(Received for publication, July 25, 1994; and in revised form, December 9, 1994)
From the
The cholecystokinin (CCK) receptor on the rat pancreatic acinar cell is a G protein-coupled receptor that is phosphorylated in response to homologous and heterologous agonist stimulation. In this work we have studied the stoichiometry of receptor phosphorylation and have utilized one-dimensional phosphopeptide mapping after cyanogen bromide cleavage to demonstrate that the third intracellular loop is the predominant domain of phosphorylation of this receptor in response to these treatments. Of the average 5 mol of phosphate/mol of receptor, greater than 95% was on the third loop, with the remainder residing on the carboxyl-terminal tail. Serine residues were the site of greater than 95% of phosphorylation, with threonine representing the remainder, and no phosphotyrosine was detected. Further, we have utilized two-dimensional phosphopeptide mapping after subtilisin cleavage to identify differing sites of CCK receptor phosphorylation which are dependent on the agonist utilized to stimulate this cell. Both qualitative and quantitative differences in phosphorylation sites were observed after acinar cell stimulation with different protein kinase C agonists. Further, distinct phosphopeptides on the map were identified as representing substrate(s) of a staurosporine-insensitive kinase activity stimulated only by receptor occupation with native CCK and were felt to represent site(s) of action of a member of the G protein-coupled receptor kinase family. This represents a sensitive and powerful approach that is applicable to sparse receptors residing in their native cellular environment to assess possible differences in patterns of phosphorylation which may be important in agonist-specific receptor regulation.
Phosphorylation of receptors is a well recognized molecular
mechanism for eliciting desensitization, an important process for
protecting the cell from agonist overstimulation. For the heptahelical
receptor proteins, phosphorylation has been implicated in regulating
coupling with guanine nucleotide-binding proteins (G
proteins)()(1, 2, 3) , binding of
arrestin-like molecules(4, 5) , and as a possible
signal for endocytosis and recycling of receptors(6) .
Agonist-stimulated desensitization varies among different receptors on the same cell and among the same type of receptor on different cells (1, 2, 3) . To understand this process clearly it will be critical to determine directly the amino acid residues that are phosphorylated on a specific receptor protein residing in a particular cell under specific stimulatory conditions. This represents a technically challenging process because of the relatively sparse number of most receptor proteins and because of difficulties in purifying these molecules to homogeneity. The typical approach has been to identify ``consensus'' sites for phosphorylation by kinases by analyzing the primary sequence of the receptor and to investigate the functional impact of the mutagenesis of those sites. This approach, however, is by necessity limited to the analysis of recombinant receptors residing in a non-native cell. Little attention has been focused on the non-native stoichiometric ratio of receptor to its regulatory molecules and even on the existence of relevant kinases and phosphatases in this cellular environment.
In this work, we have taken an alternate approach. We have established two-dimensional phosphopeptide mapping methodology that is applicable to a native G protein-coupled receptor that resides in the pancreatic acinar cell and which is phosphorylated in response to a variety of cellular agonists. This has provided direct evidence that the patterns of receptor phosphorylation are distinct from agonist to agonist, and even agonists that are thought to act via similar signaling cascades can lead to distinct patterns of receptor phosphorylation. Observed differences in receptor phosphorylation were both qualitative and quantitative and provided direct evidence that different groups of kinases phosphorylate different sites within this receptor protein.
For this work, we have chosen to focus on the rat pancreatic cholecystokinin (CCK) receptor, a type A CCK receptor. We have previously reported the agonist-stimulated phosphorylation of this molecule(7) . This required the development and application of methodology to purify this phosphoreceptor to radiochemical purity after stimulation of a preparation of dispersed pancreatic acinar cells (7) . In this preparation, receptor phosphorylation was observed after stimulation by both homologous and heterologous agonists(8, 9) . These reports suggest the existence of at least two groups of kinase activities acting on this receptor(8) . CCK receptor phosphorylation by carbamylcholine, bombesin, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), and low concentrations of CCK is entirely inhibited by the protein kinase C inhibitor staurosporine, whereas high concentrations of CCK also stimulate a staurosporine-insensitive receptor kinase activity(8) . The latter is postulated to represent a member of the G protein-coupled receptor kinase family(8) .
