Muscle cells were treated with VIP (1 µM) for 30 min in the presence or absence of ACh or phorbol myristate acetate (PMA) and then with PBS at 4°C followed by treatment with 10 mM sodium periodate for 30 min in the dark. After the removal of periodate by a wash with PBS, cells were incubated for 30 min at 4°C with 2 mM biotin LC-hydrazide (membrane-impermeable derivative of biotin) to conjugate glycoproteins with biotin (6). Cells were then solubilized in lysis buffer and incubated with antibody to VPAC2 receptors. The VPAC2 receptor immunoprecipitates were separated by electrophoresis on SDS-PAGE and then transferred to a nitrocellulose membrane. After incubation of membrane with horseradish peroxidase-conjugated streptavidin, the proteins were visualized by ECL reagent.

Phosphorylation of RKIP, VPAC2 receptor, and GRK2. Phosphorylation of VPAC2, RKIP, or GRK2 was measured in cells labeled with 32P followed by immunoprecipitation with antibody to RKIP, VPAC2, or GRK2. Samples (3 x 106 ml) were incubated with VIP (1 µM) or ACh (plus methoctramine) for 5 min and homogenized in lysis buffer [10 mM Tris (pH 7.5), 50 mM NaCl, 1% Triton X-100, and 60 mM octyl glucoside]. The cell lysates were incubated with antibody to RKIP, VPAC2, or GRK2 for 2 h at 4°C and with 40 µl of protein A-Sepharose for another 1 h. The immunoprecipitates were extracted with Laemmeli sample buffer, boiled for 5 min, and separated by SDS-PAGE. After transfer to nitrocellulose membranes, [32P]RKIP, [32P]GRK2, or [32P]VPAC2 was visualized by autoradiography, and the amount of radioactivity in the protein band was measured. The results were expressed as counts per minute (cpm) (19, 20).

RKIP-GRK2 and RKIP-Raf-1 association. RKIP-GRK2 and RKIP-Raf-1 association were measured by immunoblotting with antibody to GRK2 or Raf-1 in RKIP immunoprecipitates. Smooth muscle cells (3 x 106 cells/ml) treated with ACh (0.1 µM) in the presence of methoctramine (0.1 µM) were homogenized in lysis buffer, and then the lysates were centrifuged at 15,000 g for 30 min. The supernatant was precleared by an incubation with 40 µl of protein A-Sepharose for 4 h and then incubated overnight with the antibody to RKIP and then with protein A-Sepharose for another 2 h. The immunoprecipitates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibody to GRK2 or Raf-1. After incubation with secondary antibody, the proteins were visualized by enhanced chemiluminescence (11, 20).

Assay for adenylyl cyclase activity. Adenylyl cyclase activity was measured by using [{alpha}-32P]ATP as a substrate, as described previously (21). Muscle cell homogenates were incubated for 15 min at 37°C in 50 mM Tris·HCl (pH 7.4), 2 mM cAMP, 0.1 mM ATP, 1 mM isobutylmethyl xanthine, 5 mM MgCl2, 100 mM NaCl, 5 mM creatine phosphate, 50 U/ml creatine phosphokinase, and 0.5 mM [{alpha}-32P]ATP. The [32P]cAMP produced was collected by sequential chromatography on Dowex AG50W-4X and alumina columns. The results were expressed as picomoles of cAMP per milligram of protein per minute.

Radioimmunoassay for cAMP. cAMP levels were measured by radioimmunoassay as described previously (17, 21). Muscle cells were stimulated with 1 µM VIP for 60 s in the presence of 100 µM isobutylmethyl xanthine, and the reaction was terminated with 10% trichloroacetic acid. The samples were centrifuged and supernatant was extracted with diethyl ether and then lyophilized. The lyophilized samples were resuspended in 50 mM Na acetate (pH 6.2) and acetylated with triethylamine-acetic anhydride for 30 min. cAMP was measured in duplicate using 100-µl aliquots, and the results were expressed as picomoles per milligram of protein.

In vitro kinase assay for ERK1/2 activity. ERK1/2 activity was determined by immunokinase assay as described previously (30). Freshly dispersed or cultured muscle cells were treated with ACh (plus methoctramine) in the presence or absence of the PKC inhibitor bisindolylmaleimide for 5 min, and cell lysates were prepared. The cell lysates were precleared by centrifugation, and an aliquot was incubated with ERK1/2 antibody. The immunoprecipitates were washed twice with a phosphorylation buffer containing 10 mM MgCl2 and 40 mM HEPES (pH 7.4) and then incubated for 5 min with 5 µg of myelin basic protein. Kinase assay was initiated by the addition of 10 µCi of [{gamma}-32P]ATP (3,000 Ci/mmol) and 20 µM ATP. 32P-labeled myelin basic protein was adsorbed onto phosphocellulose disks. The extent of phosphorylation was determined from the radioactivity on phosphocellulose disks by liquid scintillation.

