©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Gastrin-releasing Peptide Receptor Is Rapidly Phosphorylated by a Kinase Other Than Protein Kinase C After Exposure to Agonist (*)

(Received for publication, September 23, 1994; and in revised form, January 25, 1995)

Glenn S. Kroog (1)(§) Eduardo Sainz (1) Peter J. Worland (1) Mark A. Akeson (1) Richard V. Benya (2) Robert T. Jensen (2) James F. Battey (1)

From the  (1)Laboratory of Biological Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, NCI and (2)Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-4255

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Several guanine nucleotide-binding protein-coupled receptors are known to be rapidly phosphorylated after agonist exposure. In this study we show that the gastrin-releasing peptide receptor (GRP-R) is rapidly phosphorylated in response to agonist exposure. When [P]orthophosphate-labeled cells were exposed to bombesin, the receptor was maximally phosphorylated on serine and threonine residues within 1 min. Although addition of 12-O-tetradecanoylphorbol 13-acetate also resulted in phosphorylation of the GRP-R, elimination of protein kinase C activity using the inhibitor 7-hydroxystaurosporine did not prevent bombesin-induced GRP-R phosphorylation. We conclude that a kinase other than protein kinase C is principally responsible for the rapid, agonist-induced phosphorylation of the GRP-R.


INTRODUCTION

Several members of the guanine nucleotide-binding protein (G protein)(^1)-coupled receptor superfamily have been shown to be rapidly phosphorylated after the addition of agonist (1, 2, 3, 4, 5, 6, 7, 8, 9) . This phosphorylation is dependent upon two classes of kinases. Second messenger dependent kinases include (cAMP-dependent) protein kinase A and (calcium- and diacylglycerol-dependent) protein kinase C (PKC). Second messenger independent kinases called G protein-coupled receptor kinases or GRKs (reviewed by Inglese et al.(10) ), include rhodopsin kinase and the beta-adrenergic receptor kinases 1 and 2. The kinase or kinases involved in receptor phosphorylation depends upon both the specific G protein-coupled receptor and the cellular milieu.

In several other receptor systems, this phosphorylation has been related to the acute diminution of responsiveness seen following continuous or repeated exposure to agonist (acute desensitization)(8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) . For the gastrin-releasing peptide receptor (GRP-R) little is known about the molecular mechanisms involved in acute desensitization. Several groups have studied the role of PKC using phorbol esters to stimulate PKC activity(21, 22) . They found that short exposures to 12-O-tetradecanoylphorbol 13-acetate (TPA) caused desensitization of bombesin-induced inositol phosphate generation without diminishing I-Tyr^4-bombesin binding, implying that PKC can cause acute desensitization without reducing the number of GRP-Rs on the cell surface. More recently, while studying desensitization of intracellular calcium ([Ca]) generation, both Frankel and Viallet (23) and Walsh et al.(24) have shown that inhibition of PKC prevented phorbol ester- but not bombesin-induced desensitization. This suggests that PKC activation may not be a physiologically relevant mechanism for agonist-induced acute desensitization of the GRP-R, raising the possibility that other kinases may be involved in acute desensitization of the GRP-R. Neither study, however, directly shows that the GRP-R itself is a target for these kinases.

In this study, we examined whether or not the GRP-R is actually phosphorylated in response to agonist binding, and if this phosphorylation is dependent upon PKC. The results presented here show that the GRP-R is rapidly phosphorylated after agonist binding and that this phosphorylation does not require PKC.


EXPERIMENTAL PROCEDURES

Materials

Balb 3T3 mouse fibroblasts and Swiss 3T3 mouse fibroblasts were obtained from the American Type Culture Collection (Rockville, MD). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and aminoglycoside G-418 were from Life Technologies, Inc. Polymerase chain reactions were performed using a GeneAmp polymerase chain reaction system 9600 from Perkin-Elmer. Protein gel electrophoresis equipment, SDS-PAGE gels, running buffer, and transfer buffer were from Novex (San Diego, CA). Peptide:N-glycosidase F (N-glycanase) was purchased from Genzyme Corp. (Cambridge, MA). Bombesin was purchased from Peninsula Laboratories (Belmont, CA). TPA was purchased from Sigma.

Mutant GRP-R Construction

Mutant cDNAs with a myc epitope (EQKLISEEDLN) (25, 26) at the 5` (5`ET) or 3` (3`ET) end were constructed by using mouse GRP-R cDNA (27) as a template for the polymerase chain reaction with synthetic oligonucleotide primers using standard methodology. The epitope was added immediately after the initiator methionine (5`ET) or immediately before the termination codon (3`ET). The resultant polymerase chain reaction products were digested with EcoRI and ligated into a modified version of the mammalian expression vector pCD2(28) . The nucleotide sequences of 5`ET and 3`ET mutant GRP-R constructs were confirmed by sequencing.

Stable Expression of Receptor Constructs in Balb 3T3 Fibroblasts

Balb 3T3 fibroblasts were transfected with 25 µg of recombinant pCD2 plasmids by calcium phosphate precipitation(29) . Selection with the aminoglycoside G-418 (800 µg/ml) was begun 48 h after transfection and continued for 2-3 weeks. Clonal cell lines were screened for I-Tyr^4-bombesin binding as described previously(30) . Clones expressing greater than 10^6 receptors/cell were chosen for use in further experiments (5`ET4 and 3`ET2). Stably transfected cell lines were maintained in DMEM containing 300 µg/ml G-418 and supplemented with 10% fetal bovine serum (FBS).