Up to the present, there were no data on the domain(s) or residues of this receptor which are phosphorylated. The recent cloning of the cDNA encoding this receptor (10) demonstrates the presence of several potentially accessible consensus sites for kinase action: four for protein kinase C, one for cyclic AMP-dependent kinase (protein kinase A), and two for casein kinase II. The current report demonstrates that the third intracellular loop is the predominant site of phosphorylation, with consensus sites on the tail of the receptor not typically targeted. Despite the presence of a consensus site, protein kinase A agonists do not phosphorylate this substrate. Protein kinase C agonists led to both quantitative and qualitative differences in their sites of action on the CCK receptor.
The CCK analogue
Gly-[(Nle)CCK-26-32]-phenethyl ester
(OPE) was synthesized as we described (11) and conjugated to an
agarose resin via its
-amino group to produce a CCK
analogue-affinity resin for receptor enrichment(12) .
Male Sprague-Dawley rats weighing between 125 and 150 g provided the source of pancreatic tissue for all protocols. These experimental protocols were reviewed and approved by the Mayo Clinic Institutional Animal Care and Use Committee.
The cellular ATP pool was radiolabeled by incubating
dispersed acini in phosphate-free Krebs-Ringer-Hepes medium containing
2 mCi of HPO
in a 100% oxygen
atmosphere at 37 °C for 30 min. Cells were then stimulated with
various agonists using conditions established previously to provide
optimal CCK receptor phosphorylation(7, 9) . All
incubations were terminated by the addition of iced inhibitor buffer
containing (in mM) 25 Hepes, 104 NaCl, 10 NaF, 2 EDTA, 2 EGTA,
20 sodium pyrophosphate, 0.1 sodium orthovanadate, 0.1
phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin, 0.2% bovine
serum albumin, and 0.01% soybean trypsin inhibitor.
After identification of the phosphoreceptor by
autoradiography, the protein was eluted by homogenization of the gel
slice with a Dounce homogenizer and subsequent shaking for 45 min. Gel
fragments were removed by centrifugation at 15,000 g.
Repetition of this process two times provided yields in excess of 95%.
Products of cyanogen bromide cleavage were separated on a urea-SDS gel system adapted from the method of Swank and Munkres(16) . In this, a Laemmli-like stacking gel was added, and the separating gel incorporated 8 M urea, 0.1% SDS, and 12.5% acrylamide (incorporating 10% bisacrylamide).
As reported previously(7) , stimulation of rat pancreatic acinar cells with 0.1 µM CCK resulted in a substantial increase in phosphorylation of the CCK receptor, representing an average 14-fold increase over its basal phosphorylation state. With the phosphorylation response to CCK considered to be 100%, 1 mM carbamylcholine (27 ± 6%) and 1 µM TPA (122 ± 8%) also resulted in marked phosphorylation of the CCK receptor above basal levels (considered to be 0%). This provided adequate amounts of CCK phosphoreceptor for the proposed mapping studies. In contrast, stimulation of acinar cells with 1 µM VIP (1 ± 4%) and 1 µM secretin (1 ± 1%) gave no significant increase in CCK receptor phosphorylation above basal levels.
As demonstrated in Fig. 1, cyanogen bromide cleavage of the CCK receptor
theoretically results in a relatively small number of fragments of
divergent sizes ranging from 0.7 to 9.9 kDa, which include a cytosolic
serine, threonine, or tyrosine residue, potential sites of receptor
phosphorylation. Of note, autoradiography of the urea-SDS gel used to
separate small fragments of the CCK phosphoreceptor after stimulation
with CCK or TPA and cleavage with cyanogen bromide demonstrated a
clearly predominant band at approximately M = 9,900 (Fig. 2). This major band migrated at the
position expected for the fragment representing the third intracellular
loop of the receptor. Consistent with this identification was the
absence of effect of endoglycosidase F to shift this band, as would be
expected of a glycoprotein. With extreme overexposure of the
autoradiogram, a light band began to appear at approximately M
= 4,200. Based on its apparent size, this
likely represents the larger fragment of the carboxyl-terminal tail of
the receptor. Densitometric analysis has demonstrated that the major
band represents greater than 95% of total receptor labeling. All
agonists studied to date produce this pattern of phosphorylation,
suggesting that the third intracellular loop is the clearly predominant
site of phosphorylation of this receptor in the pancreatic acinar cell.
