Inhibition of Protein Kinase C Prevents Rapid Desensitization of Type 1B Angiotensin II Receptor
Abstract The type 1B angiotensin II (AT1B) receptor cloned from rat kidney was stably expressed in Chinese hamster ovary cells. The stably expressed receptor was characterized by radioligand binding studies and functional coupling to inositol 1,4,5-triphosphate (IP3) formation. Exposure of cells expressing the AT1B receptor to angiotensin II (Ang II) resulted in a rapid and dose-dependent homologous desensitization of receptor-mediated production of IP3, with an essentially complete desensitization at an agonist concentration >10 nmol/L. Binding studies revealed no significant change in the number of AT1B receptors in transfected cells exposed to 1 nmol/L Ang II, whereas exposure to 100 nmol/L Ang II caused a rapid decrease of cell surface receptors, with a 75% loss of receptor number seen at 1 hour. Rapid desensitization occurred in the absence of receptor internalization. Blockade of receptor internalization with concanavalin A had at most only a slight effect on the agonist-induced desensitization. This indicates that factors other than internalization are chiefly responsible for the rapid agonist-induced desensitization. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, caused rapid desensitization of the receptor-mediated IP3 response. Neither tyrosine kinase inhibitors nor a protein kinase A activator affected the receptor-mediated IP3 response. The specific PKC inhibitor GF109203X or PKC depletion by prolonged treatment with 1 μmol/L PMA completely blocked the PMA-dependent desensitization. Desensitization evoked by a low Ang II agonist concentration (1 nmol/L) was reversed by the PKC-specific inhibitor GF109203X or PKC depletion, whereas the desensitizing effect at a high agonist concentration (100 nmol/L) is only partially prevented by PKC inhibitory treatment. These results demonstrate that PKC plays a crucial role in the desensitization of the AT1B receptor. They also suggest that receptor internalization and an additional PKC-independent pathway also contribute to desensitization of the AT1B receptor in transfected cells.
- angiotensin II
- AT1B receptor
- inositol 1,4,5-triphosphate
- protein kinase C
- Chinese hamster ovary cells
Plasma membrane Ang II receptors couple the extracellular peptide to intracellular signals that mediate the diverse physiological actions of the renin-angiotensin system.1 2 Recent pharmacological studies with selective nonpeptide receptor antagonists uncovered the presence of two major receptor isoforms, AT1 and AT2.1 2 3 The cDNA clones for these two types have been isolated.4 5 6 At present, the AT1 receptor is thought to mediate almost all the known functions of Ang II on target tissues.7 However, the precise functions of the AT2 receptor are unknown. Furthermore, subtypes of AT1 (AT1A and AT1B) receptor have been identified.8 9 Stimulation of the AT1 receptor results in the activation of phospholipase Cβ to generate the second messengers IP3 and diacylglycerol, which in turn release Ca2+ from intracellular stores and activate PKC, respectively.10 11 Previous studies1 12 13 14 15 have already shown that activation of AT1 receptor may result in its desensitization, downregulation, and internalization. In rat cardiomyocytes, consequent to incubation with agonist, the AT1 receptor undergoes agonist-induced desensitization, and this process is independent of loss of surface receptor binding capacity.15 In the adrenal gland2 and vascular smooth muscle cells,13 Ang II (>100 nmol/L) rapidly internalizes by receptor-mediated endocytosis, and this has been suggested as one of the mechanisms for receptor desensitization.16
The cellular basis for agonist-induced desensitization of G protein–coupled receptors has been best studied for the β-adrenergic receptor. Two biochemical mechanisms have been proposed for the process of desensitization.17 18 19 20 One mechanism is sequestration or internalization. The second mechanism involves a rapid functional uncoupling of the receptor from the guanine nucleotide–binding protein, mediated by phosphorylation of the receptor by cAMP-mediated kinases and second messenger–independent G protein–coupled receptor kinases. This latter mechanism provides a major route for desensitization of the β-adrenergic receptor.
Much less is known about the processes involved in mediating the desensitization of the Ang II receptor that activates phospholipase C. The desensitization of the β2-adrenergic receptor caused by exposure to low concentrations of agonists is believed to be mediated via protein kinase A.20 Phospholipase C–activating Ang II receptors do not concomitantly increase protein kinase A; thus, this second messenger is probably not involved in the desensitization of the phospholipase C–linked Ang II receptor. In contrast, PKC is activated by Ang II.10 11 In rat vascular smooth muscle cells21 and cardiac cells,15 activation of PKC with PMA leads to desensitization of the Ang II response; and in Xenopus oocytes, the PKC inhibitor staurosporine suppressed Ang II receptor–mediated desensitization.14 These observations suggest a role for PKC in the regulation of receptor desensitization.