Binding and Internalization Assays

I-Tyr^4-bombesin (2000 Ci/mmol) was prepared using IODOGEN and purified by high pressure liquid chromatography as described previously(31) . Binding studies were performed as described previously (31) in a binding buffer composed of 98 mM NaCl, 6 mM KCl, 5 mM pyruvate, 6 mM fumarate, 5 mM glutamate, 11 mM glucose, 25 mM HEPES, 2.2 mM KH(2)PO(4), 0.1% bovine serum albumin, 0.02% bacitracin, 1.5 mM CaCl(2), and 1 mM MgCl(2) adjusted to pH 7.4. Incubations were performed with disaggregated cells suspended at 2 times 10^6 cells/ml in binding buffer with 75 pMI-Tyr^4-bombesin for 30 min at 25 °C. Nonspecific binding of I-Tyr^4-bombesin was defined as the amount of radioactivity associated with cells in the presence of 1 µM bombesin. Analysis of binding data was performed using the least squares curve-fitting program LIGAND(32) .

Internalization of receptors was performed as described previously(31, 33) , defined as the percentage of specific cell-associated I-Tyr^4-bombesin resistant to acid wash and is reported as the standard error of the mean percent of internalization of four (BNR-11 and 5`ET4) or five (3`ET2) separate experiments done in triplicate. Curve fitting was done using first order kinetics.

Measurement of Intracellular Calcium

3 times 10^6 cells were plated into 100 times 20-mm tissue culture dishes. The following day the medium was removed, and the dishes were washed twice with DMEM supplemented with 0.5% FBS. The dishes were then incubated at 37 °C for 14 min in 5 ml of DMEM + 0.5% FBS containing 1 µM Fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR). The dishes were washed twice with ice-cold binding buffer, 3 ml of binding buffer were added to the dish, and the cells were detached by scraping. The cells were disaggregated by pipetting, pelleted in a tabletop centrifuge, and resuspended in 6 ml of binding buffer. The cell suspension was placed on ice until ready for use. When ready, 2 ml of cell suspension were repelleted and resuspended in binding buffer. The cells were warmed in a 37 °C water bath for 2.5 min, then added to a quartz cuvette which was placed into a Perkin-Elmer LS-5B luminescence spectrometer for fluorescence monitoring using an excitation wavelength of 336 nm and emission wavelength of 510 nm. The cells were continually stirred using a Hellma Cuv-o-stir model 333 (Hellma, Jamaica, NY), and maintained at 37 °C by connecting the cuvette holder to a HAAKE water bath (HAAKE, Inc., Paramus, NJ). After 3 min (to allow the cells to equilibrate), hormones were added as indicated in the text and figure legends. [Ca](i) was calculated using the formula [Ca](i) in mM = K(F - F(min))/(F(max) - F), where F was the fluorescence at the unknown [Ca](i), F(max) the fluorescence after addition of 0.05% Triton X-100, F(min) the fluorescence after addition of 25 mM EGTA, and K = 220 nM(34) . Increases in [Ca](i) were calculated as peak values minus baseline and reported as mean ± S.D. for representative triplicates performed over the course of 2 days.

Generation of GRP-R Antiserum

A peptide was synthesized (CVEGNIHVKKQIESRKR) by Peptide Technologies (Gaithersburg, MD) corresponding to a portion of the third intracellular loop of the mouse and human GRP-R. The peptide was conjugated to keyhole limpet hemocyanin as described previously (35) for the production of polyclonal antisera from three rabbits. The results described throughout this publication were obtained with the antiserum from rabbit no. 3.

Membrane Preparation

After the cells were treated as described below and in the figure legends, the cell culture dishes were washed twice with ice-cold phosphate-buffered saline. When used for phosphorylation studies, the saline was supplemented with 10 mM NaF, 2 mM EDTA, 2 mM EGTA, 18.4 µg/ml sodium vanadate, 1 mM PMSF, 0.01% soybean trypsin inhibitor, and 1 µg/ml leupeptin. Next, 1 ml of homogenization buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 5 mM MgCl(2), and 330 mM sucrose) per 20 cm^2 of cell culture dish surface area was added to the dish. For phosphorylation assays, this buffer was supplemented as was the phosphate-buffered saline wash buffer on the day of use. For Western blotting, only the protease inhibitors were added. The cells were scraped off the dish, placed in a 15-ml tube, and homogenized for 15 s twice at setting no. 6 with a Polytron model PT 10/35 homogenizer (Brinkmann) using Polytron's PTA 7 probe. The cell suspension was centrifuged at 4 °C for 8 min at 1500 rpm in a tabletop centrifuge. The supernatant was removed and spun at 100,000 times g for 30 min at 4 °C in a tabletop ultracentrifuge. The resulting supernatant was discarded, and the pellet was redissolved in the appropriate buffer.

