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Am J Physiol Cell Physiol 279: C925-C934, 2000;
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Vol. 279, Issue 4, C925-C934, October 2000

Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of Gi-1 and Gi-2

K. S. Murthy, J. R. Grider, and G. M. Makhlouf

Departments of Medicine and Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0711


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study examined the ability of protein kinase C (PKC) to induce heterologous desensitization by targeting specific G proteins and limiting their ability to transduce signals in smooth muscle. Activation of PKC by pretreatment of intestinal smooth muscle cells with phorbol 12-myristate 13-acetate, cholecystokinin octapeptide, or the phosphatase 1 and phosphatase 2A inhibitor, calyculin A, selectively phosphorylated Galpha i-1 and Galpha i-2, but not Galpha i-3 or Galpha o, and blocked inhibition of adenylyl cyclase mediated by somatostatin receptors coupled to Gi-1 and opioid receptors coupled to Gi-2, but not by muscarinic M2 and adenosine A1 receptors coupled to Gi-3. Phosphorylation of Galpha i-1 and Galpha i-2 and blockade of cyclase inhibition were reversed by calphostin C and bisindolylmaleimide, and additively by selective inhibitors of PKCalpha and PKCvarepsilon . Blockade of inhibition was prevented by downregulation of PKC. Phosphorylation of Galpha -subunits by PKC also affected responses mediated by beta gamma -subunits. Pretreatment of muscle cells with cANP-(4-23), a selective agonist of the natriuretic peptide clearance receptor, NPR-C, which activates phospholipase C (PLC)-beta 3 via the beta gamma -subunits of Gi-1 and Gi-2, inhibited the PLC-beta response to somatostatin and [D-Pen2,5]enkephalin. The inhibition was partly reversed by calphostin C. Short-term activation of PKC had no effect on receptor binding or effector enzyme (adenylyl cyclase or PLC-beta ) activity. We conclude that selective phosphorylation of Galpha i-1 and Galpha i-2 by PKC partly accounts for heterologous desensitization of responses mediated by the alpha - and beta gamma -subunits of both G proteins. The desensitization reflects a decrease in reassociation and thus availability of heterotrimeric G proteins.

G proteins; smooth muscle; signal transduction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE TRANSDUCTION OF SOME EXTERNAL signals into internal signals involves sequential activation of three membrane proteins: a membrane-spanning receptor, and a GTP-binding protein (G protein) that couples the receptor to specific effector enzymes (13). The latter act on membrane or cytoplasmic substrates to generate regulatory signals (second messengers) that activate various protein kinases. The internal signal is attenuated or terminated by mechanisms that target receptors, G proteins, and/or effector enzymes. The strength and duration of the signal is determined by G protein-specific GTPase-activating proteins known as "regulators of G protein signaling" (RGS) and by the intrinsic GTPase activities of the alpha -subunit and some effector enzymes [e.g., phospholipase C (PLC)-beta ] (1, 2, 9). GTP hydrolysis promotes the reassociation of the alpha - and beta gamma -subunits that impede further activation of effector enzymes by alpha - or beta gamma -subunits.

Considerable attention has been devoted to mechanisms that induce short-term or long-term desensitization of receptors. Rapid homologous desensitization of agonist-occupied receptors is initiated by specific receptor protein kinases (G protein-coupled receptor kinases) and the binding of beta -arrestins to the phosphorylated receptor, which uncouples the receptor from the G protein as a prelude to receptor endocytosis and recycling (3, 10, 35-37). The process of desensitization may be complemented by feedback phosphorylation via second messenger-activated protein kinases, chiefly protein kinase C (PKC), and cAMP- and cGMP-dependent protein kinases (PKA and PKG). Phosphorylation by these kinases does not require receptor occupancy and can thus target both homologous and heterologous receptors (35, 37). Phosphorylation of some receptors (e.g., beta 2-adrenergic receptors by PKA) can alter the specificity of receptor coupling to G proteins and initiate a different signaling cascade (7). Second messenger-activated kinases can induce desensitization also by targeting effector enzymes and attenuating their activity [e.g., phosphorylation of PLC-beta (12, 18, 39) and phospholipase A2 (28) by PKG and PKA, and adenylyl cyclase type II by PKC] (41, 43, 46).