To quantitatively assess the role of PKC in the agonist-induced desensitization of the Ang II receptor, we investigated the effect of PKC on the Ang II receptor–mediated IP3 response in CHO-K1 cells stably expressing the AT1BR. Our results showed that the PKC-specific inhibitor GF109203X and PKC downregulation by PMA completely suppressed the desensitization of AT1BR by Ang II at a low concentration (1 nmol/L). This suggests that PKC plays a major role in heterologous desensitization at low physiological agonist concentration. The desensitizing effect at higher agonist concentration (100 nmol/L) is only partially prevented by the inhibition of PKC.
Materials and Methods
Cell culture medium (Ham’s F-12), geneticin (G-418 sulfate), fetal calf serum, herbimycin-A, genistein, lavendustin-A, and anti–PKC-ζ antibody were obtained from Gibco BRL. [125I]NaI, [32P]Pi (1100 Ci · mmol−1), and [3H]IP3 radioreceptor assay kits were purchased from New England Nuclear Research Products. Monoclonal antiphosphotyrosine antibody was obtained from Upstate Biotechnology Inc. Ang II and [Sar1,Ile8]-Ang II were obtained from Peninsula Laboratories. Rad-Free kits for chemiluminescent detection of Western blots were obtained from Schleicher & Schuell. The nonpeptide antagonists losartan and PD123319 were generous gifts from Du Pont and Warner Lambert Co, respectively. CHO-K1 cells were obtained from American Type Culture Collection. The pRc/CMV expression vector was obtained from Invitrogen Corp. Restriction enzymes and calcium phosphate transfection kits were obtained from Promega Corp. GF109203X was obtained from Calbiochem. PMA, forskolin, staurosporine, ConA, and all other chemicals were purchased from Sigma Chemical Co.
Transfection and Maintenance of Cell Line
The entire coding region of rat kidney AT1B cDNA (an open reading frame encoding 359 amino acid residues) was subcloned into the BstXI site of the pRc/CMV expression vector. CHO-K1 cells were grown in Ham’s F-12 medium containing 10% fetal calf serum (complete medium) at 37°C in a humidified incubator with 5% CO2. At 40% confluence, CHO-K1 cells were transfected with 20 mg plasmid DNA by the calcium phosphate method according to the manufacturer’s protocol. After 10 hours of incubation, the medium was aspirated, and the cells were shocked with 3 mL of 15% glycerol solution for 2 minutes. The glycerol solution was aspirated, and cells were grown for 48 hours in complete medium. After that, cells were selected by 500 mg/mL geneticin (G-418). Resistant clones were subsequently isolated and transferred to 24-well plates. Based on 125I-[Sar1,Ile8]-Ang II binding, the clones with the highest level of expression for the AT1BR were used for all subsequent studies. Cells were maintained in Ham’s F-12 containing 10% fetal calf serum under a selection pressure of 150 mg/mL G-418 in an atmosphere of 95% air and 5% CO2 at 37°C.
Radioligand Binding Assay
Monoiodinated 125I-[Sar1,Ile8]-Ang II was prepared by the lactoperoxidase method22 and purified by reversed-phase HPLC as described previously23 except that a 0% to 80% acetonitrile gradient was applied for 60 minutes with a flow rate of 1.0 mL/min. Binding studies were done with cells at or near confluence by methods originally described by Gunther et al24 and Penit et al.25 Assay buffer consisted of 50 mmol/L Tris-HCl, pH 7.4, 100 mmol/L NaCl, 5 mmol/L MgCl2, 0.25% BSA, and 0.5% mg/mL bacitracin. At the beginning of each experiment, culture medium was aspirated from the wells, and cells were washed twice with ice-cold PBS. After the final wash, cells were incubated with 50 pmol/L 125I-[Sar1,Ile8]-Ang II for 2 hours at 25°C in the presence of various concentrations of unlabeled ligands (10−12 to 10−6 mol/L) for competition binding assay. The cells were washed three times with ice-cold PBS, then dissolved in 0.25N NaOH and 0.05% SDS, and the radioactivity was measured in a Beckman Compu Gamma counter. Experimental results were expressed as specific binding, defined as that portion of the total binding displaced by 10 mmol/L unlabeled Ang II. Nonspecific binding was <15% of the total binding. Values were normalized to the amount of protein. For measurement of the surface receptor binding capacity, the cells were pretreated with or without ConA (0.25 mg/mL), PMA (100 nmol/L), GF109203X (0 to 20 μmol/L), or staurosporine (3 μmol/L), then incubated with or without 1 nmol/L or 100 nmol/L Ang II for the indicated period in an atmosphere of 5% CO2/95% air at 37°C. After these treatments, cells were washed with ice-cold 50 mmol/L glycine and 150 mmol/L NaCl, pH 3.0, for 5 minutes at 4°C. After two saline washes, the binding studies were performed with near-saturating conditions with 2 nmol/L 125I-[Sar1,Ile8]-Ang II for 3 hours at 4°C.