Assay for PKC activity. PKC activity was measured in the particulate fraction as described previously (10). Muscle cells (2 x 106 cells/ml) were incubated with ACh in the presence of methoctramine, and the reaction was terminated by rapid freezing. PKC activity was measured in membrane fractions by phosphorylation of myelin basic protein, and the results are expressed as cpm per milligram of protein.

Measurement of relaxation in dispersed smooth muscle cells. Inhibition of KCl-induced contraction (i.e., relaxation) by VIP was expressed as the decrease in maximal cell contraction induced by 20 mM KCl. The mean cell length of 50 muscle cells treated with KCl alone or KCl in the presence of VIP (1 µM) before and after desensitization VPAC2 receptors with VIP or VIP and ACh (plus methoctramine) was measured by scanning micrometry and was compared with the length of untreated muscle cells (mean control cell length: 105 ± 4 µm) (17–19).

Materials. 125I-labeled VIP, [{alpha}-32P]ATP, 125I-labeled cAMP, and [32P]orthophosphate were obtained from NEN Life Sciences Products (Boston, MA); polyclonal antibodies to VPAC2 receptors, RKIP, GRK2, and Raf-1 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); and all other reagents were from Sigma.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Inhibition of GRK2-mediated VPAC2 receptor phosphorylation by PKC. Treatment of freshly dispersed smooth muscle cells with VIP caused phosphorylation of VPAC2 receptor. Previous studies have shown that agonist-induced VPAC2 receptor phosphorylation was blocked in cells expressing dominant negative GRK2, but not in cells expressing dominant GRK3 or GRK5 (14, 31), suggesting that phosphorylation was mediated exclusively by GRK2. Stimulation of PKC by PMA or ACh in the presence of the M2 receptor antagonist methoctramine (i.e., by activation of M3 receptors) inhibited VPAC2 receptor phosphorylation (Fig. 1). The effect of PMA and ACh was blocked by the PKC inhibitor bisindolylmaleimide (1 µM) (Fig. 1), but not by the ERK1/2 inhibitor PD98059 (10 µM) or the p38 MAPK inhibitor SB203580 (1 µM) (data not shown). Activation of PKC by PMA, however, did not induce GRK2 phosphorylation (data not shown). These results imply that GRK2-mediated VPAC2 receptor phosphorylation was inhibited by concomitant activation of PKC and that the inhibitory effect of PKC was not due to phosphorylation of GRK2. Treatment of cells with ACh in the absence of methoctramine had no significant effect on VPAC2 receptor phosphorylation (data not shown), suggesting that activation of M2 receptors masks the effect of the M3 receptors on VPAC2 receptor phosphorylation.


Figure 1
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  		Fig. 1. Inhibition of VPAC2 receptor (VPAC2 R) phosphorylation by PKC. Freshly dispersed gastric smooth muscle cells labeled with 32P were incubated with VIP (1 µM) or VIP plus ACh (0.1 µM in the presence of methoctramine) or PMA (1 µM) for 5 min in the absence or presence of the PKC inhibitor bisindolylmaleimide (Bisindol; 1 µM). Immunoprecipitates derived from 500 µg protein by use of VPAC2 receptor antibody were separated on SDS-PAGE. Bands corresponding to VPAC2 receptor were identified by autoradiography. Radioactivity in the bands was expressed as counts per minute (cpm). Immunoblot analysis showed equal amounts of loaded protein (not shown). Values are means ± SE of 4 experiments. **P < 0.01, significant decrease in VPAC2 phosphorylation by VIP in the presence of ACh or PMA compared with phosphorylation by VIP alone. p-VPAC2 R, phosphorylated VPAC2 receptor.

 
Inhibition of VPAC2 receptor internalization by PKC. VPAC2 receptor internalization was assessed in freshly dispersed muscle cells by the decrease in 125I-labeled VIP binding to the surface receptors after treatment with VIP. Pretreatment with VIP caused a decrease in 125I-labeled VIP binding, suggesting agonist-induced internalization of VPAC2 receptors. Stimulation of PKC by PMA or ACh in the presence of methoctramine inhibited VPAC2 receptor internalization (Fig. 2A). The effect of PMA and ACh on VPAC2 receptor internalization was blocked by bisindolylmaleimide (Fig. 2A). In addition, the selective M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine also blocked the effect of ACh on VPAC2 receptor internalization (Fig. 2A). The results imply that VPAC2 receptor internalization was inhibited by concomitant activation of M3 receptors via PKC.


