(Received for publication, September 21, 1995; and in revised form, December 7, 1995)
From the
Phosphorylation of G protein-coupled receptors is an established mechanism for desensitization in response to agonist stimulation. We previously reported phosphorylation of the pancreatic acinar cell cholecystokinin (CCK) receptor and the establishment of two-dimensional phosphopeptide mapping of its sites of phosphorylation (Ozcelebi, F., and Miller, L. J.(1995) J. Biol. Chem. 270, 3435-3441). Here, we use similar techniques to map sites of phosphorylation of the same receptor expressed on a stable receptor-bearing Chinese hamster ovary (CHO)-CCKR cell line. Like the native cell, the CHO-CCKR cell receptor was phosphorylated in response to agonist stimulation in a concentration-dependent manner; however, the time course was quite different. CHO-CCKR cell receptor phosphorylation increased progressively to a plateau after 15 min, while in the acinar cell it peaks within 2 min and returns to baseline over this interval. There were distinct qualitative and quantitative differences in the sites of phosphorylation of the two receptor systems. One site previously attributed to action of a staurosporine-insensitive kinase in the acinar cell was absent in the CHO-CCKR cell. Site-directed mutagenesis was utilized to eliminate predicted sites of protein kinase C action, but only two of four such sites affected the phosphopeptide map of this receptor. Chemical and radiochemical sequencing were performed on these and other phosphopeptides which were present in both the CHO-CCKR cells and agonist-stimulated pancreatic acinar cells to provide direct evidence for the phosphorylation sites actually utilized. Thus, these data support the usefulness and limitations of a model cell system in studying receptor phosphorylation and desensitization.
Receptor phosphorylation in response to agonist stimulation is a well established mechanism for desensitization, an important and ubiquitous process to protect the cell from overstimulation. This covalent modification of the receptor has been implicated in uncoupling the receptor-G protein interaction, mediating binding of arrestin-like proteins, and even signaling receptor sequestration, internalization, and resensitization(2, 3) .
The molecular details of these events, however, are often implied based on indirect data, due to difficulties in the direct identification of phosphorylation sites in sparse membrane proteins. This is often based on the presence of consensus sites in primary sequence analysis, and on the change in receptor behavior observed after truncations, deletions, or substitutions, often of multiple residues. Nonspecific and indirect effects can clearly result from such approaches.
We recently
reported the ability to generate a detailed two-dimensional
phosphopeptide map for radiochemically pure cholecystokinin (CCK) ()receptor present as an extremely sparse plasmalemmal
protein in the pancreatic acinar cell(1) . Because of its
sparsity and physicochemical properties, this receptor has been
extremely difficult to purify to scale suitable for direct sequencing.
The only successful report of such an effort utilized pancreata from
250 rats as source of this receptor(4) .
With the cloning of the cDNA encoding this receptor(4) , it has been possible to establish tissue culture cell lines expressing large numbers of receptors. We have established a CHO cell line which expresses approximately 25 times the normal receptor density (CHO-CCKR line expressing 125,000 receptors per cell)(5) . This type of cell line has been extensively utilized to determine the impact of mutagenesis on the function and regulation of other receptors. It is unclear, however, how closely such a cell line parallels the native cell in these activities.
In this work, we have demonstrated that the CCK receptor expressed on the CHO-CCKR cell line is phosphorylated in response to CCK stimulation. Like the native cell receptor, the predominant domain for phosphorylation was the third intracellular loop; however, the time course of phosphorylation and dephosphorylation of the recombinant receptor was quite distinct, suggesting differences in the equilibrium between relevant kinases and protein phosphatases. Application of two-dimensional phosphopeptide mapping demonstrated both qualitative and quantitative differences in sites of phosphorylation in these two receptor-bearing cells. After mutagenesis of consensus sites was insufficient to fully explain the identity of the sites of phosphorylation, we increased the scale of the phosphopeptide mapping and obtained direct sequence evidence for several sites of receptor phosphorylation utilized by both of these cells.
