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
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ABSTRACT |
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 G
i-1 and G
i-2,
but not G
i-3 or G
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 G
i-1 and G
i-2 and blockade of cyclase
inhibition were reversed by calphostin C and bisindolylmaleimide, and
additively by selective inhibitors of PKC
and PKC
. Blockade of
inhibition was prevented by downregulation of PKC. Phosphorylation of
G
-subunits by PKC also affected responses mediated by

-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)-
3 via the 
-subunits of Gi-1 and
Gi-2, inhibited the PLC-
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-
) activity. We conclude that selective phosphorylation of
G
i-1 and G
i-2 by PKC partly accounts for
heterologous desensitization of responses mediated by the
- and

-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
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INTRODUCTION |
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
-subunit and some effector enzymes [e.g., phospholipase C
(PLC)-
] (1, 2, 9). GTP hydrolysis promotes the
reassociation of the
- and 
-subunits that impede further
activation of effector enzymes by
- or 
-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
-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.,
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-
(12, 18,
39) and phospholipase A2 (28) by PKG and PKA, and adenylyl cyclase type II by PKC] (41, 43,
46).
Phosphorylation of the
- or
-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
G
z and G
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
G
z and G
12 appears to decrease the
affinity of G
for G
, 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.
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MATERIALS AND METHODS |
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 G
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 G
i-1, G
i-2,
G
i-3, or G
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]GTP
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 G
i-1 or G
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]GTP
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 PKC
,
PKC

, PKC
, and PKC
were gifts from Drs. D. A. Dartt and D. Zoukhri, Harvard Medical School.
 |
RESULTS |
Selective phosphorylation of G
i-1 and
G
i-2 by PKC.
The ability of PMA to induce phosphorylation of inhibitory G proteins
(G
i-1, G
i-2, G
i-3, and
G
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 G
i-1 (178 ± 10% above basal;
P < 0.001) and G
i-2 (181 ± 11%; P < 0.001), but not G
i-3 or
G
o (Fig. 1). The increase
in phosphorylation of G
i-1 and G
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 G
i-1
and G
i-2 antibodies (n = 3), or
immunoprecipitation with G
i-1 and G
i-2
antibodies and Western blot with phosphotyrosine antibodies
(n = 3), did not disclose any evidence of tyrosine
phosphorylation, implying that PKC phosphorylation of
G
i-1 and G
i-2 was direct and did not involve tyrosine phosphorylation.

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Fig. 1.
Selective phosphorylation of G i-1 and
G 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). G proteins were immunoprecipitated and subjected to
SDS-PAGE. 32P-labeled G 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 G i-1 and G i-2, but not
G i-3 or G 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
G i-1 and G 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 G i-1 and G i-2
were identified by autoradiography, and the measured radioactivity was
expressed as percentage increase above basal level (G i-1
721 ± 89 and G i-2 789 ± 125 cpm). Values are
means ± SE of 3 experiments.
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A similar pattern of selective phosphorylation of G
i-1
and G
i-2, but not G
i-3 or
G
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 G
i-1 and
G
i-2 was inhibited by calphostin C and by myristoylated pseudosubstrate peptide inhibitors of various PKC isozymes, including Ca2+-dependent PKC
and Ca2+-independent
PKC
; a selective inhibitor of PKC
had no effect on
phosphorylation of either G protein (Fig.
3). The effects of PKC
and PKC
inhibitors were additive.

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Fig. 3.
Inhibition of CCK-induced phosphorylation of
G i-1 and G 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 G i-1 and
G 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.
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Treatment of the muscle cells with the phosphatases 1/2A inhibitor,
calyculin A (10 µM), also caused a time-dependent increase in
phosphorylation of G
i-1 (211 ± 26%) and
G
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 G i-1 and
G 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
G i-1 and G 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.
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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]GTP
S to G
i-1 and
G
i-2, respectively. Pretreatment of the cells for 10 min
with PMA (1 µM) before membrane isolation inhibited the increase in
[35S]GTP
S binding to G
i-1 and
G
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]GTP S binding to
G i-1 and G 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.
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Blockade by PMA of G
i-1- and
G
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, 4
-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 G i-1- and
G 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.
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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 G i-1-
and G 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.
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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.
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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 PKC
and PKC
, but not PKC
(Figs. 6 and 10) (47). The
effectiveness of these inhibitors paralleled their ability to block
CCK-induced phosphorylation of G
i-1 and
G
i-2 (Fig. 3).