Measurement of IP3 Mass
Cells were subcultured into multiwell dishes (12 wells per plate) and at or near confluence were exposed to Ang II; the treatment was stopped by the addition of 1/5 volume of 100% ice-cold trichloroacetic acid to the plates. The cells were then harvested by scraping and transferred to polypropylene tubes. The cell extract was vortexed thoroughly, then centrifuged for 10 minutes at 6000g at 4°C. The supernatant was removed and warmed to room temperature for 15 minutes. Levels of IP3 in each supernatant were determined by use of a radioimmunoassay kit from Du Pont NEN following instructions supplied by the manufacturer.
Desensitization of AT1BR Subtype
Cells at or near confluence in 12-well plates were washed once with serum-free medium containing 0.1% BSA and treated or untreated with ConA (0.25 mg/mL), PMA (100 nmol/L), GF109203X (0 to 80 μmol/L), staurosporine (3 μmol/L), and various concentrations of forskolin or tyrosine kinase inhibitors, then incubated in the absence or presence of 1, 20, or 100 nmol/L Ang II for indicated periods. The reaction was stopped by washing the cells with 150 mmol/L NaCl/50 mmol/L glycine (pH 3.0) for 5 minutes at 4°C and prewarmed PBS at room temperature twice. For measurement of the IP3 level in response to Ang II, the cells were again stimulated with Ang II (100 nmol/L), and 15 seconds later the IP3 mass was assayed as described above.
Protein Phosphorylation Assay
Protein (p80) phosphorylation assay was performed essentially as described.26 Briefly, the cells expressing AT1BR were labeled with [32P]Pi (1 mCi/plate) for 4 hours in phosphate-free medium. Different concentrations of the PKC inhibitor GF109203X were added 30 minutes before stimulation with Ang II (100 nmol/L) or PMA (100 nmol/L). After stimulation with Ang II for 15 minutes or PMA for 10 minutes, cells were lysed for 5 minutes at room temperature in a buffer containing 20 mmol/L Tris-HCl, pH 7.4, 2 mmol/L EDTA, 10 mmol/L EGTA, 0.5% Triton X-100, and 10 mmol/L NaF. The lysate was then boiled for 15 minutes and centrifuged 5 minutes at 15 000 rpm. Supernatants, which contained p80, were mixed with SDS sample buffer and boiled for 5 minutes. Phosphorylated protein (pp80) was analyzed by SDS–PAGE and autoradiography.
For determination of protein tyrosine phosphorylations induced by Ang II, confluent cells were cultured for 48 hours in Ham’s F-12 medium without serum to induce quiescence. PKC inhibitor GF109203X was added 30 minutes before stimulation with Ang II (100 nmol/L). After stimulation with Ang II for 1 minute, cells were lysed on ice in 0.5 mL of P-TYR lysis buffer (50 mmol/L HEPES, pH 7.5, 1% Triton X-100, 50 mmol/L NaCl, 50 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 5 mmol/L EDTA, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, plus 10 μg/mL of aprotinin and leupeptin) according to the method described previously.27 Equal protein aliquots were fractionated on an 8.0% SDS-PAGE and electrophoretically transferred to Rad-Free membrane (Schleicher & Schuell). The blocked membranes then were incubated with antiphosphotyrosine monoclonal antibody, and the immunoreactive bands were visualized with chemiluminescent reagents as recommended by the manufacturer.