Figure 1: Diagram of the amino acid sequence predicted for the rat CCK-A receptor, with theoretical sites of cyanogen bromide cleavage illustrated. Predicted transmembrane segments are shown within the gray bar; cytoplasmic components of the receptor are below this area. All serine and threonine residues within this domain are highlighted in black, and tyrosine residues within this domain are in dark gray. Five cyanogen bromide fragments of the receptor which possess distinct masses ranging from 0.7 to 9.9 kDa can theoretically include sites of phosphorylation.
Figure 2:
Panel A, one-dimensional phosphopeptide
map of the cyanogen bromide fragments of the rat pancreatic acinar cell
CCK receptor which was phosphorylated in response to TPA or CCK. The
major band migrated on the urea-SDS acrylamide gel at M = 9,900, corresponding to the size of the third
intracellular loop. As expected, deglycosylation with endoglycosidase F
did not modify the migration of this band. A lane from an overexposed
autoradiograph which was run longer is shown to demonstrate that a
minor band migrating at M
= 4,200 was also
present. Panel B, phosphoamino acid analysis of the rat
pancreatic CCK phosphoreceptor always demonstrated a predominance of
phosphoserine (greater than 95%), a small amount of phosphothreonine,
and never any phosphotyrosine. Illustrated autoradiographs are typical
of more than six similar experiments.
Phosphoamino acid analysis of the major cyanogen bromide fragments of the CCK phosphoreceptor revealed that phosphoserine represents greater than 95% of residues (Fig. 2). With extreme overexposure, a small amount of phosphorylation became apparent at threonine residues, but no phosphotyrosine was ever observed.
Typical two-dimensional phosphopeptide maps of the CCK phosphoreceptor are shown in Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7. These maps were generated from acinar cells stimulated with CCK, TPA, carbamylcholine or from control incubations without secretagogue. These patterns have been reproducible, with unambiguous identification of distinct spots with a standard template. As seen in Fig. 3, CCK stimulation of the acinar cells generated the largest number of phosphopeptides observed under any condition. The phosphopeptide map of the CCK receptor in the basal state from cells incubated under similar conditions in the absence of any secretagogue is also shown, to demonstrate the minimal level of receptor phosphorylation observed after a similar exposure of the autoradiogram. Coincubation with CCK and the CCK receptor antagonist L-364,718 produced a map indistinguishable from the nonstimulated control map (data not shown).
Figure 3: Typical two-dimensional phosphopeptide map of the rat pancreatic CCK receptor that was phosphorylated in response to CCK, as well as a similar exposure of a map generated after a control incubation in the absence of acinar cell secretagogue. Radiochemically pure CCK receptor was cleaved with subtilisin to yield this highly reproducible pattern, with the identification code illustrated in the lower panel. This map is typical of 12 similar experiments.
Figure 4: Typical two-dimensional phosphopeptide map of the rat pancreatic CCK receptor phosphorylated in response to TPA. TPA stimulated all of the CCK-stimulated phosphopeptides except 2, 4, and 12 (shown hatched) and stimulated a disproportionate increase in phosphorylation of 6 (shown filled). All of these sites were significantly increased relative to a basal control and relative to a staurosporine-treated control to eliminate sites of action of protein kinase C. This map is typical of four similar experiments, with bar graphs illustrating densitometric quantification (mean ± S.E.) of CCK receptor phosphorylation in those experiments.
