From the
The parallel agonist-induced phosphorylation, alteration in
electrophoretic mobility, and loss of ligand binding of a guanine
nucleotide-binding regulatory protein (G protein)-coupled
chemoattractant receptor from Dictyostelium (cAR1) depend upon
a cluster of five C-terminal domain serine residues (Caterina, M. J.,
Hereld, D., and Devreotes, P. N. (1995) J. Biol. Chem. 270,
4418-4423). Analysis of mutants lacking combinations of these
serines revealed that either Ser
For
the wild-type receptor, we found that the stability of phosphorylation
depends upon the concentration and duration of agonist pretreatment.
This suggests that, following phosphorylation of Ser
Receptors that couple to G proteins
cAR1, the
chemoattractant cAMP receptor of Dictyostelium, is a G
protein-coupled receptor which, upon nutrient depletion, coordinates
the aggregation of 10
The cAR1 C-terminal cytoplasmic domain
contains 18 serine residues organized into four clusters. Agonist
binding results in the addition of 3-4 phosphates to serines
within two of these clusters. The majority of this phosphorylation
occurs within cluster 1 and results in a marked reduction in cAR1
electrophoretic mobility. A mutant in which the five serines of cluster
1 were substituted by site-directed mutagenesis exhibits reduced
cAMP-stimulated phosphorylation and fails to undergo the
electrophoretic mobility shift
(15) . Furthermore, this mutant
is markedly impaired in cAMP-induced LLB
(32) . In contrast,
elimination of the other 13 serines of the cytoplasmic domain (clusters
2, 3, and 4) had little effect on either the mobility shift or LLB.
These findings suggest that phosphorylation of cluster 1 and LLB are
related.
In the present study, we describe a detailed mutational
analysis of cluster 1 and identify the specific residues required for
each of these processes. We analyzed both the onset and reversal of
these two processes as exhibited by mutant and wild-type cAR1s under a
variety of cAMP pretreatment conditions. Our results indicate that LLB
and phosphorylation of specific residues within cluster 1 are
coordinately regulated upon the introduction and removal of cAMP and
suggest a causal relationship between these two processes.
To evaluate
the kinetics of the stabilization, we completely converted receptors to
the mobility-shifted form with 10
We have previously shown that agonist binding induces the
addition of 3 or 4 mol of phosphate per mol of cAR1 to serine residues
of the C-terminal cytoplasmic domain. Phosphorylation within cluster 1,
which includes Ser
We have also previously demonstrated that
agonist-induced LLB is largely due to a reduction in the binding
affinity of cAR1 for cAMP. While under some circumstances receptor
internalization can result from cAMP treatment, this process need not
occur for LLB to be observed. Furthermore, like the electrophoretic
mobility shift, LLB is markedly impaired in mutant receptors lacking
all five serines of cluster 1
(32) . Some of the substitutions
of individual cluster 1 serines, described in the present report,
initially appeared to break this correlation between LLB and the
mobility shift. For example, substitution of Ser
This correlation implies that the phosphorylation-dependent
electrophoretic shift and LLB are sequential processes. Phosphorylation
might occur first and induce a low affinity conformation either
allosterically or by promoting the binding of a protein such as an
arrestin homolog. Alternatively, LLB might represent the adoption of a
low affinity receptor conformation that is necessary but not sufficient
for subsequent phosphorylation. This possibility is supported by the
observation that ( R
We
identified a variety of factors which affect the stability of the
electrophoretically shifted form of the receptor. These include the
dose and time of cAMP pretreatment as well as mutations within serine
cluster 1 and the receptor's third cytoplasmic loop. For the
wild-type receptor, low cAMP concentrations (
The cAR1 mutations described in this report that impair stability of
the shifted form might do so by diminishing the extent of receptor
phosphorylation. For instance, mutations within cluster 1 might
directly eliminate phosphorylation sites or, alternatively, might
diminish phosphorylation or enhance the dephosphorylation of other
cluster 1 residues. How mutations in the putative third intracellular
loop might influence phosphorylation of serine cluster 1 is more
puzzling. Perhaps juxtaposition of these two regions in the native
receptor permits these mutations to affect the accessibility of cluster
1 to the kinase or phosphatase. Alternatively, mutations in the third
loop could reduce the extent of receptor phosphorylation if, like
It has been proposed that
Our findings suggest that an important role
of phosphorylation might be to bring about (or maintain) a low affinity
state of cAR1, possibly serving to broaden the sensitive range of cells
to micromolar cAMP concentrations. Such high concentrations are
transiently produced by aggregating cells and are thought to persist in
multicellular structures (reviewed in Ref. 31). It is also tempting to
speculate that loss of ligand binding reflects the interaction of cAR1
to another protein, perhaps one involved in the desensitization of G
protein-mediated signaling. Whatever their functions might be, the
concentration-dependent stabilization of cAR1 phosphorylation and LLB,
described here, is likely to be most relevant to the multicellular
stage. Experiments directed at distinguishing these possibilities are
in progress.