Western Blotting Studies

Cells were grown to confluence in 175-cm^2 flasks. Membranes were prepared as described above, redissolved in sample buffer (2% SDS, 10% sucrose, 10 mM dithiothreitol, 60 mM Tris, pH 6.8), and protein content was determined with the BCA protein assay by Pierce using the manufacturer's instructions. 50 µg of protein from each sample were added per lane in a precast 1 times 10-mm thick well polyacrylamide gel (Novex) and electrophoresed in running buffer (25 mM Tris, pH 8.5, 192 mM glycine, 0.1% SDS) at 120 V until the dye front reached the bottom of the gel. Next, the protein was transferred onto a 0.2-µm nitrocellulose membrane at 25 V overnight in transfer buffer (25 mM Tris, pH 8.5, 192 mM glycine, 20% MeOH) at 4 °C. Immunoblotting was performed at room temperature as described previously(36) . Briefly, nitrocellulose membranes were blocked for 1 h in Blotto (47.4 mM Tris, pH 8, 1.9 mM CaCl(2), 75.8 mM NaCl, 5% (w/v) non-fat milk, 0.2% (v/v) Nonidet P-40, 0.02% (w/v) sodium azide) plus 20% goat serum, incubated for 1 h in antiserum diluted 1:300 with Blotto, washed twice with Blotto for 15 min each, incubated for 1 h in horseradish peroxidase-labeled donkey anti-rabbit antibody (Amersham Life Science, Buckinghamshire, UK) diluted 1:1000 with Blotto, washed twice with Blotto, and then twice with buffer A (50 mM Tris, pH 8, 2 mM CaCl(2), 80 mM NaCl). All washes were for 15 min. Detection was by enhanced chemiluminescence (ECL, Amersham Corp.) using the manufacturer's instructions.

Deglycosylation of the GRP-R

Membranes were prepared, and protein content was determined as described for Western blotting studies. Cell membranes were resuspended in a microcentrifuge tube at 2 mg/ml in N-glycanase buffer (50 mM Tris/HCl, pH 7.7, 50 mM EDTA, 0.5% SDS, 1 mM PMSF, and 0.4% 2-mercaptoethanol) and denatured at 95 °C for 5 min. 60 µl of denatured sample were combined with 30 µl of 7.5% Nonidet P-40, 7.2 µl of N-glycanase, and 82.8 µl of H(2)O. The mixture was incubated at 37 °C for the indicated time, and the reaction was stopped with 60 µl of 4 times sample buffer.

Immunoprecipitation Protocol and Phosphorylation Assay

3 times 10^6 cells/dish were seeded into 100 times 20-mm tissue culture dishes and incubated in DMEM with 10% FBS at 37 °C overnight (unless otherwise noted). The following day, the cells were incubated in phosphate-free DMEM with 5% dialyzed fetal bovine serum for 30 min. Next, this medium was removed and replaced with 5 ml of the same medium supplemented with 250 µCi of [P]orthophosphate for 3 h (or the time noted in the figure legends). Compounds were then added as described in the figure legends. Cell membranes were prepared as described above, redissolved in 300 µl of radioimmune precipitation buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8) supplemented with 10 mM NaF, 18.4 µg/ml sodium vanadate, 1 mM PMSF, 0.01% soybean trypsin inhibitor, and 1 µg/ml leupeptin. All further steps were performed at 4 °C. The solution was precleared by adding 5 µl of normal rabbit serum and 7.5 µl of protein A/G-agarose beads (Oncogene Science, Manhasset, NY) and mixing in a rotator for 30 min. Precipitated material was then removed by centrifuging at 15,000 rpm for 5 min. Preclearing was then repeated. The supernatant was placed in a new tube, 5 µl of rabbit no. 3 antiserum added, and the solution was mixed for 1 h. 20 µl of protein A/G-agarose were added and mixed overnight. The next day the tubes were centrifuged for 10 min at 15,000 rpm, the supernatant was discarded, and the agarose beads were washed by incubating with 600 µl of radioimmune precipitation buffer with mixing for 10 min. The tubes were then centrifuged at 15,000 rpm for 5 min, the radioimmune precipitation buffer removed, and the wash repeated a total of four times. A final wash followed with 600 µl of a 10 mM Tris and 140 mM NaCl solution. After the final wash was removed, 30 µl of 2 times sample buffer were added to each sample. The tube was vortexed, incubated at 95 °C for 5 min to dissociate the bound proteins from the beads, and then centrifuged for 5 min at 15,000 rpm. 25 µl of the supernatant from each sample were loaded into the precast gels. After SDS-PAGE (as described above) the gel was fixed in a 30% MeOH, 10% acetic acid solution, dried for 2 h, and exposed to x-ray film or a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) screen.

Quantification of phosphorylated GRP-R was accomplished by use of a PhosphorImager. For each gel analyzed, a two-row grid was created long enough to include all the samples. Each rectangle in the lower row encompassed the receptor signal from one sample, and the upper row, which encompassed the same area above the receptor signal, was used as an intrasample background. Each lane's background was subtracted from the corresponding receptor signal to generate the signal reported in the figures.

Phosphoamino Acid Analysis

Phosphorylation of the GRP-R, membrane preparation, immunoprecipitation and SDS-PAGE were performed as described above. Phosphoamino acid analysis was performed as described previously(37, 38) . Relative positions of the phosphoamino acids were determined following ninhydrin (0.2% in acetone) visualization, and the plate was exposed to either x-ray film or a PhosphorImager screen.

Quantification of the phosphoamino acids was accomplished using a PhosphorImager. Six equally sized elliptical regions were created which were large enough to individually include the area covered by the most diffuse phosphoamino acid. Ellipses were placed so as to encompass each of the three phosphoamino acid signals. In addition, the three other ellipses were placed adjacent to the phosphoamino acid signals for background determinations. These backgrounds were averaged, and the average was subtracted from the values of the scanned phosphoamino acids.