Figure 2
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  		Fig. 2. Inhibition of VPAC2 receptor internalization by PKC. A: freshly dispersed gastric muscle cells were incubated for 30 min with VIP (1 µM) or VIP plus ACh (in the presence of methoctramine) or PMA (1 µM) for 5 min in the absence or presence of bisindolylmaleimide (1 µM). After being washed, muscle cells were incubated for 60 min at 4°C with 50 pM 125I-labeled VIP. VPAC2 receptor internalization was assessed by the decrease in 125I-VIP binding to the surface receptors after pretreatment with VIP. Nonspecific binding was determined as binding of 125I-labeled VIP in the presence of 10 µM unlabeled VIP. Specific 125I-labeled VIP binding was expressed as counts per minute. Values are expressed as means ± SE of 4 experiments. **P < 0.01 significant inhibition of VPAC2 receptor internalization by ACh or PMA. B: freshly dispersed gastric muscle cells were labeled with biotin as described in MATERIALS AND METHODS. Cells were treated with VIP (1 µM) or VIP plus ACh (in the presence of methoctramine) or PMA (1 µM) for 30 min and biotin-labeled VPAC2 receptors were analyzed by Western blot in VPAC2 receptor immunoprecipitates. VPAC2 receptor internalization was assessed by the decrease in biotin-labeled VPAC2 receptors after pretreatment with VIP. Values are expressed as means ± SE of 3 experiments. **P < 0.01, significant inhibition of VPAC2 receptor internalization by ACh or PMA. 4-DAMP, 4-deoxyphenylacetoxy-N-methylpiperidine.

 
A similar pattern of VPAC2 receptor internalization was obtained by measurements of biotin labeling of surface VPAC2 receptors. Treatment of muscle cells with VIP for 30 min induced a significant decrease in surface VPAC2 receptors. Stimulation of PKC with PMA or ACh in the presence of methoctramine significantly attenuated the decrease in surface VPAC2 receptors (Fig. 2B).

Inhibition of VPAC2 receptor desensitization by PKC. VIP caused an increase in adenylyl cyclase activity in a concentration-dependent fashion (EC50 4 ± 1 nM), with a maximal stimulation at 1 µM VIP. After treatment of cells with VIP, there was a significant decrease in adenylyl cyclase activity at all concentration of VIP, suggesting that exposure of muscle cells to VIP induced desensitization of VPAC2 receptors (Fig. 3A). Treatment of muscle cell with VIP in the presence of ACh plus methoctramine attenuated VIP-induced desensitization of VPAC2 receptors (Fig. 3A). The effect of ACh was blocked by bisindolylmaleimide (data not shown), suggesting that attenuation was mediated by PKC. PMA or ACh, however, had no effect on adenylyl cyclase activity stimulated directly by forskolin (forskolin: 35.1 ± 5.0 pmol·mg protein–1·min–1; forskolin plus ACh: 33.2 ± 5.2 pmol·mg protein–1·min–1; forskolin plus PMA: 34.4 ± 3.8 pmol·mg protein–1·min–1). The results imply that the attenuation of desensitization by PKC was not due to its direct effect on adenylyl cyclase activity but rather to inhibition of agonist-induced phosphorylation and internalization of VPAC2 receptors.


Figure 3
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  		Fig. 3. Inhibition of VIP-induced desensitization by ACh. A: adenylyl cyclase (AC) activity in response to VIP (0.1 nM to 1 µM) was measured in freshly dispersed control muscle cells and in cells treated with VIP (1 µM) or VIP plus ACh (in the presence of methoctramine) for 30 min. AC activity was expressed as picomoles of cAMP formed per milligram protein per minute. B: cAMP formation in response to VIP (1 µM) was measured in control cells and in cells treated with VIP (1 µM) or VIP plus ACh (in the presence of methoctramine) or PMA (1 µM) in the absence or presence of bisindolylmaleimide for 30 min. cAMP was measured by radioimmunoassay, and results are expressed as pmol/mg protein. Values are expressed as means ± SE of 4 experiments. **P < 0.01, significant inhibition of VPAC2 receptor desensitization by ACh or PMA.

 
Similarly, VIP-induced cAMP generation was greatly inhibited after pretreatment of cells with VIP; inhibition was attenuated by costimulation with ACh or PMA and the effect of ACh or PMA was blocked by bisindolylmaleimide (Fig. 3B). The basal levels of cAMP were similar before and after pretreatment of cells with VIP.

Further corroboration of M3 receptor-induced attenuation of VPAC2 receptor desensitization was obtained in cells where only the M3 receptor type was preserved by selective receptor protection (17). In cells where only M3 receptors were preserved, VIP-induced cAMP generation was inhibited after pretreatment of cells with VIP and the extent inhibition (70 ± 4%) was similar to naive cells (72 ± 5%). Pretreatment of M3 receptor-preserved cells with VIP in the presence of ACh attenuated VPAC2 receptor desensitization (36 ± 2% inhibition), suggesting that coactivation of M3 receptors attenuates VPAC2 receptor desensitization.