Cluster
1 mutants used in this study and the amino acid substitutions in each
are indicated. In the mutants, preserved wild-type residues are
indicated by dashes (-). A, alanine; G, glycine; S, serine.
We are grateful to Dr. Peter J. M. Van Haastert for
valuable discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
or Ser
is
required; mutants lacking both serines are defective in all of these
responses. Interestingly, several mutants, including those substituted
at only Ser
, Ser
, or Ser
or
at non-serine positions within the third cytoplasmic loop, displayed an
unstable mobility shift; the alteration was rapidly reversed upon cAMP
removal. These mutants also exhibited subnormal extents of loss of
ligand binding, which is assessed after removal of the ligand.
or
Ser
, cAR1 undergoes a further transition (EC
140 n
M, t
4 min) to a
relatively phosphatase-resistant state. We used this insight to show
that, under all conditions tested, the extent of loss of binding is
correlated with the fraction of cAR1 in the altered mobility form. We
discuss possible relationships between cAR1 phosphorylation and loss of
ligand binding.
(
)
undergo a series of changes upon agonist binding, including
serine/threonine phosphorylation of cytoplasmic domains as well as a
rapid, reversible decrease in ligand binding (reviewed in Ref. 1). The
latter has been attributed in some cases to a reduction in binding
affinity
(32) and in others to internalization of receptors
(often referred to as ``sequestration''). The exact
relationship between phosphorylation and reduction of binding is
unclear, and may vary among different G protein-coupled receptors.
Studies of acetylcholine, gastrin-releasing peptide, and cAMP receptors
indicate that phosphorylation and decreases in ligand binding are
coordinately regulated
(2, 3, 4, 5, 32) . In contrast,
the sequestration of
-adrenergic receptors is
preserved despite inhibiting receptor phosphorylation or eliminating
putative phosphorylation sites by mutagenesis
(1, 6) .
In addition, in this latter system, it has been proposed that, while
phosphorylation does not appear to be a prerequisite for sequestration
of the
-adrenergic receptor, sequestration is
necessary for receptor dephosphorylation
(7) .
individual amoebae into a
multicellular structure (Ref. 9; reviewed in Ref. 8). Like other
members of the seven transmembrane domain receptor family, cAR1
exhibits both agonist-induced phosphorylation and loss of ligand
binding (LLB)
(10, 11, 12, 13) . While
cAR1 internalization can occur under some circumstances
(14) ,
we have shown that LLB results largely from a reduction in binding
affinity
(32) .
Site-directed Mutagenesis of Serine Cluster
1
Using site-directed mutagenesis and three previously described
oligonucleotides
(15) , Serand Ser
were independently substituted with Ala and
Ser
Ser
Ser
was replaced with
Gly-Ala-Gly. Alternatively, the same replacements of Ser
,
Ser
, and Ser
were made independently using
the degenerate antisense oligonucleotide,
5`-GTACCACGAC XTG YAC XATATGGTG-3` (where
X= T or C and Y = A or C). The cluster
1 mutants are summarized in .
Cell Lines Expressing cAR1 Mutants
Mutated cAR1
cDNAs, subcloned into an expression vector (either pB18 or pJK1)
(15) , were introduced into AX-3 cells or cells lacking cAR1
(JB4 cells)
(16) by electroporation as described previously
(17) . Similar results were obtained when wild-type cAR1 and
selected mutants were expressed in each cell type. Clonal, stably
transformed cell lines were selected and maintained in HL-5 medium
(18) containing 20 µg/ml G418 (Life Technologies, Inc.).
Cells expressing wild-type cAR1 (MC36 cells) and mutant cm1 are
described elsewhere
(15, 16) . Previously described
third intracellular loop mutants
(16) used in this study and
their amino acid substitutions (in parentheses) are as follows: A3
(R184L, Y185N, Y187S); A5 (T182S, Y187H); A22 (V188F, V189L); A42
(V189D); A53 (T182S, V188T, V189R); A60 (T186A, Y187D); and A62 (R184C,
V188A, V189A).
Induction and Measurement of the Electrophoretic Mobility
Shift
Cells, grown as shaken suspensions to densities of 5
10
cells/ml, were washed with PB (5 m
M Na
HPO
, 5 m
M KH
PO
, pH 6.1) prior to each experiment.