Cell Extraction and Immunoblot Analysis for PKC-ζ
After the treatments with Ang II (1 or 100 nmol/L), the cells expressing AT1BR were washed with ice-cold PBS and scraped into lysis buffer (20 mmol/L Tris-HCl, pH 7.4, 5 mmol/L EDTA, 5 mmol/L EGTA, 5 mmol/L β-mercaptoethanol, 10 mmol/L benzamidine, 25 μg/mL leupeptin, 25 μg/mL aprotinin, and 1 mmol/L PMSF). All subsequent steps were carried out at 4°C. The cells were homogenized for 30 strokes in a type A Dounce homogenizer and centrifuged at 100 000g for 30 minutes in a Beckman TL 100 ultracentrifuge. The supernatant (cytosol) was removed, and the pellet was washed and extracted in the above buffer containing 0.5% Triton X-100. After a 30-minute incubation, the samples were centrifuged at 12 000g for 20 minutes, yielding in the supernatant the detergent-soluble fraction (membranes). Samples containing equal amounts of protein (70 to 100 mg) were separated by 8.0% SDS-PAGE and electrophoretically transferred from the gel onto Rad-Free membrane. The blocked membranes then were incubated for 30 minutes with PKC-ζ antibody, and immunoreactive bands were visualized with chemiluminescent reagents.
Data are given as mean±SEM. Statistical analysis was performed by analysis of variance and unpaired Student’s t test as appropriate. Significance was accepted at P<.05.
Pharmacological Properties of Stably Expressed Rat AT1BR in CHO-K1 Cells
CHO-K1 cells with no detectable Ang II binding (data not shown) were stably transfected with rat AT1BR plasmid. The cells were assessed for the ability to bind several Ang II receptor agonists and antagonists. The binding of 125I-[Sar1,Ile8]-Ang II, a peptidic Ang II receptor antagonist, to the cells was dose-dependently inhibited by Ang II, [Sar1,Ile8]-Ang II, and the AT1 receptor–selective antagonist losartan with the potency order [Sar1,Ile8]-Ang II>Ang II>losartan. In contrast, PD123319, an AT2 receptor–selective antagonist, had no effect on the binding of 125I-[Sar1,Ile8]-Ang II (not shown). This displacement pattern was similar to those for the rat, bovine AT1A, and rat AT1BRs that had been transiently expressed in Cos-7 cells,4 5 8 demonstrating that the recombinant AT1BR exhibited typical pharmacological characteristics of the AT1 receptor. Scatchard plots transformed from the competitive displacement study revealed binding parameters with a dissociation constant (Kd) of 2.08 nmol/L and a binding site number (Bmax) of 970 fmol/mg protein. This Kd value was in good agreement with that previously reported for the rat AT1BR transiently expressed in Cos-7 cells (Kd=2.3 nmol/L).8
CHO-K1 Cells Transfected With AT1BR Desensitize in Response to Ang II
Binding of Ang II to its receptor results in the activation of the Ca2+/IP3 signal transduction pathway.10 11 To characterize functional coupling to the signal transduction pathway, receptor-mediated phosphatidylinositol hydrolysis was determined by measuring the time-dependent generation of IP3 with a radioreceptor binding assay. The IP3 level was maximal at 15 seconds, with a level more than threefold above the basal level. The stimulation of IP3 formation was dependent on the Ang II concentration. The threshold concentration of Ang II that generated a detectable increase in IP3 (measured at 15 seconds) was between 0.1 and 1 nmol/L. At 1 nmol/L of Ang II, the IP3 level was significantly greater than the basal value (P<.05) (data not shown). Nontransfected CHO-K1 cells showed no IP3 response to Ang II.
To ascertain whether homologous desensitization of AT1BR occurs, we determined the effect of a pretreatment with Ang II on the subsequent ability to generate an inositol phosphate in response to 100 nmol/L Ang II. The pretreatment with Ang II resulted in a subsequent attenuation of Ang II–induced IP3 production. As shown in Fig 1A⇓, the AT1BR is desensitized rapidly as early as 1 minute (P<.05) on preincubation with 100 nmol/L Ang II; complete desensitization occurred within 15 minutes. However, the extent of homologous desensitization induced by 1 nmol/L Ang II did not change significantly with time over 60 minutes of preincubation. Fig 1B⇓ shows a concentration-effect relation for the inhibition of subsequent Ang II (100 nmol/L)–induced IP3 formation. A maximal desensitization occurred only at concentrations of the agonist >10 nmol/L. These findings indicate that the AT1BR expressed in CHO-K1 cells can undergo homologous desensitization.
Receptor Sequestration or Internalization Does Not Completely Explain the Reduced Ang II Response
Receptor sequestration or internalization was first considered as a potential mechanism for the reduced Ang II response. As shown in Fig 2⇓, no significant change was seen in the amount of ligand bound to the surface of cells treated with 1 nmol/L Ang II for 15 minutes. Also, the receptor affinity for 1 nmol/L Ang II was not significantly altered after 15 minutes of the preincubation (Kd=1.98±0.21 nmol/L pretreated cells versus 2.08±0.17 nmol/L control cells). In contrast, the maximal binding of 125I-[Sar1,Ile8]-Ang II to cells exposed to 100 nmol/L Ang II rapidly decreased with time, with a 55% loss after 15 minutes of preincubation (Fig 2⇓). There was no significant change in the affinity of receptors before (2.08±0.17 nmol/L) and after (1.79±0.25 nmol/L) exposure to 100 nmol/L Ang II.