Treatment of freshly dispersed smooth muscle cells with 20 mM KCl caused a peak contraction within 30 s. Contraction was measured as the mean decrease in control cell length (control cell length: 105 ± 4 µm; mean decrease in cell length: 36 ± 2 µm). Treatment of muscle cells for 60 s with VIP (1 µM) inhibited KCl-induced contraction (i.e., relaxation) by 72 ± 5%. Pretreatment of cells with VIP for 30 min to desensitize VPAC2 receptors significantly decreased relaxation (28 ± 5%) in response to subsequent addition of VIP, suggesting desensitization of functional response. Pretreatment of cells with VIP and ACh (in the presence of methoctramine) attenuated desensitization and partly restored relaxation (56 ± 3%).

Phosphorylation of RKIP by PKC Switches RKIP from Raf-1 to GRK2

In the absence of GRK2 phosphorylation by PKC, we examined the mechanism for the PKC-mediated attenuation of agonist-induced phosphorylation, internalization, and desensitization of VPAC2 receptors. Previous studies have shown that RKIP is a physiological inhibitor of Raf kinase (14, 28, 29). Phosphorylation of RKIP at Ser153 by PKC results in the dissociation of RKIP from its known target Raf-1 to associate with GRK2 and inhibition of GRK2 activity (14). ACh induced phosphorylation of RKIP, and the phosphorylation was blocked by bisindolylmaleimide (Fig. 4). In the basal state, RKIP was associated with Raf-1 but not with GRK2. Treatment with ACh induced RKIP-GRK2 association and decreased RKIP-Raf-1 association; the effect of ACh was blocked by bisindolylmaleimide, suggesting that PKC-induced phosphorylation switches RKIP from Raf-1 to GRK2 (Fig. 4). ACh-induced phosphorylation of RKIP and its switching from RKIP to GRK2 were blocked in cells expressing RKIP(S153A) (Fig. 4).


Figure 4
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  		Fig. 4. ACh-induced dissociation of Raf kinase inhibitory protein (RKIP) from Raf-1 and association with G protein-coupled receptor kinase 2 (GRK2) is mediated by PKC-dependent phosphorylation of RKIP. Cultured smooth muscle cells expressing wild-type RKIP or PKC phosphorylation-deficient RKIP [RKIP(S153A)] were treated with ACh (in the presence of methoctramine). In some experiments, bisindolylmaleimide (1 µM) was included with ACh. Cells were lysed and immunoprecipitated with antibody to RKIP and association of RKIP with GRK2 or Raf-1 was analyzed by Western blot using antibody to GRK2 or Raf-1. Phosphorylation of RKIP was identified in immunoprecipitates by autoradiography in cells labeled with 32P as described in MATERIALS AND METHODS. The experiments were repeated more than 3 times with similar results. Representative blots are shown.

 
Phosphorylation of RKIP by PKC Inhibits GRK2-Mediated VPAC2 Receptor Phosphorylation, Internalization, and Desensitization

The effect of phosphorylated RKIP on GRK2-mediated VPAC2 receptor phosphorylation, internalization, and desensitization was examined in cells expressing RKIP(S153A).

As in freshly dispersed smooth muscle cells, VIP induced VPAC2 receptor phosphorylation in cultured smooth muscle cells. Activation of M3 receptor (ACh in the presence of M2 receptor antagonist) inhibited VIP-induced VPAC2 receptor phosphorylation (Fig. 5). ACh-induced decrease in VPAC2 receptor phosphorylation was blocked in cells expressing RKIP(S153A) (Fig. 5).


Figure 5
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  		Fig. 5. Inhibition of VPAC2 receptor phosphorylation by ACh was blocked in cells expressing RKIP(S153A). Cultured smooth muscle cells expressing wild-type RKIP or RKIP(S153A) were treated with VIP (1 µM) or VIP plus ACh (in the presence of methoctramine) for 5 min. Cells were lysed and immunoprecipitated with antibody to VPAC2 receptor. Phosphorylation of VPAC2 receptors was identified in immunoprecipitates by autoradiography in cells labeled with 32P as described in MATERIALS AND METHODS. The radioactivity in the protein bands was measured and expressed as counts per minute. Immunoblot analysis showed equal amounts of loaded protein (not shown). Values are expressed as means ± SE of 4 experiments. **P < 0.01 significant decrease in VPAC2 receptor phosphorylation by ACh compared with phosphorylation by VIP alone.