Figure 5: Typical two-dimensional phosphopeptide map of the rat pancreatic CCK receptor phosphorylated in response to carbamylcholine. This agent stimulated all of the CCK-stimulated sites except 4 and 12, with all of the TPA-stimulated sites observed as well as peptide 2. All of these sites were significantly increased relative to a basal control and were inhibited by pretreatment with the protein kinase C inhibitor staurosporine. This map is typical of four similar experiments, with bar graphs illustrating densitometric quantification (mean ± S.E.) of the CCK receptor phosphorylation in those experiments.
Figure 6: Two-dimensional phosphopeptide maps of rat pancreatic CCK receptor phosphorylated in response to 0.1 nM and 0.1 µM CCK. Spots 4 and 12 were observed only after stimulation with the higher concentration of native agonist. Over this range of secretagogue concentration, the degree of phosphorylation of some other peptides (2 and 6) was also increased. Patterns illustrated are typical of four similar experiments.
Figure 7: Two-dimensional phosphopeptide maps of rat pancreatic CCK receptors phosphorylated in response to 0.1 µM CCK in the presence of the protein kinase C inhibitors staurosporine and calphostin C revealed relative enrichment in phosphorylation of spots 2, 4, and 12. Patterns of phosphopeptides in control maps were similar to those in Fig. 3. In the presence of inhibitors phosphorylation represented 30% of that seen on the CCK-stimulated control map, and the basal control represented 3% of the phosphorylation signal.
Stimulation with TPA (Fig. 4) and carbamylcholine (Fig. 5) produced only subsets of the phosphopeptides observed with CCK stimulation. Phosphorylation of all of these sites within the CCK receptor returned to basal levels in the presence of the protein kinase C inhibitor staurosporine. Of note, phosphopeptides 2, 4, and 12, which were observed with CCK stimulation, were consistently absent after TPA stimulation. All of the other major sites observed after CCK stimulation were also seen after TPA stimulation. Also noteworthy was a major quantitative difference between the TPA and CCK maps for phosphopeptide 6. This was dramatically phosphorylated in response to TPA and represented only a very minor site relative to the other apparent substrates of protein kinase C in response to CCK stimulation. As expected, coincubation of cells with TPA and staurosporine produced a map indistinguishable from the basal map, whereas the CCK receptor antagonist L-364,714 had no effect on the TPA-stimulated receptor phosphorylation pattern.
Carbamylcholine is believed to stimulate an intracellular activation cascade much like CCK, mediated by phospholipase C activation, phosphatidylinositol hydrolysis, increased intracellular calcium, and translocation of protein kinase C. Carbamylcholine-stimulated acinar cells resulted in a phosphopeptide map of the CCK receptor which demonstrated all of the sites observed in response to TPA, with a reduced yield of the very basic phosphopeptide stimulated heavily by TPA (Fig. 5). In this regard, carbamylcholine was very much like CCK. In addition to the sites observed in response to TPA, carbamylcholine also stimulated phosphorylation of peptide 2, which was also observed with CCK. In contrast to CCK, however, carbamylcholine did not stimulate phosphorylation of peptides 4 or 12. Further, none of the CCK receptor peptides that were phosphorylated in response to cellular stimulation with carbamylcholine was affected in any way by coincubation with the CCK receptor antagonist L-364,718.
Two-dimensional phosphopeptide maps were also generated after stimulation of acinar cells with 0.1 µM secretin or 0.1 µM VIP to provide a more sensitive assessment of a potential site of action of cyclic AMP-dependent kinase than the previously performed quantitation of intact receptor phosphorylation (9) . Even after long exposures, however, these secretagogues produced maps identical to those observed in the basal state (data not shown).
Based on the two-dimensional phosphopeptide map patterns observed after stimulation with different agonists, we have identified all of the phosphopeptides except for peptides 2, 4, and 12 as likely representing sites of action of protein kinase C. Consistent with this interpretation, all of these phosphopeptides were markedly reduced when cells were pretreated with staurosporine or calphostin C to inhibit protein kinase C activity ( Fig. 4and Fig. 5). The number of distinct sites of phosphorylation is unclear since the same site may appear in more than one phosphopeptide of differing lengths. The site represented by peptide 6 appears to be distinct from the sites represented by the other protein kinase C substrates since the intensity of the signal from phosphopeptide 6 varies in a disproportionate manner from the intensity of the other sites.