We investigated further the possible role of receptor sequestration or internalization in the Ang II–stimulated desensitization. ConA has been reported to block the internalization of a number of receptors.18 28 Cells were preincubated with ConA (0.25 mg/mL) for 30 minutes at 37°C. Radioligand binding experiments with 2 nmol/L 125I-[Sar1,Ile8]-Ang II (a near-saturating concentration) revealed that ConA can obviously inhibit the internalization of AT1BR induced by Ang II (100 nmol/L) (Fig 2⇑). ConA did not markedly affect the IP3 formation in response to Ang II (Fig 3⇓) and basal IP3 levels (data not shown). As shown in Fig 3⇓, the IP3 response of the ConA-treated cells was desensitized by 15 minutes of incubation with Ang II (1 nmol/L) to the same extent as the response of the control cells. Additionally, the desensitization of AT1BR also occurred in response to a 1-minute incubation with 100 nmol/L Ang II even when the receptor internalization was almost completely blocked by ConA (percent of basal IP3, 247±15.2% Ang II–treated cells versus 335±11.2% control cells). However, the complete desensitization induced by 15 minutes of incubation with 20 or 100 nmol/L Ang II can be slightly reduced by the treatment with ConA. The results with ConA indicated that Ang II–induced desensitization of AT1BR occurs in the absence of the receptor internalization. Additional factors are mainly responsible for the rapid agonist-induced desensitization.
PMA-Dependent Desensitization of AT1BR Requires PKC
Recently, Ali et al29 30 reported that PKC plays an important role in mediating the agonist-evoked desensitization by C5a, a chemoattractant receptor, and by platelet-activating-factor receptor in transfected RBL-2H3 cells. One plausible hypothesis is that PKC may also mediate the Ang II–evoked desensitization of AT1BR. To test this hypothesis, cells were treated with PMA (100 nmol/L) for 5 or 10 minutes. This leads to a translocation of PMA-sensitive PKC activity from the cytosol to the particulate fraction of transfected cells (data not shown). As shown in Fig 5⇓, PMA treatment mimicked the effects of Ang II in desensitizing the cells to the peptide, with no effect on the basal level of IP3. To determine whether this decrease in IP3 response was due to a decrease in receptor number or affinity after PMA (100 nmol/L) administration, radioligand binding studies were performed. No significant alteration was seen in the amount of ligand bound to the surface of cells treated with PMA (100 nmol/L, 5 minutes) (98 303±19 003 versus 92 507±10 094 cpm without PMA treatment) or in receptor affinity (Kd=1.78±0.26 versus 2.08±0.17 nmol/L without PMA treatment).
To determine further whether PMA-dependent desensitization requires PKC, we took advantage of the newly tested in vitro and in vivo nontoxic PKC-specific inhibitor GF109203X.26 To establish the effectiveness of this PKC inhibitor, we assayed its effect on the in vivo phosphorylation of MARCKS-p80, a well-known endogenous PKC substrate,31 or on the protein tyrosine phosphorylation induced by Ang II. Fig 4⇓ shows that the PKC-dependent phosphorylation of MARCKS-p80 was markedly inhibited by GF109203X at a concentration of 10 μmol/L. The same result was observed when PKC was activated by PMA (100 nmol/L) treatment of cells (not shown). In contrast, the Ang II (100 nmol/L)–induced protein tyrosine phosphorylation was not affected by GF109203X (Fig 4⇓). These results demonstrated the specific PKC-inhibitory effect of GF109203X. Prior incubation with either GF109203X (10 μmol/L) or the nonspecific PKC inhibitor staurosporine (3 μmol/L) completely blocked the PMA-induced desensitization of AT1BR (Fig 5⇓). Neither compound affected the receptor binding affinity or number or the basal IP3 level (data not shown). Consistent with the effects of PKC inhibitors, downregulation of PKC by overnight (24-hour) incubation with PMA (1 μmol/L)32 completely blocked the desensitization of the AT1BR by PMA (Fig 5⇓).
To show that these effects are due to inhibition of PKC, we examined the dose-response relation of the suppression of the AT1BR desensitization by the PKC- specific inhibitor GF109203X. As shown in Fig 5⇑ (inset), the PMA-induced desensitization of the AT1BR in transfected cells was inhibited by GF109203X with an IC50 of 3.5 μmol/L, and inhibition of desensitization was virtually complete at a concentration >10 μmol/L.