The rat type A CCK receptor cDNA we previously cloned (5) was subcloned into the pBK-CMV expression vector (Stratagene, La Jolla, CA), and was mutagenized by the method of Sayers et al.(7) . Correct sequences of all constructs were confirmed by DNA sequencing using the dideoxynucleotide chain termination method(8) . COS-7 and CHO-K1 cells were acquired from ATCC (Rockville, MD), and were cultured in Dulbecco's modified Eagle's medium with 5% Fetal Clone 2 supplement (Hyclone Laboratories). They were transfected with 2-4 µg of DNA using DEAE-dextran or Lipofectin(9) . Transient transfectants were harvested for study 48 to 72 h after transfection. Stable receptor-bearing cell lines were established as we described(5) .
In
experiments requiring chemical rather than radiochemical purity of the
CCK phosphoreceptor, the eluted samples were then desalted and buffer
was exchanged over a 0.6 8-cm size exclusion column (Sephadex
G-50) run with buffer containing 50 mM ammonium bicarbonate
and 0.01% digitonin. The relevant fractions were then loaded onto a
wheat germ agglutinin-agarose affinity column, washed with 0.5 M sodium chloride, and eluted by electrophoresis in SDS-containing
buffer. This procedure was then followed by another desalting step and
the relevant fractions were dried under vacuum. They were then ready
for further purification by HPLC or mapping procedures described below.
When chemical purity had not been attained, the eluates from the thin layer plates were further purified on a 40% alkaline polyacrylamide gel using the technique of West et al.(14) . Bands identified by autoradiography were then eluted and again separated by reversed phase HPLC prior to Edman degradation sequencing.
Two types of Edman
degradation sequencing were utilized. These included automated pulsed
liquid sequencing in the Applied Biosystems Instrument and manual Edman
degradation radiochemical sequencing. In peptides having more than one
potential site of phosphorylation, the phosphoserines were identified
indirectly by modification with an alkanethiol prior to automated Edman
sequencing using a method based on the procedure of Meyer et
al.(15, 16) . The manual sequencing was performed
after binding the purified peptides to 20 mg of N-(2-aminoethyl)-3-aminopropyl glass beads in buffer
containing 10 mg/ml 1ethyl-3-(3-dimethylaminopropyl)carbodiimide for 2
h at room temperature. This was alkalinized with triethylamine and
dried under vacuum, with the whole cycle repeated three times. Thirty
µl of triethylamine:methanol:phenylisothiocyanate (1:7:1) was added
and incubated at 50 °C for 5 min. The sample was then dried under
vacuum and washed three times with methanol. Twenty µl of
trifluoroacetic acid was then added for 5 min at room temperature,
before the next cycle was begun. In each cycle, the methanol extract
was counted in a spectrometer.
Like our previous observations in pancreatic acinar cells(10) , agonist stimulation of the CHO-CCKR cells resulted in increased phosphorylation of the CCK receptor (Fig. 1). This occurred in a concentration-dependent manner for both CCK and the phorbol ester, TPA. When stimulated similarly and treated in the same way, no phosphorylation was observed in this region of an SDS-polyacrylamide gel used to separate products of phosphorylation of the parent cell line, CHO-K1 cells (data not shown).
Figure 1:
CCK and
TPA stimulated phosphorylation of the CHO-CCKR cell CCK receptor in a
concentration-dependent manner. Shown are typical autoradiographs of
the M = 85,000-95,000 region of a
SDS-polyacrylamide gel used to purify the CCK receptor from cells
stimulated with the noted secretagogues, as well as means ± S.E.
for three independent experiments. Basal receptor phosphorylation is
considered to represent 0% and the maximal response to CCK is
considered to represent 100%.
However, unlike the acinar cell experience in which treatment with the protein kinase C inhibitor, staurosporine, inhibited only approximately 50% of receptor phosphorylation stimulated by CCK(18) , in the CHO-CCKR cells this treatment reduced receptor phosphorylation by 75 ± 7% (Fig. 2). Of further interest, the time course of CCK receptor phosphorylation in response to CCK stimulation was quite different in the CHO-CCKR cell than in the acinar cell. The phosphorylation of the recombinant receptor occurred rapidly upon agonist stimulation, reaching its maximal level in 15 min and maintaining that level through the 30-min time point, while in the acinar cell receptor phosphorylation peaked within 2 min and returned to its basal state over the same interval (18) (Fig. 3).