Phosphorylation of the alpha - or beta -subunits of some G proteins has been proposed as a mechanism of desensitization that targets G proteins and limits their ability to transduce signals (11, 15, 16, 19-22, 45). PKC-dependent phosphorylation of Galpha z and Galpha 12 has been demonstrated (11, 16), but evidence for phosphorylation of other G proteins, including various isoforms of Gi, has been conflicting (4-6, 16, 22, 40). Phosphorylation of Galpha z and Galpha 12 appears to decrease the affinity of Galpha for Gbeta gamma , impeding reassociation of the subunits and decreasing the availability of the heterotrimeric G protein in the plasma membrane. The present study examined whether Gi-1, Gi-2, Gi-3, and Go were targets of phosphorylation by PKC in intestinal smooth muscle, and whether phosphorylation of one or more of these G proteins resulted in heterologous desensitization. The results indicate that Gi-1 and Gi-2, but not Gi-3 or Go, are phosphorylated by PKC, resulting in attenuation of subsequent responses mediated by both G proteins.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of dispersed smooth muscle cells. Smooth muscle cells were isolated from the circular muscle layer of rabbit intestine by sequential enzymatic digestion, filtration, and centrifugation as described previously (23-26). Muscle strips were incubated for 30 min at 31°C in 15 ml of HEPES medium containing 0.1% collagenase and 0.1% soybean trypsin inhibitor. The partly digested strips were washed with 100 ml of enzyme-free medium, and the muscle cells were allowed to disperse spontaneously for 30-60 min. The cells were harvested by filtration through a 500-µm Nitex mesh followed by two 10-min centrifugations at 350 g.

Measurement of cAMP in dispersed smooth muscle cells. cAMP was measured in dispersed cells by radioimmunoassay as described previously (23, 24, 26). Aliquots (0.5 ml) containing 106 cells/ml were incubated with various agents, and the reaction was terminated after 60 s with 60% cold trichloroacetic acid (vol/vol). The mixture was centrifuged at 2,000 g for 15 min at 4°C. The supernatant was extracted three times with 2 ml of diethyl ether and lyophilized. The samples were reconstituted for radioimmunoassay in 500 µl of 50 mM sodium acetate (pH 6.2) and acetylated with triethylamine/acetic anhydride (2:1 vol/vol) for 30 min. cAMP was measured in duplicate with the use of 100-µl aliquots and expressed as pmol/106 cells.

Measurement of adenylyl cyclase activity in muscle membranes. Smooth muscle cells were incubated with phorbol 12-myristate 13-acetate (PMA, 1 µM) for 10 min, and a crude homogenate was prepared in 50 mM Tris · HCl (pH 7.4), 1 mM ATP, 2 mM cAMP, 100 µM isobutyl methyl xanthine, 5 mM MgCl2, 100 mM NaCl, 5 mM creatine phosphate, and 50 U/ml creatine kinase. Adenylyl cyclase activity was measured in the presence of [32P]ATP and 1 mM GTP by an adaptation of the method of Salomon et al. (38). The reaction was terminated after 15 min by addition of stop solution containing 2% SDS, 45 mM ATP, and 1.5 mM cAMP. [32P]cAMP was separated by sequential chromatography on Dowex AG50W-4X and alumina columns. The samples were measured by scintillation counting, and the results were expressed as picomoles of cAMP per milligram of protein per minute.

Measurement of inositol phosphates. Total inositol phosphate formation in smooth muscle cells was measured by ion-exchange chromatography as described previously (31). Ten milliliters of cell suspension (2× 106 cells/ml) were labeled with myo-[3H]inositol (15 µCi/ml) for 3 h at 31°C. After centrifugation at 350 g for 10 min, the cells were resuspended in 10 ml of fresh HEPES medium. After treatment with a cANP-(4-23) for 10 min, the cells were centrifuged at 350 g for 5 min. [D-Pen2,5]enkephalin (DPDPE), somatostatin, or cyclopentyladenosine (CPA) was then added to 0.5 ml of cell suspension and the samples incubated for 30 s. The reaction was terminated with 940 µl of chloroform:methanol:HCl (50:100:1, vol/vol/vol). After addition of chloroform (310 µl) and water (310 µl), the samples were vortexed, and the phases were separated by centrifugation at 1,000 g for 15 min. The upper aqueous phase was applied to a column that contained 1 ml of 1:1 slurry of Dowex AG1-X8 resin (100-200 mesh in formate form) and distilled water. The column was washed with 10 ml of water followed by 10 ml of 5 mM sodium tetraborate-60 mM ammonium formate. Inositol phosphates were eluted with 5 ml of 0.8 M ammonium formate-0.1 M formic acid. The eluates were collected into scintillation vials and counted in gel phase after addition of 10 ml of scintillant. The results were expressed as counts per minute (cpm)/106 cells or percentage increase above basal levels.

Immunoblotting of 32P-labeled Galpha subunits. Ten milliliters of smooth muscle cell suspension (2-3 × 106 cells/ml) were incubated with [32P]orthophosphate for 4 h at 31°C. One-milliliter samples were then incubated with PMA for 10 min and microfuged for 2 min. The samples were washed three times with phosphate-buffered saline and incubated in lysis buffer that contained 150 mm NaCl, 50 mM sodium phosphate (pH 7.2), 2 mM EDTA, 1 mM dithiothreitol, 10 mg/ml aprotinin, 0.2 mg/ml leupeptin, 0.5% SDS, 1% sodium deoxycholate, 1% Triton X-100, 5 mM NaF, 2 mM Na4P2O7, and 2 mM Na3VO4. The cell lysate was boiled for 5 min and incubated on ice for 1 h. The supernatant was precleared by incubation with 40 µl of protein A-Sepharose for 6 h at 4°C and incubated overnight with Galpha i-1, Galpha i-2, Galpha i-3, or Galpha o antibody at a final concentration of 4 µg/ml, and then with 40 µl of Protein A-Sepharose for another 1 h. The immunoprecipitates were collected, washed five times with 1 ml of wash buffer (0.5% Triton X-100, 150 mM NaCl, 10 mM Tris · HCl, pH 7.4), extracted with Laemmli buffer, boiled for 5 min, and separated by 12% SDS-PAGE. After transfer to nitrocellulose membranes, 32P-labeled G proteins were visualized by autoradiography, and radioactivity was measured and expressed as cpm.