Recently, Molloy et al27 and Schorb et al33 reported that Ang II can induce rapid protein tyrosine phosphorylation and tyrosine protein kinase activation in rat aortic smooth muscle cells and rat cardiac fibroblasts, respectively. To determine whether the protein tyrosine kinase was able to affect the Ang II–induced formation of IP3, we measured IP3 response in cells treated with three different tyrosine kinase inhibitors: genistein, herbimycin-A, or lavendustin-A. The results (not presented) showed that neither compound affected the IP3 response to Ang II in intact cells. In other experiments, the results showed that the homologous desensitization of the AT1BR was not affected by pretreatment of cells with genistein (500 μmol/L) along with Ang II (100 nmol/L) for 15 minutes (percent of basal IP3, 125±12.1% genistein+Ang II versus 118±8.9% Ang II). It is known that protein kinase A plays a major role in the heterologous desensitization at a low agonist concentration in the β-adrenergic receptor system.20 To investigate the possible role of protein kinase A in the Ang II–induced desensitization, cells were treated with the protein kinase A activator forskolin at various concentrations for 20 minutes, and the IP3 level evoked by Ang II was determined. It was found that forskolin even at 50 μmol/L had no effect on the IP3 formation (data not shown). This suggested that protein kinase A is not involved in the Ang II–evoked desensitization of AT1BR in the transfected cells.
Inhibition of PKC Completely Blocks the Ang II–Induced Rapid Homologous Desensitization of AT1BR at Low Agonist Concentration
As shown in Fig 1A⇑, Ang II–induced desensitization of AT1BR occurs rapidly. The pretreatment of AT1BR-transfected cells with 1 nmol/L Ang II for 15 minutes desensitized the response to Ang II (100 nmol/L). The inhibitors of PKC GF109203X (10 μmol/L) and staurosporine (3 μmol/L) or depletion of PKC completely abolished the rapid desensitization of AT1BR evoked by 1 nmol/L Ang II (Fig 6⇓). To confirm that these effects are due to inhibition of PKC, we demonstrated that the suppression of the Ang II (1 nmol/L)–induced desensitization by GF109203X was concentration-dependent, with an IC50 (for GF109203X) of 4.0 μmol/L and maximal inhibition occurring at concentrations >10 μmol/L. Taken together, these data suggest that PKC is the major mediator of heterologous desensitization of the AT1BR at a low agonist concentration.
Inhibition of PKC Partially Prevents the Rapid Homologous Desensitization of AT1BR at a High Agonist Concentration
The addition of Ang II at a high concentration (100 nmol/L) to the AT1BR-transfected cells resulted in subsequent complete attenuation of Ang II–induced IP3 elevations (Fig 7⇓). However, in the presence of PKC inhibitors, the desensitization of the AT1BR evoked by 100 nmol/L Ang II was partially prevented by GF109203X (10 μmol/L) or staurosporine (3 μmol/L), whereas neither compound, GF109203X (0 to 20 μmol/L) or staurosporine (3 μmol/L), affected the internalization of AT1BR in intact cells (data not shown). It has been reported that PKC is markedly activated by Ang II (100 nmol/L) in several types of cells.10 11 To determine whether these effects are due to the insufficient inhibition of PKC, we examined the concentration dependence for the inhibition of AT1BR desensitization by GF109203X. As shown in Fig 7⇓, the desensitization of the AT1BR evoked by Ang II (100 nmol/L) was inhibited by GF109203X with an IC50 of 7.0 μmol/L, and inhibition was only partially prevented at a high dose of this compound (20 μmol/L) or even at a still higher concentration (80 μmol/L). The desensitization of the AT1BR induced at high agonist concentration was only partially attenuated in PKC-depleted cells. The extent of this attenuation was greater than that achieved with GF109203X. We also found that ConA had only a slight effect on homologous desensitization of AT1BR in PKC-depleted transfected cells (Fig 7⇓).
The diverse effects traditionally attributed to Ang II are mediated primarily through the type 1 Ang II receptor. Since the two subtypes of AT1 receptor are often expressed in the same tissues and cells, studies of the mechanisms in the regulation of individual subtypes are often impossible. To circumvent this problem, particularly in studying the problem of receptor desensitization at the molecular level, we established stable expression of a single Ang II receptor subtype (AT1BR) into CHO-K1 cells, which are devoid of endogenous Ang II receptors.