Washed, growth-stage cells were stimulated with the indicated
concentrations of cAMP (22 °C, shaking at 200 rpm, durations
indicated in legends) in the presence of 10 m
M DTT to inhibit
phosphodiesterase
(19) . Where indicated, 5 m
M caffeine
was added to inhibit endogenous cAMP production
(20) . Aliquots
of cells were combined with sample buffer
(21) , mixed, and
stored at -20 °C. These samples were subjected to SDS-PAGE on
10% or 12% low bispolyacrylamide gels
(22) , transferred to
polyvinylene difluoride filters (Immobilon, Millipore), blocked with 3%
bovine serum albumin, and immunoblotted using cAR1 C terminus-specific
antiserum and chemiluminescence as described elsewhere
(15) .
Quantitation of immunoblots was performed using a digitizing scanner
(Logitech) and SigmaScan/Image software (Jandel).
Induction and Measurement of LLB
Unless indicated
otherwise, washed, growth-stage cells were stimulated with cAMP as
described for the induction of the electrophoretic mobility shift.
Unless indicated otherwise, cells were then diluted 10-fold in ice-cold
PB, pelleted by centrifugation at 0-4 °C (Sorvall SS34 rotor,
2000 rpm, 3 min), washed three times with ice-cold PB, and resuspended
at 10cells/ml. Binding of 16 n
M [
H]cAMP was then measured in PB by either a
sedimentation assay
(23) or by centrifugation through silicone
oil
(24) as indicated in the figure legends.
Reversal of LLB and Mobility Shift
In order to
synchronize the reversal of mobility shifting and LLB for cell samples
treated at various cAMP concentrations, the initial two PB washes
sometimes contained cAMP and dithiothreitol, as indicated in the figure
legends. Reversal time is therefore defined as beginning with the
initiation of the third wash, which was always with PB alone. By this
convention, the 20 min time point illustrated in the figures occurs
immediately after completion of the fourth wash and resuspension of the
cells.
Phosphorylation of the Central Three Serines of Cluster
1 Causes the Electrophoretic Mobility Shift
We have previously
demonstrated that agonist-induced phosphorylation of cAR1 within the
five serines of cluster 1 (Ser, Ser
,
Ser
, Ser
, and Ser
) causes a
discrete shift to a lower electrophoretic mobility form
(15) .
This is illustrated in Fig. 1by the inability of mutant cm1,
which lacks these five serines, to undergo the transition in mobility.
To identify the specific serine residues required, we selectively
substituted the serines of cluster 1 as indicated in . As
shown in Fig. 1, when the flanking residues (Ser
and
Ser
) were both substituted ( mut2), the ability
of the receptor to exhibit the mobility shift was unaffected. In
contrast, substitution of the central three serine residues
(Ser
, Ser
, and Ser
) in
mut4, eliminated the shift. The failure of cm1 or mut4 to
shift is not related to levels of expression (Fig. 1) or due to
impaired binding (data not shown). These results indicate that one or
more of the central three serines, but not the flanking serines,
mediate the electrophoretic mobility shift.
Figure 1:
Electrophoretic mobility shift of cAR1
serine cluster 1 mutants. Cells expressing wild-type ( wt) or
mutant cAR1s were incubated without (-) or with (+) cAMP as
indicated (15 min, 22 °C, shaken at 200 rpm). Caffeine (5
m
M) and DTT (10 m
M) were added to inhibit cAMP
synthesis and degradation, respectively. Samples of whole cells
stimulated with 1 µ
M cAMP ( wt, cm1, and
mut2) or CHAPS-extracted cells (15) stimulated with 10
µ
M cAMP (all others shown) were analyzed by SDS-PAGE on
12% low bisacrylamide gels and immunoblotting with anti-cAR1
antiserum.
We then mutated the
central three serines in various combinations. We found that each could
be mutated individually without impairing the electrophoretic mobility
shift (Fig. 1, mut14, mut15, and
mut16). Thus, none of these serines is essential for this
property. The mobility shift was markedly impaired when both
Serand Ser
were mutated (Fig. 1,
mut18). Furthermore, the preservation of either Ser
or Ser
by itself (Fig. 1, mut17 and
mut19) resulted in receptors which shifted like wild-type
cAR1. These results suggest that either Ser
or
Ser
can be phosphorylated and cause the shift. The small
fraction of shifted receptor in the cAMP-stimulated mut18 sample
suggests that Ser
can also mediate the shift but with
lower efficiency than either Ser
or Ser
.