Agonist-stimulated G protein activity. Agonist-stimulated G protein activity was measured as previously described (23, 26, 34). Membranes were obtained from control muscle cells, and cells were treated for 10 min with PMA (1 µM). The membranes were incubated at 37°C with 60 nM [35S]GTPgamma S alone or with somatostatin (1 µM) or DPDPE (1 µM) in a medium containing 10 mM HEPES (pH 7.4), 100 µM EDTA, and 10 mM MgCl2. After the reaction was stopped, the solubilized membranes were placed in wells precoated with specific antibodies to Galpha i-1 or Galpha i-2. After 2 h, the wells were washed with phosphate buffer that contained 0.05% Tween 20, and the radioactivity was counted.

Radioligand binding. Radioligand binding to dispersed smooth muscle cells was measured as described previously (23). Muscle cells were suspended in HEPES medium containing 1% bovine serum albumin (BSA). For competitive binding, triplicate aliquots (0.5 ml) of cell suspension (106/ml) were incubated for 15 min with 50 pM [125I]somatostatin-14 alone or in the presence of unlabeled somatostatin-14 (10 µM). Saturable [125I]somatostatin binding was examined with the use of radioligand concentrations in the range of 10-400 pM in the presence or absence of unlabeled somatostatin (10 µM). Bound and free radioligand were separated by rapid filtration under reduced pressure through 5-µm polycarbonate Nucleopore filters followed by repeated washing (4 times) with 3 ml of ice-cold HEPES medium that contained 0.2% BSA. Nonspecific binding was measured as the amount of radioactivity associated with the muscle cells in the presence of 10 µM of unlabeled ligand. Specific binding was calculated as the difference between total and nonspecific binding (24 ± 6%).

Materials. [125I]somatostatin, [32P]ATP, [125I]cAMP, [32P]H3PO4, [35S]GTPgamma S, and [2-3H]inositol were obtained from NEN Life Sciences Products (Boston, MA); Dowex AG50W-4X and Dowex AG1-X8 resin were from Bio-Rad (Hercules, CA); and G protein and phosphotyrosine antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Myristoylated peptide inhibitors of PKCalpha , PKCalpha beta gamma , PKCvarepsilon , and PKCdelta were gifts from Drs. D. A. Dartt and D. Zoukhri, Harvard Medical School.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Selective phosphorylation of Galpha i-1 and Galpha i-2 by PKC. The ability of PMA to induce phosphorylation of inhibitory G proteins (Galpha i-1, Galpha i-2, Galpha i-3, and Galpha o) was examined in dispersed intestinal smooth muscle cells labeled with 32Pi. Treatment of the cells with PMA (1 µM) for 10 min caused a significant increase in phosphorylation of Galpha i-1 (178 ± 10% above basal; P < 0.001) and Galpha i-2 (181 ± 11%; P < 0.001), but not Galpha i-3 or Galpha o (Fig. 1). The increase in phosphorylation of Galpha i-1 and Galpha i-2 was time and concentration dependent and was abolished by calphostin C (Figs. 1 and 2). Experiments in which muscle cells were treated with PMA followed by immunoprecipitation with phosphotyrosine antibodies and Western blot with Galpha i-1 and Galpha i-2 antibodies (n = 3), or immunoprecipitation with Galpha i-1 and Galpha i-2 antibodies and Western blot with phosphotyrosine antibodies (n = 3), did not disclose any evidence of tyrosine phosphorylation, implying that PKC phosphorylation of Galpha i-1 and Galpha i-2 was direct and did not involve tyrosine phosphorylation.


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  		Fig. 1.   Selective phosphorylation of Galpha i-1 and Galpha i-2 by protein kinase C (PKC). Intestinal smooth muscle cells labeled with 32P were incubated with phorbol 12-myristate 13-acetate (PMA; 1 µM, top) or cholecystokinin octapeptide (CCK-8; 1 nM, bottom) for 10 min in the presence (hatched bars) or absence (solid bars) of calphostin C (Cal. C). Galpha proteins were immunoprecipitated and subjected to SDS-PAGE. 32P-labeled Galpha proteins were identified by autoradiography, and the measured radioactivity was expressed as counts per minute (cpm). PMA and CCK induced significant increases in phosphorylation of Galpha i-1 and Galpha i-2, but not Galpha i-3 or Galpha o; the increase was abolished by calphostin C. Values are means ± SE of 4 experiments.