It has been noted that Ang II action is accompanied by a rapid and profound desensitization known as tachyphylaxis. The desensitization and internalization are seen to occur simultaneously. The present results clearly indicate that AT1BR-mediated IP3 production is rapidly desensitized even in the absence of receptor internalization. This desensitization was dependent on the dose of added Ang II; a maximal desensitization occurred only at an agonist concentration >10 nmol/L. Rapid desensitization of the IP3 response occurring within minutes of exposure to the agonist has been shown for Ang II receptors in the adrenal gland,1 vascular smooth muscle cells,1 Xenopus oocytes,14 and rat heart.15 However, the mechanisms by which Ang II receptors desensitize are currently unclear. In the adrenal gland2 and vascular smooth muscle cells,13 Ang II (>100 nmol/L) rapidly internalizes by receptor-mediated endocytosis, and this has been suggested as a mechanism for receptor desensitization.16 Recent studies suggest that the AT1 receptor undergoes agonist-induced desensitization in rat cardiomyocytes, apparently in a manner independent of loss of the surface receptor binding capacity.15 To determine the relation between physical and functional coupling of receptors to agonist and receptor sequestration, we assessed the extent of sequestration or internalization induced by a physiological (1 nmol/L) and supraphysiological (100 nmol/L) concentration of Ang II in AT1BR-transfected cells. The analysis of Ang II binding and internalization with cells pretreated with Ang II at the low and high concentrations (15 minutes) demonstrated no significant change in receptor number or affinity (Fig 2⇑) with 1 nmol/L Ang II but significant internalization (55%) with 100 nmol/L of the agonist. Rapid desensitization occurred on pretreatment with 1 nmol/L Ang II in the absence of receptor internalization. Also, rapid desensitization occurred in response to 1 minute of incubation with 100 nmol/L Ang II even when the receptor internalization was almost completely blocked by ConA treatment. Additionally, full desensitization occurred with 20 or 100 nmol/L Ang II, and this was only slightly prevented when receptor internalization was blocked by the treatment with ConA. We also found that ConA has only a slight effect on homologous desensitization of AT1BR in PKC-depleted cells. This indicates that sequestration or internalization of receptors does not seem to play a major role in causing the desensitization and is virtually absent as a cause at physiological levels of Ang II. Thus, rapid desensitization of AT1BR-mediated IP3 formation is mainly a consequence of the agonist-induced intracellular signaling mechanisms.
With the β-adrenergic and rhodopsin receptors, one of the early events after agonist stimulation is the phosphorylation of the receptor at specific regions on serine and threonine residues by nonspecific kinases such as PKC or protein kinase A or by specific G protein receptor kinases.19 Studies on the agonist-induced desensitization of β-adrenergic receptor suggest that protein kinase A plays a major role in the heterologous desensitization at a low agonist concentration, whereas β-adrenergic kinase is important in eliciting homologous desensitization at high agonist concentration.19 20 There are multiple serine and threonine residues in the intracellular loops of AT1BR, and a serine- and threonine-rich region exists at the carboxy terminal cytosolic domain of the AT1BR, as in the carboxy terminal regions in other G protein–coupled receptors, ie, β-adrenergic and rhodopsin receptors.19 34 35 There are three potential consensus sites for PKC phosphorylation (S331TK, S338YR, and S348AK) in the carboxy terminal region of the AT1BR.4 5 36 Thus, it is possible that Ang II–mediated receptor modification (phosphorylation) plays an important role in Ang II receptor desensitization.
Since the AT1BR was coupled to the phospholipid hydrolysis/intracellular calcium mobilization pathway that leads to the activation of PKC, we investigated the role of PKC in the desensitization of the AT1BR. It has been reported that acute treatment with PMA could cause desensitization of the AT1 receptors.21 Recently, Abdellatif et al15 also reported that activation of PKC with PMA leads to desensitization of AT1R-mediated IP3 formation in rat cardiomyocytes, but PMA-induced depletion of cellular PKC does not alter this homologous desensitization of the cardiac receptors for Ang II. However, several aspects of this study require clarification. No characterization of the culture was performed, and it is most likely a mixture of cardiomyocytes and other cell types, particularly cardiac fibroblasts, which have recently been shown to express high levels of Ang II receptors.37 Since multiple subtypes exist in the heart1 and in these cardiomyocyte cultures,15 it is not possible to draw definitive conclusions regarding receptor specificity when no characterization of Ang II receptor subtypes was performed. In Xenopus oocytes,14 the PKC inhibitor staurosporine suppressed Ang II receptor–mediated desensitization. In the present study, in agreement with previous reports,15 21 we found that acute treatment with PMA could induce desensitization of AT1BR. This PMA-induced desensitization of AT1BR was blocked completely by the PKC-specific inhibitor GF109203X or the nonspecific inhibitor staurosporine or by PKC depletion by prolonged exposure to PMA. Like the PMA-induced desensitization of the AT1BR, the cloned AT1BR expressed in CHO-K1 cells is rapidly desensitized on exposure to Ang II. We also found that cloned AT1AR expressed in CHO-K1 cells performed similarly in this system (data not shown). Our results presented in this article clearly indicate that the biochemical mechanisms underlying Ang II– and PMA-induced desensitization of the AT1BR are similar. The PKC-specific inhibitor GF109203X26 or the nonspecific inhibitor staurosporine or PKC downregulation by an overnight treatment with PMA (1 μmol/L)32 could completely abolish the desensitization by PMA as well as the desensitization by Ang II at a low physiological concentration of 1 nmol/L.
