(Received for publication, September 23, 1994; and in revised form, January 25, 1995)
From the
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.
Several members of the guanine nucleotide-binding protein (G
protein)()-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
-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
-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.
Internalization of receptors was performed as
described previously(31, 33) , defined as the
percentage of specific cell-associated I-Tyr
-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.
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.
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.
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 flasks. Cells were harvested and suspended at
2
10
cells/ml in binding buffer with 75 pM
I-Tyr
-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
-bombesin resistant to
acid wash. In B and C, the various cell lines were
plated into 100
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).
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 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
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
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
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).
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-Phe
,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 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
-Phe
,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.
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 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-
, -
, 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 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
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%.
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
-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
-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
-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 -adrenergic receptor, both a second messenger-dependent
kinase (protein kinase A) and second messenger-independent kinase
(
-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 (
-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 -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
-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.