Figure 2:
Staurosporine (10 µM)
partially inhibited CCK-stimulated CCK receptor phosphorylation, and
completely inhibited that stimulated by TPA. Shown is a representative
autoradiograph of the M =
85,000-95,000 region of a SDS-polyacrylamide gel used to purify
the CCK receptor after stimulating the CHO-CCKR cells under the
conditions described, as well as means ± S.E. for three
independent experiments.
Figure 3:
CCK (1 µM) stimulated the
rapid and stable phosphorylation of the CCK receptor in the CHO-CCKR
cells. Shown is a typical autoradiograph of the M = 85,000-95,000 region of a SDS-polyacrylamide gel
used to purify the CCK receptor after stimulating the CHO-CCKR cells
for the time noted, as well as means ± S.E. for four independent
experiments.
Figure 4: Shown is a diagram of the predicted amino acid sequence and membrane topography of the rat CCK-A receptor, with predicted sites of cyanogen bromide cleavage noted. The two domains in which data support the possible phosphorylation are accentuated, with consensus sites for protein kinase C action noted. Shown is a representative autoradiograph of a one-dimensional phosphopeptide map of the CCK receptor after cyanogen bromide cleavage which has been separated on a SDS-urea acrylamide gel.
Figure 5: a, shown are typical autoradiographs of two-dimensional phosphopeptide maps of the CCK receptor after subtilisin cleavage, and a numbered key for these maps. These represent the CCK receptor expressed on the pancreatic acinar cell (a) and CHO-CCKR cell (b) stimulated by CCK, and the CHO-CCKR cell stimulated by TPA (c). All of the previously described phosphopeptides are seen on the CCK-stimulated acinar cell map, while only phosphopeptide 12 is missing from the analogous CHO-CCKR cell map, and phosphopeptides 4 and 12 are not seen after stimulating the CHO-CCKR cell with TPA. b, shown are typical autoradiographs of two-dimensional phosphopeptide maps (after subtilisin cleavage) of CCK receptor site mutants which were stimulated by CCK. S275A and T424A mutants were not consistently different from control maps (seen in a). The S260A mutant consistently reduced phosphopeptide 5 by half of the expected intensity (since this phosphopeptide has two sites of phosphorylation), while the S264A mutant consistently eliminated phosphopeptide 6.
We have previously established methodology to separate sites of phosphorylation of the acinar cell CCK receptor by generating a two-dimensional phosphopeptide map after subtilisin cleavage of the radiochemically pure receptor phosphoprotein(1) . Fourteen distinct phosphopeptides were reproducibly observed on such a map of the acinar cell CCK phosphoreceptor stimulated by CCK treatment(1) . Similar treatment of the CHO-CCKR cells resulted in the phosphorylation of all but one of these phosphopeptides, identified as phosphopeptide 12 (Fig. 5a). Consistent with the staurosporine sensitivity of the receptor phosphorylation observed in Fig. 1, the site missing from the CHO-CCKR maps (phosphopeptide 12) represented one of the sites previously attributed to a staurosporine-insensitive kinase, thought to likely represent the sites of action of a member of the G protein-coupled receptor kinase family(1, 18) .
TPA stimulation of the CHO-CCKR cells resulted in a two-dimensional phosphopeptide map which was qualitatively similar to that observed for the CCK receptor on the pancreatic acinar cell after similar treatment (Fig. 5a). Like the experience in the acinar cell, phosphopeptides 4 and 12 were not observed after TPA stimulation, and phosphopeptide 2 was observed only after CCK stimulation, and not in response to TPA stimulation. Of note, phosphopeptide 6, which in the acinar cell was phosphorylated much more heavily in response to TPA than CCK(1) , was not observed to be differentially phosphorylated in response to these agonists in the CHO-CCKR cell.
Since one of the strong consensus sites for protein kinase C action
was Thr (T
-I-R), sited within the minor
phosphopeptide fragment of M
= 4,200 which
was observed after cyanogen bromide cleavage, the possibility that this
represented phosphopeptide 7 or 8 was explored by site-directed
mutagenesis. Fig. 5b illustrates representative
two-dimensional phosphopeptide maps of CCK-stimulated CHO cells
expressing a CCK receptor construct in which Ala replaced
Thr
. In comparing this with the wild type receptor
pattern seen in Fig. 5a, there were no consistent
differences between the two maps, suggesting that this was not the
phosphothreonine observed in phosphopeptides 7 or 8.