Protein Kinase C Assay

The procedure was modified from Chakravarthy et al.(39) using the MARCKS PKC site peptide FKKSFKL-NH(2) (synthesized in house). Cells were plated and treated as described in the figure legends. Membranes were prepared and redissolved in 100 µl of ice-cold 2 times buffer (50 mM Tris, pH 7.4, 10 mM MgCl(2), 2 µM CaCl(2), 0.2 mM NaVO(4), 0.2 mM sodium pyrophosphate, and 2 mM NaF supplemented with 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 2 mM PMSF just before use). After determining the protein concentration, 10 µg of membrane protein was added into a microcentrifuge tube with 30 µl of 50 mM Tris, pH 7.4, 10 µl of 1 mM MARCKS PKC site peptide in 50 mM Tris, pH 7.4, 10 µl of ATP solution containing 0.5 mM unlabeled ATP, and 0.15 mCi/ml [P]ATP in 50 mM Tris, pH 7.4, made up to a final volume of 100 µl with 2 times buffer. Experiments were performed in triplicate including a set of tubes for background determination containing 50 µl of 2 times buffer and no membrane. Reactions were incubated in a heating block at 30 °C for 10 min with vigorous shaking. The reactions were stopped with 10 µl of 5% acetic acid, and the tubes were spun in a refrigerated centrifuge for 5 min at 15,000 rpm. 50 µl of the reaction volume were spotted onto 2.25-cm^2 pieces of P81 paper (Whatman). The papers were then repeatedly washed in 200-300 ml of 5% acetic acid for 10 min until the background (no membrane) papers had counts less than 400 cpm as determined with a Geiger counter. The papers were air-dried overnight and then counted by liquid scintillation.


RESULTS

Development of Transfected Cell Lines Expressing High Numbers of the GRP-R

Several approaches were used concurrently in an attempt to create an antibody specific for the GRP-R to study acute phosphorylation. At the same time as the antisera to the peptide derived from the third loop of the GRP-R were being prepared, mutant GRP-Rs were synthesized with a myc epitope at either the 5` (5`ET) or 3` (3`ET) end of the receptor. Tagging proteins with this epitope was previously shown to be useful for Western blot analysis (26) . When stable transfectants of these constructs were made in Balb 3T3 mouse fibroblasts, clones were isolated (5`ET4 and 3`ET2) which expressed much higher levels (>10^6 receptors/cell) of the GRP-R than either BNR-11, a transfectant with the wild-type GRP-R expressed in Balb cells (2 times 10^5 receptors/cell), or Swiss 3T3 cells (5 times 10^4 receptors/cell). The 5`ET4 and 3`ET2 cell lines behaved similarly to BNR-11 in ligand affinity, effector coupling, internalization and desensitization assays. As shown in Fig. 1A, all three receptors internalized rapidly and to a similar degree, with 70-80% of cell associated radiolabeled bombesin in acid resistant pools by 30 min. Therefore, internalization of the GRP-R is not impaired by the epitope tagging of either end of the receptor. Also, all three receptors exhibited similar patterns of ligand-induced [Ca](i) increases. Addition of 5 nM bombesin lead to an increase in [Ca](i) of 473 ± 146 nM in BNR-11, 357 ± 37 nM in 3`ET2, and 334 ± 85 nM in 5`ET4. There was complete desensitization in all cell lines to a subsequent addition of 5 nM bombesin. Furthermore, as shown in Fig. 1, B and C, sequential addition of 0.5 nM bombesin to all three cell lines resulted in similar patterns of desensitization. These studies indicate that addition of the myc epitope to either the amino or carboxyl terminus of the mouse GRP-R does not significantly effect any receptor function. Western blots of membrane preparations from 5`ET4 and 3`ET2 using the monoclonal antibody 9E10 (Oncogene Science, Manhasset, NY) which is specific for the myc epitope, provided only weak signals from the mutant receptors, and attempts at immunoprecipitation with 9E10 were unsuccessful (results not shown).


Figure 1: Comparison of the ability of wild type (BNR-11) and epitope-tagged (5`ET4 and 3`ET2) GRP-R cell lines to undergo internalization and acute desensitization. A, the various cell lines were grown to confluence in 175-cm^2 flasks. Cells were harvested and suspended at 2 times 10^6 cells/ml in binding buffer with 75 pMI-Tyr^4-bombesin for various times at 37 °C. After incubation, cell samples were added to either 10 volumes of 0.2 M acetic acid (pH 2.5) containing 0.5 M NaCl or 10 volumes of binding buffer and incubated for 5 min at 4 °C. Cells were then pelleted through oil, and cell-associated radioactivity was measured. Internalization is defined as the percentage of specific cell-associated I-Tyr^4-bombesin resistant to acid wash. In B and C, the various cell lines were plated into 100 times 20-mm tissue culture dishes. The following day the medium was removed, and the dishes washed twice with DMEM supplemented with 0.5% FBS and then incubated at 37 °C for 14 min in 5 ml of DMEM + 0.5% FBS containing 1 µM Fura-2 acetoxymethyl ester. The cells were washed twice with ice-cold binding buffer, detached by scraping into binding buffer, pelleted, and resuspended in 6 ml of binding buffer. The cell suspension was placed on ice until ready for use. When ready, 2 ml of cell suspension were repelleted and resuspended in binding buffer. The cells were warmed in a 37 °C water bath, then placed into a luminescence spectrometer for fluorescence monitoring as described under ``Experimental Procedures.'' The cells were continually stirred and maintained at 37 °C. 0.5 nM bombesin was added sequentially at 2-min intervals. B, representative tracings of each cell line. Addition of bombesin is indicated by a ``.'' C, bar graph showing change in [Ca] from baseline (mean ± S.D.). 1st, 2nd, and 3rd refer to the increase in [Ca] with the addition of 0.5 nM bombesin at the times indicated in B (0, 2, or 4 min).