 
Pretreatment of cultured muscle cells with VIP caused a decrease in 125I-VIP binding and VIP-induced cAMP formation, suggesting agonist-induced internalization and desensitization of VPAC2 receptors (Figs. 6 and 7). Activation of PKC via M3 receptors inhibited VPAC2 receptor internalization and desensitization (Figs. 6 and 7). ACh-induced decrease in VPAC2 receptor internalization and desensitization were blocked in cells expressing RKIP(S153A) (Figs. 6 and 7). These results provide evidence that the inhibitory effect of ACh on GRK2-dependent VPAC2 receptor phosphorylation, internalization, and desensitization was mediated via phosphorylation of RKIP by PKC, which in turn led to switching of RKIP from Raf-1 to GRK2 and inhibition of GRK2 activity.


Figure 6
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  		Fig. 6. Inhibition of VPAC2 receptor internalization by ACh was blocked in cells expressing RKIP(S153A). Cultured smooth muscle cells expressing wild-type RKIP or RKIP(S153A) were treated with VIP (1 µM) or VIP plus ACh (in the presence of methoctramine) for 30 min. VPAC2 receptor internalization was assessed by the decrease in 125I-labeled VIP binding to the surface receptors after pretreatment with VIP or VIP plus ACh. Specific 125I-labeled VIP binding was expressed as counts per minute. Values are expressed as means ± SE of 4 experiments. **P < 0.01, significant decrease in VPAC2 receptor internalization by ACh compared with internalization by VIP alone.

 

Figure 7
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  		Fig. 7. Inhibition of VPAC2 receptor desensitization by ACh was blocked in cells expressing RKIP(S153A). Cultured smooth muscle cells expressing wild-type RKIP or RKIP(S153A) were treated with VIP (1 µM) or VIP plus ACh (in the presence of methoctramine) for 30 min. cAMP formation in response to VIP (1 µM) was measured by radioimmunoassay and results expressed as pmol/mg protein. Values are expressed as means ± SE of 4 experiments. **P < 0.01, significant decrease in VPAC2 receptor desensitization by ACh compared with desensitization by VIP alone.

 
Phosphorylation of RKIP by PKC Augments ACh-Induced ERK1/2 Activation

We examined the possibility that ACh-induced switching of RKIP from Raf-1 could result in the augmentation of ACh-induced ERK1/2 activity. In freshly dispersed muscle cells, ACh induced activation of ERK1/2 (2,098 ± 257 cpm/mg protein above basal levels of 560 ± 89 cpm/mg protein). The effect of ACh on ERK1/2 activity was blocked by bisindolylmaleimide (438 ± 87 cpm/mg protein above basal levels). Similar PKC-sensitive activation of ERK1/2 by ACh was obtained in cultured muscle cells (Fig. 8). In cells expressing RKIP(S153A), however, activation of ERK1/2 by ACh was greatly inhibited (74 ± 5%) and bisindolylmaleimide had no significant effect on the residual ERK1/2 activity. Activation of PKC by ACh was similar in both the wild-type and RKIP(S153A), expressing cells suggesting that RKIP had no effect on PKC activity (Fig. 8). These results provide evidence that activation of ERK1/2 by ACh was mediated by PKC via phosphorylation of RKIP and subsequent switching of RKIP from Raf-1, thus unmasking activation of Raf-1-dependent pathways.


Figure 8
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  		Fig. 8. PKC-mediated activation of ERK1/2 by ACh was blocked in cells expressing RKIP(S153A). Cultured smooth muscle cells expressing wild-type RKIP or RKIP(S153A) were treated with ACh (plus methoctramine) in the presence or absence of bisindolylmaleimide for 5 min. ERK1/2 and PKC activity was measured as described in MATERIALS AND METHODS. Values are expressed as means ± SE of 4 experiments. **P < 0.001, significant decrease in ACh-induced ERK1/2 activation in cells expressing RKIP(S153A) compared with cells expressing wild-type RKIP.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study shows that activation of PKC, whether by contractile agonists (e.g., ACh) or PMA, inhibited VIP-induced phosphorylation, internalization, and desensitization of VPAC2 receptors in gastric muscle cells. The inhibition was mediated by phosphorylation of RKIP via PKC and subsequent dissociation of RKIP from Raf-1 and association with GRK2. In the basal state RKIP is associated with Raf-1. After phosphorylation by PKC, RKIP dissociates from its target Raf-1 to associate with GRK2 (14). Association of RKIP with GRK2 inhibits the ability of GRK2 to phosphorylate VPAC2 receptors leading to inhibition of VPAC2 receptor internalization and desensitization.