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Fig. 10.
Blockade of PKC-dependent reversal of SST- and
DPDPE-induced inhibition of cAMP by PKC and PKC inhibitors.
Smooth muscle cells were incubated for 10 min with CCK-8 (1 nM) in the
presence or absence of selective PKC , PKC , and PKC 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 PKC and PKC
(**P < 0.01), but not PKC . Values are means ± SE of 4 experiments.
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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 G i-1- and
G 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-
activity by PKC.
The studies described above disclosed the role of PKC in attenuating
inhibition of adenylyl cyclase activity mediated by the
-subunits of
Gi-1 and Gi-2. We next examined whether G
protein phosphorylation by PKC attenuated the ability of the

-subunits of both G proteins to activate phospholipase C-
3
(PLC-
3). Our previous studies had shown that the 
-subunits of
Gi-1, Gi-2, and Gi-3 selectively
activated PLC-
3 in smooth muscle (23-26).
DPDPE caused a significant increase in PLC-
activity (746 ± 29 cpm/106 cells [3H]inositol phosphates). The
PLC-
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-
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-
3 via the 
-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-
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-
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- (PLC- ) 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-
activity was expressed as total [3H]inositol phosphates
above basal levels (360 ± 62 cpm/106 cells).
Pretreatment with cANP-(4-23) inhibited PLC-
activity stimulated by SST and DPDPE; inhibition was partly reversed by
Cal. C. Values are means ± SE of 4 experiments.
|
|
 |
DISCUSSION |
This study demonstrates that PKC-dependent phosphorylation of the
-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 G
i-1 and
G
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
G
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 G
i-1 and G
i-2, but not
G
o or G
i-3, and reversed the inhibition of adenylyl cyclase mediated by somatostatin receptors that couple to
Gi-1 and Go (23) and opioid
-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
G
i-1 and G
i-2, but not G
o.
Suppression of dephosphorylation with calyculin A increased
phosphorylation of G
i-1 and G
i-2 but not
G
i-3; the increase in phosphorylation of
G
i-1 and G
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
-subunits of various G proteins have
shown that G
12, a ubiquitous G protein, and
G
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
G
Z and G
12 phosphorylation
(Ser27 and Ser16) are located in the
NH2-terminal domain of G
that determines the binding of
- and 
-subunits; phosphorylation of these residues impedes the
reassociation of
- and 
-subunits (11, 16, 20). Ser16 in G
i-1 and G
i-2 is
analogous to Ser16 in G
Z and
Ser38 in G
12; its phosphorylation might,
therefore, be expected to impede reassociation of
- and

-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
-subunits, impede the reassociation of
- and 
-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 G
i-1 and G
i-2 also led
to a decrease in responses mediated by 
-subunits. The notion was
tested with the NPR-C agonist, cANP-(4-23), which
selectively activates Gi-1 and Gi-2 and
stimulates PLC-
3 activity via the 
-subunits of both G proteins
(32, 33). Treatment of smooth muscle with cANP-(4-23) inhibited the PLC-
response to
subsequent activation of Gi-1 by somatostatin or activation
of Gi-2 by DPDPE. The PLC-
response was only partly
restored when PKC activity was blocked with calphostin C. We postulate
that residual inhibition reflects sequestration of G
i-1
and G
i-2 by binding to caveolin-3 (8, 17, 29). In contrast, inhibition of the
PLC-
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 G
i-1
and G
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-
3 activity stimulated by somatostatin-3 and opioid
-receptors coupled to G
i-1 and
G
i-2 (23, 26, and this study), but had no effect on
PLC-
3 activity stimulated by A1 and M2
receptors coupled to G
i-3 (24, 27, 29).
The pattern implied that inhibition of PLC-
activity by PKC in
smooth muscle was G protein specific and was not exerted directly on PLC-
isozymes. This is in contrast to other cell types, where PKC
was shown to inhibit directly mammalian or avian PLC-
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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