Characterization of Rabbit No. 3 Antiserum

After unsuccessful attempts with the 9E10 anti-myc monoclonal antibody, attention was then focused on polyclonal antisera derived from rabbits immunized with a peptide derived from the GRP-R third intracellular loop linked via an amino-terminal cysteine to keyhole limpet hemocyanin. The rabbit producing the highest titer antiserum detected the GRP-R as a broad band of approximately 70-90 kDa in a variety of GRP-R-expressing cells (Fig. 2A). The intensity of the signal in each cell line appeared proportional to the number of receptors (noted above) in these cell lines and no signal was detected in untransfected Balb cells. Two other bands (23 and 45 kDa) were present in all cell lines with equal intensity and are presumed to be nonspecific since they are unrelated to levels of GRP-R expression and are not efficiently competed with the immunizing peptide (Fig. 3C).


Figure 2: Identification of a specific GRP-R peptide antiserum: Western blots of membrane preps from various cell lines and effect of N-glycanase treatment. A, various cell lines were grown to confluence in 175-cm^2 flasks. Cells were harvested and membranes were prepared. 50 µg of membrane protein from each cell line were resolved on a 4-20% SDS-PAGE gel, transferred overnight onto a nitrocellulose membrane, probed with a 1:300 dilution of rabbit no. 3 antiserum, and developed by chemiluminescence. Lane 1, Balb, Balb 3T3 mouse fibroblasts which have no detectable GRP-R mRNA or binding sites; lane 2, 5`ET4, a stable transfectant of Balb cells expressing highest levels of the GRP-R modified by 5` addition of the myc epitope; lane 3, BNR-11, a stable transfectant of Balb cells expressing intermediate levels of the wild type GRP-R; and lane 4, Swiss 3T3, Swiss 3T3 mouse fibroblasts which express lower levels of the GRP-R. Positions of prestained molecular mass markers are indicated in kilodaltons (kDa). B, membranes were prepared as in A and treated with (+) or without(-) N-glycanase (to remove oligosaccharides) prior to loading on a 10% SDS-PAGE gel, transferred overnight onto a nitrocellulose membrane, probed with a 1:300 dilution of rabbit no. 3 antiserum and developed by chemiluminescence. 3`ET2 is a stable transfectant of Balb cells with the GRP-R modified by 3` addition of the myc epitope. Position of prestained molecular mass markers are indicated in kilodaltons (kDa).




Figure 3: Bombesin-induced phosphorylation of the GRP-R. A, different cell lines were plated in 100 times 20-mm dishes. The following day the cells were labeled with 250 µCi of [P]orthophosphate for 3 h then incubated in the presence (+) or absence(-) of 100 nM bombesin for 10 min at 37 °C. Membranes were prepared and immunoprecipitations performed using the GRP-R antiserum (as under ``Experimental Procedures''). Immunoprecipitates were resolved on a 4-20% SDS-PAGE gel. The gel was fixed and dried and then exposed to an x-ray film for 50 h. The position of GRP-R is indicated. B, GRP-R-transfected Balb cells (5`ET4) were plated in 150 times 25-mm dishes. The next day they were incubated in the presence (+) or absence(-) of 100 nM bombesin for 10 min at 37 °C. Membranes were prepared, and 50 µg of membrane protein from each sample were resolved on a 4-20% SDS-PAGE, transferred overnight onto a nitrocellulose membrane, probed with a 1:300 dilution of rabbit no. 3 antiserum, and developed by chemiluminescence. C, GRP-R transfected Balb cells (5`ET4) were plated in 150 times 25-mm dishes. The following day the cells were labeled with 250 µCi of [P]orthophosphate for 3 h, then incubated in the presence (+) or absence(-) of 100 nM bombesin for 10 min at 37 °C. Membranes were prepared and immunoprecipitations performed using the GRP-R antiserum which had been preincubated overnight at 4 °C with the indicated amounts of the peptide used for the generation of the antiserum. Immunoprecipitates were resolved on a 4-20% SDS-PAGE gel. The gel was fixed and dried and then exposed to an x-ray film for 9 h.



Treatment with N-glycanase to remove oligosaccharides eliminated the broad 70-90-kDa band from the lane with the GRP-R (-, 3`ET2), produced a doublet at approximately 40 kDa, and had no effect on the nonspecific bands expressed in untransfected Balb cells (Fig. 2B). Although in this experiment the cumulative intensities differed between the N-glycanase-treated (40 kDa) and untreated (70-90 kDa) receptor bands, shorter incubations in N-glycanase (30-60 min) produced a 40-kDa receptor band of similar cumulative intensity to the 70-90-kDa receptor band in the untreated lanes (not shown). The N-glycanase-induced decrease in receptor mass is consistent with previous studies establishing that the GRP-R is a glycoprotein with asparagine-linked sugar chains(30, 40) . The molecular weight of the deglycosylated receptor is comparable to the predicted molecular weight of the GRP-R from the cDNA sequence(27, 41) . The existence of a doublet GRP-R suggests two mobilities for the receptor, possibly differentiated by phosphorylation status.