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  		Fig. 2.   Time- and concentration-dependent phosphorylation of Galpha i-1 and Galpha i-2 induced by PMA. Intestinal smooth muscle cells labeled with 32P were incubated with 1 µM PMA for different intervals (bottom) or with various concentrations of PMA (top) for 10 min. 32P-labeled Galpha i-1 and Galpha i-2 were identified by autoradiography, and the measured radioactivity was expressed as percentage increase above basal level (Galpha i-1 721 ± 89 and Galpha i-2 789 ± 125 cpm). Values are means ± SE of 3 experiments.

A similar pattern of selective phosphorylation of Galpha i-1 and Galpha i-2, but not Galpha i-3 or Galpha o, was observed after a 10-min treatment of smooth muscle cells with cholecystokinin octapeptide (CCK-8; 1 nM), which activates Gq/11 and stimulates phosphoinositide hydrolysis and PKC activity (25, 31) (Fig. 1). CCK-induced phosphorylation of Galpha i-1 and Galpha i-2 was inhibited by calphostin C and by myristoylated pseudosubstrate peptide inhibitors of various PKC isozymes, including Ca2+-dependent PKCalpha and Ca2+-independent PKCvarepsilon ; a selective inhibitor of PKCdelta had no effect on phosphorylation of either G protein (Fig. 3). The effects of PKCalpha and PKCvarepsilon inhibitors were additive.


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  		Fig. 3.   Inhibition of CCK-induced phosphorylation of Galpha i-1 and Galpha i-2 by isoform-selective pseudosubstrate peptide inhibitors of PKC. Smooth muscle cells labeled with 32P were incubated for 10 min with CCK (1 nM) in the presence or absence of isoform-selective PKC inhibitors (1 µM) or Cal. C (1 µM). 32P-labeled Galpha i-1 and Galpha i-2 were identified by autoradiography, and the measured radioactivity expressed as cpm. Values are means ± SE of 3 experiments. *P < 0.05, **P < 0.01 inhibition of CCK-induced phosphorylation.

Treatment of the muscle cells with the phosphatases 1/2A inhibitor, calyculin A (10 µM), also caused a time-dependent increase in phosphorylation of Galpha i-1 (211 ± 26%) and Galpha i-2 (216 ± 14%) that was maximal within 5 min (Fig. 4). Calyculin-induced phosphorylation was abolished by bisindolylmaleimide (1 µM), a competitive inhibitor of the ATP-binding site of PKC.


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  		Fig. 4.   Time-dependent phosphorylation of Galpha i-1 and Galpha i-2 induced by calyculin A. Smooth muscle cells labeled with 32P were incubated for various time intervals with calyculin A (10 µM) in the presence or absence of the PKC inhibitor bisindolylmaleimide (1 µM). 32P-labeled Galpha i-1 and Galpha i-2 were identified by autoradiography, and the measured radioactivity was expressed as cpm. Values are means ± SE of 3 experiments. **P < 0.01 inhibition of calyculin-induced phosphorylation.

Blockade by PMA of agonist-stimulated activation of Gi-1 and Gi-2. The effect of pretreatment with PMA on activation of Gi-1 by somatostatin and Gi-2 by DPDPE was determined in solubilized smooth muscle membranes. As previously shown (23, 26), somatostatin and DPDPE increased the binding of [35S]GTPgamma S to Galpha i-1 and Galpha i-2, respectively. Pretreatment of the cells for 10 min with PMA (1 µM) before membrane isolation inhibited the increase in [35S]GTPgamma S binding to Galpha i-1 and Galpha i-2 induced by the corresponding agonists (Fig. 5).


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  		Fig. 5.   Blockade by PMA of agoninst-stimulated activation of Gi-1 and Gi-2. The effect of pretreatment with PMA on activation of Gi-1 by somatostatin and Gi-2 by [D-Pen2,5]enkephalin (DPDPE) was determined in smooth muscle membranes. Pretreatment of the cells for 10 min with PMA (1 µM) before membrane isolation inhibited the increase in [35S]GTPgamma S binding to Galpha i-1 and Galpha i-2, induced by the corresponding agonists. The results are expressed in counts per minute per milligram of protein. Values are means ± SE of 3 experiments. **P < 0.01 from control.

Blockade by PMA of Galpha i-1- and Galpha i-2-mediated inhibition of adenylyl cyclase. To determine whether phosphorylation was associated with a decrease in signaling by Gi-1 and Gi-2, smooth muscle cells were treated with PMA for 10 min and then with forskolin (10 µM) for 1 min, either alone or in combination with various agonists. Four agonists were used: somatostatin to activate Gi-1 and Go (23), DPDPE to activate Gi-2 and Go (26), acetylcholine to activate Gi-3 (via M2 receptors), and CPA to activate Gi-3 (via A1 receptors) (23, 24, 26, 27).