The essential role of RKIP and the inhibitory phosphorylation by PKC were reflected in measurements of VPAC2 receptor phosphorylation, internalization, and desensitization. ACh-induced inhibition of VPAC2 receptor phosphorylation, internalization (determined from the decrease in 125I-labeled VIP binding to VPAC2 receptors after VIP treatment), and desensitization (determined from the decrease in adenylyl cyclase activity and cAMP formation) was reversed by pretreatment of cells with the PKC inhibitor bisindolylmaleimide as well as in cells expressing RKIP(S153A).

In addition, ACh-induced dissociation of RKIP from Raf-1 and association with GRK2 were inhibited by bisindolylmaleimide and in cells expressing RKIP(S153A). After activation of PKC, phosphorylation-deficient RKIP remained bound to Raf-1 and did not switch to GRK2. These studies also provide evidence for the mechanism by which PKC derived from activation of Gq-coupled receptors activates ERK1/2 and an explanation of how PKC can physiologically regulate ERK1/2 in smooth muscle cells. The mechanism involves phosphorylation of RKIP by PKC on Ser153 and loss of inhibition on Raf-1 by switching phosphorylated RKIP from Raf-1 to GRK2. Previous studies have shown that although Raf-1 was directly phosphorylated by PKC, mutation of the phosphorylation sites had no effect on PKC-induced activation Raf-1, suggesting that phosphorylation of Raf-1 is not necessary for activation (4, 14).

RKIP is a member of the phosphatidylethnolamine-binding protein family and a physiological inhibitor of Raf-1 and GRK2 (14). In the basal state, RKIP binds specifically Raf-1, but not Ras, and thus inhibits the activation of ERK1/2 pathway (4, 14, 28, 29). RKIP is not a kinase or a substrate for Raf-1, but it specifically inhibits Raf-1 interaction with its downstream target MEK and thus suppresses ERK1/2 activity. Translocation and activation of PKC results in phosphorylation of membrane-bound (phospholipid-bound) RKIP on Ser153 and removal RKIP's inhibition of Raf-1 (14). In vitro studies have shown that RKIP is a substrate for both classical and atypical PKCs, including PKC-{alpha}, -betaI, -betaII, -{gamma}, -{zeta}, but not novel PKCs, including PKC-{delta}, -{epsilon}, and -{eta}, suggesting that RKIP phosphorylation by PKC is isoform specific (4). Phosphorylation by PKC also switches RKIP's association from Raf-1 to GRK2, leading to inhibition of GRK2 activity (14). It was estimated that the cellular stoichiometries are sufficient for Raf-1 to bind all RKIP molecules under basal conditions and for RKIP to bind all GRK2 molecules upon stimulation. In vitro studies have shown that GRK2, but not GRK5, PKA, or PKC, was specifically inhibited by RKIP (14). Studies using purified RKIP have also shown that RKIP inhibited GRK2-mediated phosphorylation of the rhodopsin receptor (14). A physiological role for RKIP as a GRK2 inhibitor was demonstrated in cardiomyocytes where downregulation of endogenous RKIP expression by a RKIP antisense or short interfering RNA restrained beta-adrenergic signaling and contractile activity (14).

GRK2 plays an important role in agonist-induced phosphorylation of the receptor resulting in internalization and desensitization of G protein-coupled receptors (2, 7, 8). GRK2 requires Gbeta{gamma} for its recruitment to the membrane and activation. Recent studies (31) have shown that, in gastric smooth muscle cells, Gs-coupled VPAC2 receptor phosphorylation, internalization, and desensitization were exclusively mediated by activation of GRK2 and that the effect of GRK2 is augmented by feedback phosphorylation of GRK2 on Ser685 by PKA. The mechanism by which PKA-induced phosphorylation augments GRK2 activity appears to be by increasing its binding to Gbeta{gamma} and recruitment to the membrane (3, 31). GRK2, however, is not directly phosphorylated by PKC (data not shown). In contrast to PKA, PKC inhibits GRK2 activity and the mechanism of inhibition does not appear to involve inhibition of its interaction with Gbeta{gamma}. Since the binding domain for RKIP is in the NH2-terminus domain of GRK2, which is important for catalytic activity and for interaction with the receptor, association of RKIP with GRK2 could inhibit the catalytic activity and/or interaction with the receptor for phosphorylation (14).

In summary, the present studies provide evidence that in gastrointestinal smooth muscle concomitant activation of Gq-coupled M3 receptors cross-regulates signaling by Gs-coupled VPAC2 receptors. Activation of PKC by Gq-coupled receptors inhibits VPAC2 receptor phosphorylation, internalization, and desensitization and the mechanism involves PKC-dependent switching of RKIP from Raf-1 to GRK2 and inhibition of GRK2 activity (Fig. 9). The functional significance of this mechanism may reside where there is overlap in the release of Gq-coupled excitatory transmitters (e.g., ACh) with the Gs-coupled inhibitory transmitters (e.g., VIP) during the peristalsis of the gut. These findings are also important in the interpretation of the results in experiments designed to characterize the inhibitory function of VIP in muscle preparations pretreated with contractile agonists.