Perhaps the most interesting phosphopeptides on the map are 4 and 12. These correlate with the kinase activity reported previously after CCK stimulation, which was felt to represent action of a member of the G protein-coupled receptor kinase family(8) . Consistent with that identification was the concentration dependence of stimulation of phosphorylation of these spots. As shown in Fig. 6, low concentrations of CCK stimulated only the apparent sites of action of protein kinase C, whereas high concentrations were necessary to stimulate phosphorylation of these two candidate phosphopeptides (4 and 12). Additionally, as seen in Fig. 7, stimulation with CCK in the presence of the protein kinase C inhibitors staurosporine and calphostin C produces enrichment of these sites relative to the protein kinase C substrates.
Analysis of the stoichiometry of CCK receptor phosphorylation after stimulation with 0.1 µM CCK suggested the presence of 5 mol of phosphate/mol of CCK receptor (range 3-7 mol). This is quite consistent with the two-dimensional phosphopeptide maps observed. There were at least four major covariate patterns within the map. As discussed above, the presumed sites of action of protein kinase C represent two covariate groups, one representing phosphopeptides 1, 3, 5, 7, 8, 9, 10, 11, 12, 13, and 14, and one representing phosphopeptide 6. Phosphopeptide 2, which is stimulated by carbachol but not TPA, represents a third group; and phosphopeptides 4 and 12, which are only stimulated by CCK, represent a fourth group. Although each group must represent a minimum of one phosphoamino acid, any group may represent more than one site as well. This would be the situation if the subtilisin did not separate each distinct site of phosphorylation of the receptor.
To be able to begin to understand the role of phosphorylation in the mechanisms of receptor regulation, it is critical to understand the molecular details of agonist-stimulated receptor phosphorylation. In a native cellular environment, this has rarely been accomplished because of the sparse numbers of receptors, which are difficult to purify. Rather, most of our insights have come from the mutagenesis of recognized consensus sites for known kinases, with the expression of mutant receptors in a tissue culture cell. This approach has many potential problems.
The accuracy and completeness of choosing consensus phosphorylation sites are unclear. Although the preferred sites of action of major kinases such as protein kinase C are well established, these sites are not always phosphorylated by active enzyme, and a large number of less well appreciated amino acid sequences may be recognized by this kinase (21) . Many cellular kinases do not yet have well established consensus sequences for their sites of action. Also, there is always the possibility that a mutation will affect receptor biosynthesis, its transport to the plasma membrane, or have a disruptive effect on conformation, rather than simply acting as a nonphosphorylated normal substrate.
Further, the processes of receptor regulation may be peculiar to a given cell. Clearly, the most relevant cellular environment is the native cell expressing the receptor and all relevant kinases, phosphatases, and other regulatory molecules in appropriate stoichiometry. Tissue culture cells used for expression of the recombinant receptor may have critical, yet unrecognized, differences from the native cellular environment.
The approach used in the current report overcomes all of these potential problems. Since receptor phosphorylation is performed by agonist stimulation of the pancreatic acinar cell, all regulatory events relevant to the native receptor in that cell take place. The one-dimensional and two-dimensional phosphopeptide mapping approaches taken provide direct evidence for the receptor domain(s) phosphorylated in this setting and for the specific sites of phosphorylation stimulated by different agonists. Further, groups of kinases can be pharmacologically manipulated in such a system to gain insights into their contribution to net receptor phosphorylation stimulated by each agonist.
For the current approach to be meaningful, it is critical to be able to purify the receptor of interest to radiochemical homogeneity. Ideally, this should be possible in a simple, rapid procedure that separates the receptor from potential reaction with cellular protein phosphatases, kinases, and proteases. We previously established and validated this type of methodology for the CCK receptor on the pancreatic acinar cell (7) .