Forskolin-stimulated cAMP (22.2 ± 1.8 pmol/106 cells above a basal level of 4.5 ± 0.2) was inhibited 80 ± 5% by somatostatin, 82 ± 2% by DPDPE, 75 ± 4% by CPA, and 76 ± 2% by acetylcholine (Fig. 6). Pretreatment of the cells with PMA (1 µM) had no significant effect on basal (4.4 ± 0.6 pmol/106 cells) or forskolin-stimulated (21.3 ± 1.7 pmol/106 cells) cAMP levels, but it partially reversed the inhibition induced by DPDPE to 33 ± 5% (P < 0.01 from control inhibition) and somatostatin to 43 ± 7% (P < 0.01) without affecting inhibition induced by either CPA or acetylcholine (Fig. 6). The effect of PMA was suppressed by calphostin C (1 µM) (Fig. 6). Pretreatment with the inactive phorbol ester, 4alpha -phorbol, had no significant effect on inhibition of forskolin-stimulated cAMP induced by all four ligands (76 ± 2% to 82 ± 2% inhibition). The reversal of cAMP inhibition mediated by Gi-1 and Gi-2 paralleled the selective phosphorylation of these two G proteins by PKC. It is worth noting that reversal of cAMP inhibition was partial, reflecting the fact that Go, which partly mediates the effects of somatostatin and opioid peptides, was not subject to phosphorylation by PKC.


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  		Fig. 6.   PKC-dependent reversal of Galpha i-1- and Galpha i-2-mediated inhibition of cAMP formation. Smooth muscle cells were incubated for 10 min with PMA (1 µM, top) or CCK-8 (1 nM, bottom) in the presence or absence of Cal. C, and then treated for 1 min with forskolin (10 µM) alone or in combination with various agonists (somatostatin, SST, 1 µM; DPDPE, 1 µM; cyclopentyladenosine, CPA, 1 µM; and acetylcholine, ACh, 0.1 µM). cAMP was expressed as pmol/106 cells above basal level (4.5 ± 0.2 pmol/106 cells). Inhibition of cAMP (open bars) induced by DPDPE and SST only was partly reversed by pretreatment with PMA or CCK-8 (solid bars); the reversal was abolished by Cal. C. Values are means ± SE of 4 experiments.

The measurements were repeated in dispersed smooth muscle cells derived from muscle strips incubated for 24 h with 0.1 µM PMA to downregulate PKC. The prolonged treatment with PMA did not affect basal (5.0 ± 0.4 pmol/106 cells) or forskolin-stimulated (23.2 ± 1.5 pmol/106 cells) cAMP levels, or the inhibition of cAMP induced by DPDPE or somatostatin. Treatment of these cells with 1 µM PMA for 10 min did not reverse the inhibition of cAMP induced by DPDPE (81 ± 4%) or somatostatin (76 ± 3%), providing further support for the notion that blockade of cAMP inhibition was mediated by PKC (Fig. 7).


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  		Fig. 7.   Absence of PKC-dependent effects on Galpha i-1- and Galpha i-2-mediated inhibition of cAMP after PKC downregulation. Smooth muscle strips were incubated with 100 nM PMA for 24 h to downregulate PKC. Smooth muscle cells were then isolated from the strips and treated for 10 min with 1 µM PMA followed by a 1 min-treatment with forskolin (10 µM) in combination with somatostatin (1 µM) or DPDPE (1 µM). Downregulation of PKC had no effect on basal or forskolin-stimulated cAMP. Comparison with results in Fig. 6 shows that inhibition of cAMP induced by somatostatin and DPDPE was not reversed after 10-min treatment with PMA. Data are means ± SE of 3 experiments.

Control studies were done to determine whether other protein targets (e.g., receptor or adenylyl cyclase) in the signaling pathway besides Gi-1 and Gi-2 were affected by PKC. Treatment with PMA had no effect on forskolin-stimulated adenylyl cyclase activity in smooth muscle membranes (Fig. 8), or as noted above, on basal and forskolin-stimulated cAMP levels in dispersed smooth muscle cells, consistent with the notion that adenylyl cyclase types V/VI expressed in gastrointestinal smooth muscle and directly activated by forskolin is not inhibited by PKC (27). However, adenylyl cyclase activity stimulated by GTP (1 mM) in membranes, which reflected net activation via Gs and inhibition via Gi/Go, was significantly augmented (72 ± 4%; P < 0.001) by pretreatment of the cells with PMA, consistent with selective inactivation of one or more inhibitory G proteins by PKC (Fig. 8). Similar results were obtained on treatment of muscle cells with pertussis toxin (200 ng/ml for 60 min; GTP alone: 7.5 ± 0.5 pmol cAMP · mg protein-1 · min-1; GTP after pertussis toxin treatment: 14.0 ± 1.4 pmol cAMP·mg protein-1 · min-1). Finally, 10-min treatment with PMA (1 µM) had no direct effect on saturation or competitive somatostatin binding to somatostatin receptors on dispersed smooth muscle cells (Fig. 9). A fit of the data to a two-site model yielded dissociation constant (Kd) values of 0.21 ± 0.03 nM and 12.6 ± 5.8 nM for high and low affinity sites, and Bmax values of 270 ± 65 and 3,225 ± 628 fmol/mg protein. The values were closely similar after treatment with PMA: Kd 0.23 ± 0.04 and 13.7 ± 6.2 nM; density of binding sites, Bmax 288 ± 54 and 3,408 ± 607 fmol/mg protein.