Figure 9
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  		Fig. 9. Cross-regulation of VPAC2 receptor desensitization by muscarinic M3 receptors (M3 R) via RKIP. Phosphorylation, internalization, and desensitization of VPAC2 receptors are exclusively mediated by GRK2. Concomitant activation of muscarinic M3 receptors leads to stimulation of PKC and phosphorylation of RKIP. Phosphorylation-dependent switching of RKIP from Raf-1 to GRK2 unmasks the stimulation of the ERK1/2 pathway by Raf-1 and inhibits phosphorylation, internalization, and desensitization of VPAC2 receptors by GRK2.

 

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This work was supported by Grant DK-28300 from the National Institute of Diabetes and Digestive and Kidney Diseases.


   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)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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  1. Bornstein JC, Costa M, Grider JR. Enteric motor and interneuronal circuits controlling motility. Neurogastroenterol Motil 16, Suppl 1: 34–38, 2004.[CrossRef][ISI][Medline]
  2. Carman CV, Lisanti MP, Benovic JL. Regulation of G protein-coupled receptor kinases by caveolin. J Biol Chem 274: 8858–8864, 1999.[Abstract/Free Full Text]
  3. Cong M, Perry SJ, Lin FT, Fraser ID, Hu LA, Chen W, Pitcher JA, Scott JD, Lefkowitz RJ. Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79. J Biol Chem 276: 15192–15199, 2001.[Abstract/Free Full Text]
  4. Corbit KC, Trakul N, Eves EM, Diaz B, Marshall M, Rosner MR. Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein. J Biol Chem 278: 13061–13068, 2003.[Abstract/Free Full Text]
  5. Fan G, Shumay E, Malbon CC, Wang H. c-Src tyrosine kinase binds the beta2-adrenergic receptor via phospho-Tyr-350, phosphorylates G-protein-linked receptor kinase 2, and mediates agonist-induced receptor desensitization. J Biol Chem 276: 13240–13247, 2001.[Abstract/Free Full Text]
  6. Ferguson G, Watterson KR, Palmer TM. Subtype-specific kinetics of inhibitory adenosine receptor internalization are determined by sensitivity to phosphorylation by G protein-coupled receptor kinases. Mol Pharmacol 57: 546–552, 2000.[Abstract/Free Full Text]
  7. Gainetdinov RR, Premont RT, Bohn LM, Lefkowitz RJ, Caron MG. Desensitization of G protein-coupled receptors and neuronal functions. Annu Rev Neurosci 27: 107–144, 2004.[CrossRef][ISI][Medline]
  8. Grady EF, Bohm SK, Bunnett NW. Turning off the signal: mechanisms that attenuate signaling by G protein-coupled receptors. Am J Physiol Gastrointest Liver Physiol 273: G586–G601, 1997.[Abstract/Free Full Text]
  9. Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P, Said SI, Sreedharan SP, Wank SA, Waschek JA. International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol Rev 50: 265–270, 1998.[Abstract/Free Full Text]
  10. Hersch E, Huang J, Grider JR, Murthy KS. Gq/G13 signaling by ET-1 in smooth muscle: MYPT1 phosphorylation via ETA and CPI-17 dephosphorylation via ETB. Am J Physiol Cell Physiol 287: C1209–C1218, 2004.[Abstract/Free Full Text]
  11. Huang J, Mahavadi S, Sriwai W, Hu W, Murthy KS. Gi-coupled receptors mediate phosphorylation of CPI-17 and MLC20 via preferential activation of the PI3K/ILK pathway. Biochem J 396: 193–200, 2006.[CrossRef][ISI][Medline]
  12. Krasel C, Dammeier S, Winstel R, Brockmann J, Mischak H, Lohse MJ. Phosphorylation of GRK2 by protein kinase C abolishes its inhibition by calmodulin. J Biol Chem 276: 1911–1915, 2001.[Abstract/Free Full Text]
  13. Kunze WA, Furness JB. The enteric nervous system and regulation of intestinal motility. Annu Rev Physiol 61: 117–142, 1999.[CrossRef][ISI][Medline]
  14. Lorenz K, Lohse MJ, Quitterer U. Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2. Nature 426: 574–579, 2003.[CrossRef][Medline]
  15. McDonald TP, Dinnis DM, Morrison CF, Harmar AJ. Desensitization of the human vasoactive intestinal peptide receptor (hVIP2/PACAP R): evidence for agonist-induced receptor phosphorylation and internalization. Ann NY Acad Sci 865: 64–72, 1998.[Abstract/Free Full Text]