The identity of
phosphopeptides can also be inferred from analysis of the receptor
sequence, since the two-dimensional phosphopeptide map provides
information regarding the expected charge of any given spot. Since both
phosphopeptides 7 and 8 are on the cathodic side of the site of
application, they are expected to be positively charged at the pH of
the buffer (3.5) used for thin layer electrophoresis. There are only
two additional threonine residues (Thr and
Thr
) which are present in candidate domains of the CCK
receptor and are near serine residues (both phosphoserine and
phosphothreonine are present in phosphopeptides 7 and 8). Both of these
residues could theoretically be present within basic peptides, although
no cleavage sites can be engineered for Thr
to
theoretically give it the expected charge. The expected charge of
K-K-P-S
(P)-T
(P) would be consistent with
the position of these phosphopeptides on the map. Of interest,
Thr
does not fit any of the established consensus motifs
for the action of protein kinase C. Due to the minor nature of these
phosphopeptides on the map, this has not yet been directly
demonstrated. Ser
, which fits the strong consensus motif
for protein kinase C action, was also mutagenized to an Ala residue,
but failed to have any consistent effect on the phosphopeptide map.
This suggests that this residue is not utilized as a site for
phosphorylation. Another predicted site for protein kinase C action
was, however, utilized by the cell. Phosphopeptide 6 was postulated to
represent K-K-S
(P)-A-K, based on similar calculation
rationale. Indeed, this was directly confirmed by mutagenesis of
Ser
to Ala, with elimination of this phosphopeptide after
CCK stimulation (Fig. 5b). Also, stimulating this
construct with the phorbol ester, TPA, failed to demonstrate
phosphopeptide 6 (data not shown).
Several of the major
phosphopeptides present on the two-dimensional map were purified to
chemical homogeneity and directly sequenced. These are shown in Table 1, along with their calculated charges at pH 3.5 and the
charge predicted by the map position. A representative example of this
process is represented by phosphopeptide 5. This spot was recovered
from 7 thin layer plates by scraping, and eluted into 0.1%
trifluoroacetic acid upon sonication and centrifugation. The
supernatant was then diluted with aqueous buffer A of the reversed
phase HPLC system, and injected onto the C-18 column. The elution
profile is shown in Fig. 6. The identity of this peak was
confirmed by re-running it on a two-dimensional phosphopeptide map to
demonstrate its migration at the position of phosphopeptide 5. The
peptide in the major radioactive peak was then applied to a
Polybrene-coated glass fiber filter which was exposed to automated
Edman degradation sequencing, as well as manual cycles of Edman
degradation with quantitation of the radioactivity eluted in each cycle (Fig. 6). This confirmed its sequence as
D-A-S(P)-Q-K-K-S
(P).
Figure 6: Purification and radiochemical sequencing of phosphopeptide 5. Shown is the HPLC profile of the final step in purification, with a repeat two-dimensional phosphopeptide map of the product, and the radioactive elution profile of the Edman degradation cycles.
For phosphopeptides which were not adequately purified by a single HPLC step, an intermediate step of alkaline polyacrylamide gel electrophoresis was introduced and they were rerun on HPLC ( Fig. 7shows representative data from phosphopeptides 9 and 10).
Figure 7: Purification of phosphopeptides 9 and 10. Shown are HPLC profiles and an autoradiograph of the alkaline polyacrylamide gel used to separate the products.
In addition to
radiochemical sequencing, the phosphoserines in phosphopeptides 9 and
10 were specifically identified by their conversion to S-propylcysteine
with 1-propanethiol using the method of Madden et al.(16) prior to sequencing. The S-propylcysteine residue is
compatible with the Edman degradation chemistry and gives an easily
identifiable phenylthiohydantoin peak on the ABI 476 protein sequencer.
Phosphopeptide 9 was identified as G-G-S(P)-R-L and
phosphopeptide 10 was identified as L-S
(P)-R-Y using this
approach.