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  		Fig. 8.   Differential effects of PMA on forskolin- and GTP-stimulated adenylyl cyclase activity in muscle membranes. Smooth muscle homogenates isolated from control (open bars) and PMA (1 µM)-treated muscle cells (solid bars) were stimulated for 15 min with 1 mM GTP or 10 µM forskolin in the presence of 100 µM isobutyl methyl xanthine and [32P]ATP. cAMP formation was measured by column chromatography and expressed as picomoles of cAMP per milligram of protein per minute above basal level (8.8 ± 1.5 pmol cAMP · mg protein-1 · min-1). Values are means ± SE of 4 experiments. **P < 0.01 from control.



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  		Fig. 9.   SST binding in control and PMA-treated muscle cells. Muscle cells were incubated with various concentrations of [125I]SST (top) or 50 pM [125I]SST (bottom) for 15 min in the presence or absence of unlabeled SST (10 µM). Specific binding was calculated as the difference between total and nonspecific binding (24 ± 6%). Top depicts saturation binding, and bottom depicts competition binding (IC50 2.5 ± 0.4 and 3.2 ± 0.2 nM). Values are means ± SE of 3-4 experiments.

Blockade by agonists and phosphatase inhibitors of Gi-1- and Gi-2-mediated inhibition of adenylyl cyclase. Activation of PKC with CCK-8 elicited similar results to those with PMA. Pretreatment for 10 min with a maximal concentration of CCK-8 (1 nM) partially reversed the inhibition of forskolin-stimulated cAMP induced by DPDPE to 30 ± 8% (P < 0.01 from control inhibition) and somatostatin to 37 ± 8% (P < 0.01) without affecting inhibition induced by either CPA or acetylcholine (Fig. 6). The effect of CCK was suppressed by calphostin C and by myristoylated pseudosubstrate peptide inhibitors of PKCalpha and PKCvarepsilon , but not PKCdelta (Figs. 6 and 10) (47). The effectiveness of these inhibitors paralleled their ability to block CCK-induced phosphorylation of Galpha i-1 and Galpha i-2 (Fig. 3).


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  		Fig. 10.   Blockade of PKC-dependent reversal of SST- and DPDPE-induced inhibition of cAMP by PKCalpha and PKCvarepsilon inhibitors. Smooth muscle cells were incubated for 10 min with CCK-8 (1 nM) in the presence or absence of selective PKCalpha , PKCvarepsilon , and PKCdelta inhibitors, and then were treated for 1 min with forskolin (10 µM) alone or in combination with SST (1 µM, top) or DPDPE (1 µM, bottom). cAMP was expressed as pmol/106 cells above basal level. Inhibition of cAMP (open bars) induced by DPDPE and SST was partly reversed by pretreatment with CCK-8 (solid bars); the reversal was abolished by inhibitors of PKCalpha and PKCvarepsilon (**P < 0.01), but not PKCdelta . Values are means ± SE of 4 experiments.

The role of PKC in reversing the inhibition of cAMP mediated by Gi-1 and Gi-2 was corroborated by studies with calyculin A. Pretreatment of muscle cells with 10 µM calyculin A for 10 min partially reversed the inhibition of forskolin-stimulated cAMP induced by DPDPE to 34 ± 3% (P < 0.01 from control inhibition) and somatostatin to 45 ± 5%, but not that induced by CPA or acetylcholine (Fig. 11). The effect of calyculin A was suppressed by bisindolylmaleimide, which blocks the catalytic site of PKC, but not by calphostin C, which blocks the regulatory diacylglycerol-binding site (Fig. 11).


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  		Fig. 11.   Calyculin-induced reversal of Galpha i-1- and Galpha i-2-mediated inhibition of cAMP formation. Smooth muscle cells were incubated for 10 min with calyculin A (Cal. A, 10 µM) in the presence or absence of bisindolylmaleimide (BIM, 1 µM) and then treated for 1 min with forskolin (10 µM) alone or in combination with various agonists (SST, 1 µM; DPDPE, 1 µM; CPA, 1 µM; and ACh, 0.1 µM). cAMP was expressed as pmol/106 cells above basal level. Inhibition of cAMP (open bars) induced by DPDPE and SST only was partly reversed by pretreatment with Cal. A (solid bars); the reversal was abolished by bisindolylmaleimide. Values are means ± SE of 4 experiments.