The pancreatic acinar cell is a very interesting model for this study because of the extensive data on the presence of distinct secretagogue-stimulated second messenger cascades within the cell and on potentiating and inhibitory interactions between these intracellular events(22) . CCK and carbamylcholine are prototypic of the ``calcium-mediated'' secretagogues, whereas secretin and VIP are prototypic of ``cyclic AMP-mediated'' secretagogues. This is also a cellular system in which hormone-elicited desensitization is known to occur(23) , with both homologous and heterologous components (8, 9) . Indeed, CCK responses have been shown to be desensitized after exposure to CCK(23) , as well as to carbamylcholine and TPA(24, 25) . The receptor phosphorylation events described in the current work are likely key in these processes.
Further, the absence of CCK receptor phosphorylation observed after secretin and VIP in this work is consistent with the lack of desensitization of the CCK response described previously with these cyclic AMP agonists(26) . This suggests that the consensus site for action of the cyclic AMP-dependent kinase which exists in the third intracellular loop of this receptor is not accessible.
Three consensus sites for action of protein kinase C exist within the third intracellular loop, and one site exists in the carboxyl-terminal tail of this receptor. The current data are supportive of the action of this kinase at a minimum of two such sites within the third loop, while having at most, minimal activity at the consensus site in the receptor tail. Such observations further validate the current approach of direct experimental analysis of sites of receptor phosphorylation rather than depending only on analysis of primary sequence.
It has been postulated previously that distinct isoenzymes of protein kinase C may possess distinct substrate specificities not recognized by primary amino acid sequence of their apparently uniform sites of action (27) . Consistent with that hypothesis are the qualitative differences observed between the sites of action on the CCK receptor of protein kinase C stimulated by CCK, carbamylcholine, and TPA. A preliminary report suggests that each of these pancreatic secretagogues elicits distinct responses of the isoforms of this enzyme present in the pancreatic acinar cell(28) .
There were two phosphopeptides identified in the
CCK receptor map which were only observed after CCK stimulation and not
after stimulation with any other acinar cell agonists. The
phosphorylation of these peptides was concentration-dependent, only
observed with high concentrations of CCK, and it was fully inhibited by
L-364,718. Like the prototypic G protein-coupled receptor kinase,
ARK, this activity was not inhibited by staurosporine(8) ,
and it was inhibited by heparin(8) . All of these observations
are consistent with this representing the action of a member of this
rapidly expanding family of kinases (29, 30, 31, 32) . At this time it
is unclear which member may be the relevant kinase for this action in
the pancreatic acinar cell.
It is of interest that the apparent
predominant sites of action of both protein kinase C and what is likely
a G protein-coupled kinase are within a single loop of the CCK
receptor. This is in contrast to the -adrenergic
receptor in which sites of action of cyclic AMP-dependent kinase are
predominantly within the third loop, whereas those of
ARK are on
the carboxyl-terminal tail(33) . Distinct domains of action
provide clear separation of the
ARK sites as a distinct binding
domain for
-arrestin(4, 33, 34) . If an
arrestin-like protein also binds to the CCK phosphoreceptor, it will be
of interest to determine the possible effects of sites of action of
these two groups of kinases.
At present, CCK receptor desensitization in the acinar cell can be correlated with the phosphorylation of the CCK receptor at any site(7, 23) . It will, of course, be of great interest to determine whether there are distinct mechanisms for desensitization initiated by phosphorylation at distinct sites. This would provide the cell with a broader constellation of responses with differing physiologic implications which might be appropriate for different settings.
A particularly important application of the methodology established in the current work will be the application of the receptor phosphopeptide mapping to recombinant receptor overexpressed in model cell systems. These provide the opportunity for working at a different scale in which receptor domains can be more easily purified to chemical homogeneity for direct sequencing and unambiguous identification of distinct phosphorylated residues. These can then also be mutagenized and studied in that system. By possessing the clear background data that the same site is seen in a map of native receptor, the resulting data will be much more meaningful. They can then be effectively incorporated into a model of receptor regulation and normal cell physiology.