Immunoprecipitation of [S]methionine-labeled cells with rabbit no. 3 antiserum revealed an equivalent 70-90-kDa band found only in GRP-R-expressing cell lines and not in untransfected Balb cells (data not shown).

In Vivo Phosphorylation of the GRP-R

Immunoprecipitation of the GRP-R from cell lines expressing moderate to high levels of the GRP-R (5`ET4, 3`ET2, and BNR-11) showed a faint band of 70-90-kDa before addition of bombesin, consistent with basal phosphorylation of the GRP-R (Fig. 3A). When stimulated with 100 nM bombesin for 10 min, the GRP-R in these cell lines showed a marked increase in phosphorylation. Swiss 3T3 cells, which express endogenous GRP-R at lower levels than receptor expression found in transfected cells (5`ET4, 3`ET2, and BNR-11), exhibited phosphorylation near the level of detection of the assay. As expected, no phosphorylation can be seen in untransfected Balb cells (Fig. 3A).

Since the epitope tagged receptors behaved similarly to wild type, but expressed higher levels of GRP-R protein (Fig. 2A), all further experiments were performed with these receptors. 5`ET4 was chosen instead of 3`ET2, since, in this construct, the epitope is in the amino-terminal extracellular domain, and the myc epitope includes a serine which could potentially be phosphorylated were it found in the intracellular domain. Using the receptor with the epitope tag in a presumed extracellular domain (5`ET4) rules out the possibility that this serine would become phosphorylated in vivo, or that the myc epitope would interfere with the study.

To rule out the possibility that receptor protein levels in the membrane change during the phosphorylation assay, a Western blot was performed on membranes from cells treated in the presence or absence of 100 nM bombesin for 10 min (Fig. 3B). No detectable change in receptor protein levels occurred during the 10-min phosphorylation assay.

To confirm that the 70-90-kDa phosphorylated species is the GRP-R, we incubated the samples with increasing amounts of the intracellular loop peptide (immunogen) before immunoprecipitation. Fig. 3C shows that the peptide specifically blocks immunoprecipitation of the band representing the phosphorylated receptor.

The time course of the bombesin-induced GRP-R phosphorylation is very rapid. Maximum phosphorylation occurs by 1 min (the earliest time point studied), and is maintained for at least 30 min during constant exposure to bombesin (Fig. 4A). Half-maximal GRP-R phosphorylation occurs with 3 ± 0.7 nM bombesin, and maximal phosphorylation occurs by 100 nM (Fig. 4B). Ligand-induced receptor phosphorylation requires binding by an agonist, since addition of 3 µM [D-F(5)-Phe^6,D-Ala]BN(6-13) methyl ester, a GRP-R antagonist, causes no significant increase in GRP-R phosphorylation (Fig. 4C, antagonist). Addition of this antagonist with bombesin prevents the bombesin-induced phosphorylation (Fig. 4C, both).


Figure 4: GRP-R phosphorylation: time course, dose-response, and effect of receptor antagonist. GRP-R transfected Balb cells (5`ET4) were placed in 100 times 20-mm dishes. The next day the cells were labeled with 250 µCi of [P]orthophosphate for 3 h and stimulated with bombesin or GRP-R antagonist as described below. Membranes were prepared, and immunoprecipitations were performed using rabbit no. 3 antiserum. Immunoprecipitates were resolved on a 4-20% SDS-PAGE gel. The gels were fixed and dried and then exposed to x-ray film or a PhosphorImager screen. Results shown are representative of several independent experiments. Quantification was performed by PhosphorImager analysis (as described under ``Experimental Procedures''). A, cells were stimulated with 100 nM bombesin for the indicated times at 37 °C and exposed to x-ray film for 3 h. B, cells were stimulated with the indicated concentrations of bombesin for 5 min at 37 °C and exposed to x-ray film for 9 h. C, cells were stimulated with 100 nM bombesin and/or 3 µM of the GRP-R antagonist [D-F(5)-Phe^6,D-Ala]BN(6-13) methyl ester for 10 min and exposed to x-ray film for 16 h.



To determine which amino acids were phosphorylated, two-dimensional phosphoamino acid analysis was performed. This analysis revealed that most of the phosphorylation (84%) occurred on serine, a small amount (16%) on threonine, and none on tyrosine (data not shown). Since all three PKC consensus sequences found in the mouse GRP-R contain serine and not threonine, this result indicates that at least some of the GRP-R phosphorylation must be due to a kinase, or kinases, other than PKC.

Role of PKC in Rapid Bombesin-induced Phosphorylation

To explore the potential role of PKC in rapid, ligand-dependent phosphorylation, we used the PKC activator TPA to determine whether the GRP-R is a potential substrate for PKC. As shown in Fig. 5, TPA induces phosphorylation of the GRP-R in a concentration- and time-dependent manner, with maximal phosphorylation found at 100 nM TPA, after 30 min of exposure. These studies show that GRP-R can serve as a PKC substrate. However, the time course of PKC-mediated phosphorylation is much longer than that observed for ligand-activated phosphorylation (compare Fig. 4A and Fig. 5B).