Despite the demonstrated importance of phosphorylation of G protein-coupled receptors as a molecular mechanism for receptor regulation, there are few examples of the direct demonstration of specific receptor residues which are phosphorylated in the intact cell (21) . This relates in large part to the sparsity and extreme hydrophobicity of receptor molecules which make purification difficult. While this problem can be overcome with cell lines which express larger numbers of receptors than native receptor-bearing cells, it is critical to understand that a given site of phosphorylation present in such a cell is also utilized in the native environment. Potential differences exist in the cellular complement of protein kinases and phosphatases, as well as in the microenvironment in which the recombinant receptor might reside. The present approach to this problem was addressed by comparing the two-dimensional phosphopeptide maps of the same receptor in both its native cellular environment and in a receptor-expressing cell line.
Indeed, the CCK receptor expressed on the CHO-CCKR cell line was phosphorylated in response to CCK stimulation in a concentration-dependent manner, much like the native receptor on the acinar cell. The time course of that phosphorylation was different, however. Whereas the native receptor was phosphorylated rapidly and reversibly, peaking within 2 min and rapidly returning to its basal state, the recombinant receptor established and maintained its level of phosphorylation throughout this time interval. Perhaps the receptor phosphatase activity we recently described (22) is not present in the CHO-CCKR cell line. There could also be differences in the cellular complement of kinases which act on the receptor.
Consistent with these possibilities, there were both qualitative and quantitative differences in the phosphopeptide maps of the CCK receptor phosphorylated in the pancreatic acinar cell and in the CHO-CCKR cell. There are important insights to be derived both from these differences and from the extensive similarities proven by the maps. As we have demonstrated, a cell line expressing large numbers of receptor molecules provides an ideal substrate to directly sequence the prominent sites of receptor phosphorylation. When these are sites observed in the native environment as well, we can be certain of their relevance.
The absence of a site of phosphorylation within the same receptor molecule expressed on two different cells could be explained several ways. The intracellular signaling events previously observed suggests that the conformation of the receptor in the plasma membrane was appropriate and that at least some coupling and signaling events were intact. Given the complexity of intracellular signaling events and cross-talk, it is possible that a subset of signaling events was not stimulated in the model cell line. It is also possible that a relevant kinase might have been absent in the cell line. We believe that this is the most likely explanation for the absence of phosphorylation of peptide 12 in the CHO-CCKR cells. This should be an excellent cellular system to introduce candidate kinases in an attempt to phosphorylate the CCK receptor on the appropriate residue.
The sites of CCK receptor phosphorylation by protein kinase C provide important insights. Clearly the enzyme was translocated and activated by CCK and TPA, as previously observed(23, 24) . The predicted topology of the receptor based on hydrophobicity and on analogy with other ``heptahelical receptors'' suggests that there are 12 consensus sites for action by protein kinase C (4 of these were identified by the (S*/T*,X, R/K) motif recognized by the PROSITE data base(20) ) which are in sites within the third intracellular loop and the carboxyl-terminal tail of the receptor which would be predicted to be accessible. Despite which definition of protein kinase C consensus is chosen, there are several of these sites which are not utilized by these cells, likely reflecting inaccessibility to the activated kinase. Currently, there are no meaningful conformational models for the loop regions of receptors in this family. Perhaps such data can be built into a model of these regions in the near future.
The functional impact of phosphorylation of this receptor, and the
significance of each site of phosphorylation is clearly of interest. In
analogous G protein-coupled receptors, phosphorylation mediates binding
of arrestin-like proteins which interfere with G protein-coupling and
thereby block initiation of signaling cascades(25) . Indeed, in
work in preparation, ()we have demonstrated that
desensitization of the stimulated inositol trisphosphate response
occurs rapidly in both pancreatic acinar cells and receptor-bearing
CHO-CCKR cells, at the time of initiation of CCK receptor
phosphorylation in these cells. Of note, that report demonstrates that
this desensitization persists in both types of cells, even after the
acinar cell receptor becomes dephosphorylated. This likely reflects the
migration of this receptor into the ``insulation
compartment'' we recently described in the acinar
cell(26) . This represents a postulated mechanism for
desensitization in which G protein uncoupling occurs as a result of
receptor immobilization in a plasmalemmal compartment depleted in G
proteins, rather than requiring receptor phosphorylation to interfere
with this step in signaling. It will be quite interesting to determine
whether receptor phosphorylation plays any role in directing the
receptor into this or other cellular compartments of desensitization.