Inhibition of Gi-1- and Gi-2-mediated phospholipase C-beta activity by PKC. The studies described above disclosed the role of PKC in attenuating inhibition of adenylyl cyclase activity mediated by the alpha -subunits of Gi-1 and Gi-2. We next examined whether G protein phosphorylation by PKC attenuated the ability of the beta gamma -subunits of both G proteins to activate phospholipase C-beta 3 (PLC-beta 3). Our previous studies had shown that the beta gamma -subunits of Gi-1, Gi-2, and Gi-3 selectively activated PLC-beta 3 in smooth muscle (23-26).

DPDPE caused a significant increase in PLC-beta activity (746 ± 29 cpm/106 cells [3H]inositol phosphates). The PLC-beta response was inhibited by 39 ± 7% after activation of PKC by pretreatment of the cells for 10 min with acetylcholine; the inhibition was completely reversed by calphostin C. Similar inhibition of the PLC-beta response to DPDPE was observed after stimulation of PKC activity with CCK-8 (46 ± 8%), substance P (35 ± 7%), and CPA (33 ± 8%).

cANP-(4-23), a selective agonist of the natriuretic peptide clearance receptor, NPR-C, was recently shown to activate PLC-beta 3 via the beta gamma -subunits of Gi-1 and Gi-2 (32, 33). Pretreatment of the cells for 10 min with cANP-(4-23) (1 µM) inhibited the PLC-beta response to both DPDPE and somatostatin by 61 ± 6% and 64 ± 7%, respectively; the inhibition was partially reversed by calphostin C to 33 ± 4% and 29 ± 3%, respectively (P < 0.01 from control inhibition), implying that it was only partly mediated by PKC (Fig. 12). In contrast, the PLC-beta response to CPA was not affected by pretreatment of the cells with cANP-(4-23). The lack of effect of PKC on a response mediated by Gi-3 is consistent with the lack of PKC-dependent phosphorylation of this G protein.


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  		Fig. 12.   Inhibition of SST- and DPDPE-stimulated phospholipase C-beta (PLC-beta ) activity by PKC. Smooth muscle cells labeled with myo-[3H]inositol were incubated in cANP-(4-23) (1 µM) for 10 min in the presence (hatched bars) or absence (solid bars) of Cal. C (1 µM). The cells were washed and incubated for 1 min with SST (1 µM), DPDPE (1 µM), or CPA (1 µM). Control cells were treated with agonists without prior incubation with cANP-(4-23) (open bars). PLC-beta activity was expressed as total [3H]inositol phosphates above basal levels (360 ± 62 cpm/106 cells). Pretreatment with cANP-(4-23) inhibited PLC-beta activity stimulated by SST and DPDPE; inhibition was partly reversed by Cal. C. Values are means ± SE of 4 experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that PKC-dependent phosphorylation of the alpha -subunits of Gi-1 and Gi-2 in smooth muscle limits the ability of the G proteins to transduce signals. PKC-dependent phosphorylation was confined to Gi-1 and Gi-2 and was not observed with Gi-3 or Go. Selective phosphorylation of Galpha i-1 and Galpha i-2 resulted in heterologous desensitization of responses mediated by these two G proteins and could contribute to homologous desensitization.

Previous studies had shown that PKC was capable of phosphorylating Galpha i-2 in some cells only; phosphorylation of Gs or Gq/11 was not detected, and phosphorylation of Gi-3 and Go was not examined (4-6, 22, 40, 45). The present study showed that Gi-1 and Gi-2, but not Gi-3 or Go, were directly phosphorylated by PKC in smooth muscle and not via tyrosine kinase(s).

The role of PKC-dependent phosphorylation in heterologous desensitization of response was demonstrated in this study by activation of PKC with a phorbol ester (PMA) or an agonist (CCK-8), followed by treatment of the cells with agonists (somatostatin, DPDPE, CPA, and acetylcholine) known to activate specific G proteins. Pretreatment of muscle cells with PMA or CCK-8 caused rapid phosphorylation of Galpha i-1 and Galpha i-2, but not Galpha o or Galpha i-3, and reversed the inhibition of adenylyl cyclase mediated by somatostatin receptors that couple to Gi-1 and Go (23) and opioid delta -receptors that couple to Gi-2 and Go (26), but not muscarinic M2 and adenosine A1 receptors that couple to Gi-3 (24, 27). Reversal of the inhibition induced by somatostatin and DPDPE was not complete, reflecting phosphorylation of Galpha i-1 and Galpha i-2, but not Galpha o. Suppression of dephosphorylation with calyculin A increased phosphorylation of Galpha i-1 and Galpha i-2 but not Galpha i-3; the increase in phosphorylation of Galpha i-1 and Galpha i-2 was inhibited by bisindolylmaleimide, which blocks the ATP-binding site of PKC, implying that phosphorylation was mediated by receptor-independent, endogenous PKC activity normally contained by endogenous phosphatase activity.