Figure 5: TPA-induced phosphorylation of the GRP-R: effect of time and dose. GRP-R transfected Balb cells (5`ET4) were placed in 100 times 20-mm dishes. The next day the cells were labeled with 250 µCi of [P]orthophosphate for 3 h and then stimulated with bombesin or TPA as described below. Membranes were prepared, and immunoprecipitations were performed using the GRP-R antiserum. Immunoprecipitates were resolved on a 4-20% SDS-PAGE gel. The gels were fixed and dried and then exposed to x-ray film or a PhosphorImager screen. Results shown are representative of several experiments. Quantification was performed by PhosphorImager analysis. A, cells were stimulated with 100 nM bombesin for 10 min or the indicated concentration of TPA for 20 min. B, cells were stimulated with or without 100 nM TPA for the indicated times (in minutes). Both gels were exposed to x-ray film for 7 h.



To understand the role of PKC in rapid, ligand-activated GRP-R phosphorylation, we performed concurrent assays for PKC activity and GRP-R phosphorylation using combinations of TPA, bombesin, and the specific PKC inhibitor UCN-01. UCN-01 discriminates between the Ca-dependent (PKC-alpha, -beta, and -) and Ca-independent (PKC- and -) isozymes better than staurosporine with a 15-20-fold higher relative potency for the Ca-dependent isozymes(42) . The Ca-dependent isozymes are presumably the kinases activated by the GRP-R signal transduction cascade. As shown in Fig. 6, 100 nM TPA for 20 min induces a 17-fold increase in PKC activity over background, while 100 nM bombesin for 10 min induces a less than 2-fold increase in PKC activity. In contrast, TPA induces less (85%) GRP-R phosphorylation than bombesin. Additionally, a 1-h pretreatment with the selective PKC inhibitor UCN-01 lowered the basal PKC activity and the basal level of phosphorylation. Pretreatment with this inhibitor prevented the TPA-induced increase in PKC activity and inhibited 70% of TPA-induced phosphorylation. In contrast, pretreatment with UCN-01 caused a relative hyperphosphorylation of the GRP-R when the cells were subsequently treated with bombesin (compared to cells not pretreated) at the same time as the PKC assay revealed no PKC activity. This observation implies that a kinase other than PKC is involved in rapid bombesin-induced GRP-R phosphorylation.


Figure 6: The effect of inhibition of PKC on GRP-R phosphorylation. Six out of twelve 100 times 20-mm dishes previously seeded with GRP-R transfected Balb cells (5`ET4) were incubated with 250 µCi of [P]orthophosphate/dish for the phosphorylation assay (black bars). The other six dishes, for the PKC assay, received no radioactivity at this time (white bars). Next, pairs of dishes (one for the PKC assay and the other, incubating in P for the phosphorylation assay) were treated as follows: lane 1, no treatment (control); lane 2, 100 nM bombesin was added for 10 min; lane 3, 100 nM TPA was added for 20 min; lane 4, 300 nM UCN-01 was added for 1 h, and then 100 nM TPA was added for 20 min; lane 5, 300 nM UCN-01 was added for 1 h; lane 6, 300 nM UCN-01 was added for 1 h, and then 100 nM bombesin was added for 10 min. Membranes were then prepared. Cells for the phosphorylation assay had the addition of drugs timed so that these cells were all harvested after incubating in P for a total of 3.5 h. For the phosphorylation assay, immunoprecipitations were performed using rabbit no. 3 antiserum. Immunoprecipitates were resolved on a 4-20% SDS-PAGE gel. The gel was fixed and dried and then exposed to x-ray film or a PhosphorImager screen. For the PKC assay, 10 µg of membrane were combined with kinase buffer, target peptide (p80/MARCKS PKC site peptide), and [P]ATP as described under ``Experimental Procedures.'' A set for background determination was also prepared without any membrane. This mixture was then incubated at 30 °C for 10 min, spotted onto 2.25-cm^2 P81 paper, washed several times with 5% acetic acid, and scintillation counted after drying. The experiment was done in triplicate, results were averaged, and the background subtracted. To facilitate the comparison between assays, the highest value for each assay was arbitrarily defined as ``100% of maximum'' and all other values for that assay were set relative to 100%.




DISCUSSION

In this study, we show for the first time that bombesin induces rapid phosphorylation of GRP-R on serine and threonine residues. Phosphorylation of GRP-R appears to be tightly correlated to the occupation of receptor by agonist since the concentration of bombesin required to induce phosphorylation is comparable to that needed for other bombesin-induced effects, such as displacement of I-Tyr^4-bombesin binding (40, 43, 44) or ligand-stimulated inositol triphosphate accumulation (45, 46) . Ligand-induced GRP-R phosphorylation requires a GRP-R agonist, since addition of a GRP-R antagonist alone did not induce GRP-R phosphorylation, and addition of antagonist with bombesin prevented bombesin-induced phosphorylation. Finally, although the PKC activator TPA can induce phosphorylation of the GRP-R, the bombesin-dependent GRP-R phosphorylation does not require PKC activity in the rapid phase of phosphorylation (<10 min). In fact, the GRP-R appears to be more highly phosphorylated by agonist when PKC activity is completely inhibited.