  16. Murthy KS. Signaling for contraction and relaxation in smooth muscle of the gut. Annu Rev Physiol 68: 345–374, 2006.[CrossRef][ISI][Medline]
  17. Murthy KS, Jin JG, Grider JR, Makhlouf GM. Characterization of PACAP receptors and signaling pathways in rabbit gastric muscle cells. Am J Physiol Gastrointest Liver Physiol 272: G1391–G1399, 1997.[Abstract/Free Full Text]
  18. Murthy KS, Makhlouf GM. Differential coupling of muscarinic m2 and m3 receptors to adenylyl cyclases VOL/VOLI in smooth muscle. Concurrent m2-mediated inhibition via G{alpha}i3 and m3-mediated stimulation via Gbeta{gamma}q. J Biol Chem 272: 21317–21324, 1997.[Abstract/Free Full Text]
  19. Murthy KS, Makhlouf GM. Differential regulation of phospholipase A2 (PLA2)-dependent Ca2+ signaling in smooth muscle by cAMP- and cGMP-dependent protein kinases. Inhibitory phosphorylation of PLA2 by cyclic nucleotide-dependent protein kinases. J Biol Chem 273: 34519–34526, 1998.[Abstract/Free Full Text]
  20. Murthy KS, Zhou H, Grider JR, Brautigan DL, Eto M, Makhlouf GM. Differential signaling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway. Biochem J 374: 145–155, 2003.[CrossRef][ISI][Medline]
  21. Murthy KS, Zhou H, Makhlouf GM. PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase VOL/VOLI in smooth muscle. Am J Physiol Cell Physiol 282: C508–C517, 2002.[Abstract/Free Full Text]
  22. Pitcher JA, Tesmer JJ, Freeman JL, Capel WD, Stone WC, Lefkowitz RJ. Feedback inhibition of G protein-coupled receptor kinase 2 (GRK2) activity by extracellular signal-regulated kinases. J Biol Chem 274: 34531–34534, 1999.[Abstract/Free Full Text]
  23. Shetzline MA, Premont RT, Walker JK, Vigna SR, Caron MG. A role for receptor kinases in the regulation of class II G protein-coupled receptors. Phosphorylation and desensitization of the secretin receptor. J Biol Chem 273: 6756–6762, 1998.[Abstract/Free Full Text]
  24. Shetzline MA, Walker JK, Valenzano KJ, Premont RT. Vasoactive intestinal polypeptide type-1 receptor regulation. Desensitization, phosphorylation, and sequestration. J Biol Chem 277: 25519–25526, 2002.[Abstract/Free Full Text]
  25. Teng B, Murthy KS, Kuemmerle JF, Grider JR, Makhlouf GM. Selective expression of vasoactive intestinal peptide (VIP)2/pituitary adenylate cyclase-activating polypeptide (PACAP)3 receptors in rabbit and guinea pig gastric and tenia coli smooth muscle cells. Regul Pept 77: 127–134, 1998.[CrossRef][ISI][Medline]
  26. Ulrich CD II, Holtmann M, Miller LJ. Secretin and vasoactive intestinal peptide receptors: members of a unique family of G protein-coupled receptors. Gastroenterology 114: 382–397, 1998.[CrossRef][ISI][Medline]
  27. Walker JK, Premont RT, Barak LS, Caron MG, Shetzline MA. Properties of secretin receptor internalization differ from those of the beta(2)-adrenergic receptor. J Biol Chem 274: 31515–31523, 1999.[Abstract/Free Full Text]
  28. Yeung K, Janosch P, McFerran B, Rose DW, Mischak H, Sedivy JM, Kolch W. Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein. Mol Cell Biol 20: 3079–3085, 2000.[Abstract/Free Full Text]
  29. Yeung K, Seitz T, Li S, Janosch P, McFerran B, Kaiser C, Fee F, Katsanakis KD, Rose DW, Mischak H, Sedivy JM, Kolch W. Suppression of Raf-1 kinase activity and MAP kinase signaling by RKIP. Nature 401: 173–177, 1999.[CrossRef][Medline]
  30. Zhou H, Das S, Murthy KS. Erk1/2- and p38 MAP kinase-dependent phosphorylation and activation of cPLA2 by m3 and m2 receptors. Am J Physiol Gastrointest Liver Physiol 284: G472–G480, 2003.[Abstract/Free Full Text]
  31. Zhou H, Murthy KS. PKA-dependent phosphorylation of GRK2 augments VPAC2 receptor phosphorylation, internalization, and homologous desensitization in smooth muscle cells (Abstract). Gastroenterology 126: A555, 2004.



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