In vitro studies on isolated alpha -subunits of various G proteins have shown that Galpha 12, a ubiquitous G protein, and Galpha Z, which is predominantly expressed in platelets and neurons, are readily phosphorylated by PKC (11, 16, 19, 20). The functional consequences of phosphorylation were alluded to but were not examined in situ. The primary sites of Galpha Z and Galpha 12 phosphorylation (Ser27 and Ser16) are located in the NH2-terminal domain of Galpha that determines the binding of alpha - and beta gamma -subunits; phosphorylation of these residues impedes the reassociation of alpha - and beta gamma -subunits (11, 16, 20). Ser16 in Galpha i-1 and Galpha i-2 is analogous to Ser16 in Galpha Z and Ser38 in Galpha 12; its phosphorylation might, therefore, be expected to impede reassociation of alpha - and beta gamma -subunits (11, 16). Furthermore, the rate of GTP/GDP exchange in Gi-1 and Gi-2 is more rapid than in G12 and GZ. This makes it possible for PKC, whether activated by a phorbol ester or an agonist, to phosphorylate dissociated alpha -subunits, impede the reassociation of alpha - and beta gamma -subunits, and reduce the availability of the trimeric species for subsequent activation by other receptors, thereby leading to heterologous desensitization of responses mediated by a specific G protein. As shown in Fig. 5, treatment with PMA decreased the ability of somatostatin and DPDPE to activate Gi-1 and Gi-2, respectively, reflecting a decrease in the availability of the trimeric species.

The decrease in the availability of G proteins that results from phosphorylation of Galpha i-1 and Galpha i-2 also led to a decrease in responses mediated by beta gamma -subunits. The notion was tested with the NPR-C agonist, cANP-(4-23), which selectively activates Gi-1 and Gi-2 and stimulates PLC-beta 3 activity via the beta gamma -subunits of both G proteins (32, 33). Treatment of smooth muscle with cANP-(4-23) inhibited the PLC-beta response to subsequent activation of Gi-1 by somatostatin or activation of Gi-2 by DPDPE. The PLC-beta response was only partly restored when PKC activity was blocked with calphostin C. We postulate that residual inhibition reflects sequestration of Galpha i-1 and Galpha i-2 by binding to caveolin-3 (8, 17, 29). In contrast, inhibition of the PLC-beta response to DPDPE after treatment of muscle cells with acetylcholine (that activates Gq/11 and Gi-3 via M3 and M2 receptors, respectively) could be fully restored by calphostin C, because under these conditions, phosphorylation but not caveolin sequestration of Galpha i-1 and Galpha i-2 would be induced.

No evidence was obtained to suggest that other protein targets in the signaling pathways initiated by somatostatin or DPDPE (e.g., receptors or effector enzymes) were affected by PKC. Adenylyl cyclase type V/VI expressed in gastrointestinal smooth muscle is not a PKC substrate (27, 41), and its activity was not affected by pretreatment with PMA or agonists. However, GTP-stimulated adenylyl cyclase activity, which reflected the balance of activation via Gs and inhibition via Gi/Go, was significantly augmented by PKC consistent with selective inactivation or reduction in the availability of Gi-1 and Gi-2. Although PKC-dependent phosphorylation of somatostatin and opioid receptors has been reported in some cells (14, 43), no effect was detected on the affinity or density of somatostatin receptors after treatment of muscle cells with PMA.

Where the phosphoinositide pathway was concerned, prior activation of PKC inhibited PLC-beta 3 activity stimulated by somatostatin-3 and opioid delta -receptors coupled to Gbeta gamma i-1 and Gbeta gamma i-2 (23, 26, and this study), but had no effect on PLC-beta 3 activity stimulated by A1 and M2 receptors coupled to Gbeta gamma i-3 (24, 27, 29). The pattern implied that inhibition of PLC-beta activity by PKC in smooth muscle was G protein specific and was not exerted directly on PLC-beta isozymes. This is in contrast to other cell types, where PKC was shown to inhibit directly mammalian or avian PLC-beta isozymes (12, 39). The results leave open the question of whether phosphorylation by PKC could additionally target RGS proteins and enhance or attenuate their ability to accelerate GTP hydrolysis. Inhibition of response that results from acceleration of GTP hydrolysis by a cognate, phosphorylated RGS may not be readily distinguishable from inhibition that results from phosphorylation of the corresponding G protein.

PKC-dependent, G protein-specific heterologous desensitization could occur physiologically when neurotransmitters are delivered sequentially to target smooth muscle cells. During peristalsis, neurotransmitters (e.g., acetylcholine and tachykinins) that activate PKC could influence the response to other neurotransmitters, such as opioid peptide, which activates Gi-2, and vasoactive intestinal peptide, which activates Gs via VPAC2 receptors (42), and Gi-1 and Gi-2 via NPR-C (33).


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-15564.


    FOOTNOTES

Address for reprint requests and other correspondence: G. M. Makhlouf, PO Box 980711, Medical College of Virginia, Virginia Commonwealth Univ., Richmond, VA 23298.

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.

Received 14 December 1999; accepted in final form 17 April 2000.


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