In a recent study, Benya et al.(33) eliminated all potential sites of phosphorylation in the GRP-R COOH-terminal tail by either deleting the COOH-terminal domain or converting serine and threonine residues in this domain to Ala, Asn, or Gly. At all time points examined after addition of bombesin, transfectants with either of these modified receptors had a greater than 70% reduction (relative to a transfectant with the wild type GRP-R) in internalized I-Tyr^4-bombesin. Additionally, mutating only the PKC consensus sequence (and a neighboring threonine) within this region reduced internalization by only 35%. These data suggested the idea that phosphorylation of residues in the COOH-terminal tail may play a role in GRP-R internalization, and some of this phosphorylation may be mediated by a kinase other than PKC. Findings in the present study are consistent with this hypothesis.

Our data do not establish a causal relationship between GRP-R phosphorylation and regulation of GRP-R activity by either desensitization or internalization. However, the time course of phosphorylation is consistent with the possibility that phosphorylation may be a prerequisite for either internalization or acute, homologous desensitization. Swope and Schonbrunn (22) saw a 60-70% inhibition of bombesin-induced insulin release after a 5-min preincubation with 100 nM bombesin. In that study, a 3-min incubation with labeled bombesin resulted in internalization of about two-thirds of the bound I-Tyr^4-bombesin(22) . In the same transfected cell model employed here to study phosphorylation, Benya et al.(33) found 60% internalization of labeled ligand by 15 min (confirmed in Fig. 1). Furthermore, our finding that inhibition of PKC leads to a bombesin-induced hyperphosphorylation of the GRP-R correlates with the observation by Walsh et al.(24) that depletion of PKC leads to a more pronounced bombesin-induced acute desensitization than in cells with intact PKC.

Receptor hyperphosphorylation in the absence of PKC activity suggests a novel mechanism for agonist-induced acute desensitization. In the visual system, photoexcited rhodopsin is phosphorylated by a single second messenger independent kinase (rhodopsin kinase) leading to desensitization(12, 47, 48, 49) . For the beta-adrenergic receptor, both a second messenger-dependent kinase (protein kinase A) and second messenger-independent kinase (beta-adrenergic receptor kinase) can contribute to acute agonist-induced desensitization(13, 14, 50, 51) . In the olfactory system a sequential interplay of both a second messenger-dependent kinase (protein kinase A or C) and a second messenger-independent kinase (beta-adrenergic receptor kinase 2) has been proposed to be necessary for odorant-induced desensitization(17, 18) . In contrast, we speculate that perhaps a low basal level of second messenger-dependent kinase activity (PKC) prevents maximum agonist-induced phosphorylation by another kinase, thereby preventing maximum desensitization.

These data do not rule out the possibility that PKC-induced GRP-R phosphorylation may be important for GRP-R function. The physiological significance of acute phosphorylation may be a function of the specific residues phosphorylated, rather than the total number. We cannot rule out that a small number of crucial residues are rapidly phosphorylated by PKC after addition of bombesin. Alternatively, PKC may not phosphorylate the GRP-R acutely, but instead phosphorylate it under other conditions, and this phosphorylation may be important in the regulation of GRP-R activity. Indeed, our data clearly indicate that the GRP-R is a substrate for PKC when enzyme activity is elevated (after TPA induction) for a sufficient length of time (30 min). In this regard, the predicted GRP-R sequence has three evolutionarily conserved PKC phosphorylation sites(27, 52) . Perhaps receptor phosphorylation by PKC occurs after prolonged incubation in agonist, where it may be a critical factor in long term desensitization or down-regulation of the GRP-R. Further studies are needed which focus on the identities of the specific residues required for internalization and acute homologous desensitization, and which kinase or kinases are required to phosphorylate them. Also, studies of GRP-R phosphorylation after chronic exposure to agonist will be needed to explore a potential role for receptor phosphorylation by PKC in the regulation of these processes.

The kinase or kinases that are responsible for the majority of the rapid, agonist-induced phosphorylation of the GRP-R remains to be defined, as well as the serine and threonine residues on the receptor molecule that undergo phosphorylation by these kinases. Second messenger-independent kinases (GRKs), including rhodopsin kinase and beta-adrenergic receptor kinase, are likely candidates by analogy to other heptahelical transmembrane receptor systems. The NK-1 peptide receptor in phospholipid vesicles can undergo rapid, agonist-dependent phosphorylation by beta-adrenergic receptor kinases 1 and 2 in vitro(53) . Further studies will be needed to define whether or not there is a member or members of the GRK family that are critical for rapid, agonist-induced phosphorylation of the GRP-R.


FOOTNOTES

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

§
To whom correspondence should be addressed: National Cancer Institute, Bldg. 37, Rm. 5D02, Bethesda, MD 20892-4255. Tel: 301-402-3429; Fax: 301-480-2514.

(^1)
The abbreviations used are: G protein, guanine nucleotide-binding protein; PKC, protein kinase C; GRK, protein-coupled receptor kinase; GRP-R, gastrin-releasing peptide receptor; TPA, 12-O-tetradecanoylphorbol 13-acetate; N-glycanase, peptide:N-glycosidase F; PAGE, polyacrylamide gel electrophoresis; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; PMSF, phenylmethylsulfonyl fluoride; UNC-01, 7-hydroxystaurosporine.


ACKNOWLEDGEMENTS

We thank Dr. Richard Kahn for his critical review of the manuscript.


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