In the present study, the desensitization mechanisms at low (1 nmol/L) and at high (100 nmol/L) agonist concentrations are different. At 1 nmol/L, Ang II induced an incomplete desensitization that was not changed with time over 60 minutes of incubation. Furthermore, the desensitization was blocked by PKC inhibitors in a dose-dependent manner and was also completely blocked by PKC depletion. With a high-dose agonist treatment, essentially a complete desensitization occurred within 15 minutes, and this desensitization was only partially inhibited by PKC inhibition and depletion. Similar findings have recently been reported for the cloned C5a chemoattractant receptor or platelet-activating factor receptor expressed in transfected RBL-2H3 cells: PKC mediated heterologous phosphorylation and desensitization of C5a receptor at low levels of stimulation by C5a, whereas the PKC inhibitor staurosporine only partially inhibited the platelet-activating-factor receptor phosphorylation that correlated with desensitization in response to a high level of platelet-activating factor.29 30 Our observations indicated that a PKC-dependent mechanism plays a primary role in the desensitization of AT1BR at low agonist concentration. Our observations also suggest that an additional non–PKC dependent mechanism mediating the homologous complete desensitization of the receptor at the high agonist concentration may not function upon treatment with a low agonist concentration. Clearly, more work is needed to fully understand the involvement of the PKC, as well as a possible PKC-independent kinase (such as a β-adrenergic receptor kinase) in the Ang II–induced phosphorylation of AT1BR and the impact of specific receptor phosphorylations on receptor function.
The potent and selective PKC inhibitor GF109203X had been shown to act as a competitive inhibitor of ATP but not PMA for the PKC isozymes α, β, and γ but was less active as an inhibitor for PKC-ε (fourfold to sixfold less).26 This pattern of PKC isoform selectivity suggests that the agonist-induced desensitization in our study may be mediated by PMA-sensitive PKC isozymes, in particular by α, β, or γ. Although PMA- and calcium-insensitive PKC-ζ exists in AT1BR-transfected cells, it does not appear to be activated by treatment with the low or high concentration of Ang II, as judged by the absence of the translocation of PKC-ζ from cytosol to the particulate fraction (Fig 8⇓). The identification of specific PKC isozymes implicated in the agonist-induced regulation of the AT1BR is to be clarified in further studies.
In summary, using a CHO-K1 cell line stably expressing functional rat AT1BR, we have shown that the desensitization of AT1BR occurs within minutes of initial exposure to Ang II even in the absence of receptor internalization. It is maximal at agonist concentrations >10 nmol/L. These data suggest that besides internalization, additional factors mainly contribute to the rapid agonist-induced desensitization. PKC inhibitors or PKC depletion can abolish the PMA-induced desensitization of AT1BR. Desensitization evoked by a low physiological agonist concentration of Ang II was reversed by PKC inhibition or depletion, whereas that evoked by a supraphysiological agonist concentration was only partially blocked.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1BR||=||type 1B angiotensin II receptor|
|CHO||=||Chinese hamster ovary|
|HPLC||=||high-performance liquid chromatography|
|PAGE||=||polyacrylamide gel electrophoresis|
|PKC||=||protein kinase C|
|PMA||=||phorbol 12-myristate 13-acetate|
This work was supported in part by National Institutes of Health research grants HL-14192 and HL-35323. We wish to thank Trinita Fitzgerald for superb technical assistance in cell culture. The authors thank Drs E.J. Landon and T. Hamakubo for critical reading of the manuscript.
- Received November 14, 1994.
- Accepted April 12, 1995.
- © 1995 American Heart Association, Inc.
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