Occasionally, wild-type and mutant receptors having intermediate
electrophoretic mobilities upon cAMP-stimulation are observed ( e.g.
mut2 and mut17 in Fig. 1). These possibly represent
underphosphorylated forms.
cAMP-induced LLB of Mutants and Its Relationship to the
Electrophoretic Shift
We next assessed the abilities of these
cluster 1 mutants to undergo LLB in order to further evaluate the
relationship between this property and receptor phosphorylation. As
shown in Fig. 2, mutants which did not undergo the
electrophoretic mobility shift ( e.g. cm1 and mut4)
were also impaired in LLB. However, among the mutants which displayed
the electrophoretic mobility shift, a spectrum of LLB behaviors was
observed, ranging from those with normal LLB ( e.g. mut16) to
those with dramatically impaired LLB ( e.g. mut15 and
mut17).
Figure 2:
Loss of ligand binding by cAR1 mutants.
Cells expressing wild-type ( wt) or mutant cAR1s were incubated
without or with cAMP (10
M, 15 min),
washed, resuspended to 10
/ml, and analyzed for LLB by the
sedimentation assay described under ``Materials and
Methods.'' Plotted for each cell line is the percentage of binding
exhibited by untreated cells which is lost upon cAMP pretreatment.
Values represent the means ± range of triplicate determinations
from two independent experiments. mut2 was analyzed in only one
experiment.
Wild-type cAR1 phosphorylation and the resulting
mobility shift are readily reversible at 22 °C but very slowly
reversible at 4 °C
(11, 25) . Since measurement of
LLB requires prior removal of the cAMP stimulus by repeated washing, it
was conceivable that instability of the shifted forms of mutants such
as mut15 explained the apparent incongruity of their LLB and shifting
properties. To examine this possibility, the shifted forms of selected
mutants were assessed at various times at 4 °C following cAMP
removal, including the time at which we routinely measure LLB. As shown
in Fig. 3, the reversal of the mobility shift for wild-type cAR1
is extremely slow at 0 °C. Nearly all of the receptor remained
shifted 120 min after cAMP removal. Quantitation and extrapolation (not
shown) of these data indicated that the half-time for reversal exceeds
5 h. In contrast, some of the cluster 1 mutants exhibit an increased
rate of conversion of the shifted to the unshifted form. Mutation of
Ser(mut15) dramatically reduced the stability of the
shifted form ( t
< 20 min). Mutation of either
Ser
(mut1) or Ser
(mut14) had a moderate
destabilizing effect ( t
60-120 min).
Surprisingly, mutation of Ser
(mut16), one of the two
serines able to mediate the shift, had no apparent effect on stability.
Mutants substituted at two cluster 1 serines exhibited stabilities
consistent with those observed for single-serine substitutions. Those
lacking Ser
(mut17 and mut18) were predictably unstable
( t
< 20 min, data not shown). The
intermediate stability of mut19 ( t
20 min)
can be attributed to mutation of Ser
.
Figure 3:
Stability of the electrophoretic mobility
shift of cAR1 mutants. Cells expressing wild-type or mutant cAR1s were
incubated with cAMP (10
M, 15 min) washed
four times with ice-cold PB, resuspended to 5
10
/ml, and incubated on ice for the indicated times.
Aliquots of cells were transferred to sample buffer before the addition
of cAMP (-), following the 15 min stimulation and at 20, 60, and
120 min after the initiation of the third PB wash. (See
``Materials and Methods'' for explanation of time scale.)
Cluster 1 mutants are defined in Table I. A62 and A53 are third intracellular loop mutants (16). The data shown are
representative of at least two independent
experiments.
In light of the
instabilities of the shifted forms of cluster 1 mutants, we revised our
strategy and compared the extent of LLB with the residual mobility
shift of each mutant at the time of the LLB measurement. As
demonstrated in Fig. 4, this analysis now revealed a significant
correlation between the extent of LLB and the fraction of receptors in
the shifted state, suggesting an interrelationship of these two
properties.
Figure 4:
Correlation between cAMP-induced loss of
ligand binding and electrophoretic mobility shift in cAR1 mutants. The
percentage loss of cAMP binding exhibited by wild-type cAR1
( wt, filled square), cluster 1 mutants ( filled
circles) and third intracellular loop mutants ( open
circles) immediately following pretreatment with cAMP
(10
M, 15 min) and four washes with
ice-cold PB (20-min time point in Fig. 3) is plotted against the
percentage of receptor in the lower mobility form, determined under the
same conditions. LLB data for wild-type cAR1 and cluster 1 mutants is
taken from Fig. 3 and that for the third intracellular loop mutants is
from Caterina et al. (16). Mobility shift data was obtained by
scanning the 20-min time point from autoradiographs generated as in
Fig. 3 and represents the mean determination from two independent
experiments. LLB and mobility shift for a given mutant were usually
assayed in separate experiments but at the same time after cAMP
removal. Comparison of data points with the illustrated correlation
line (generated by linear regression) yields r
= 0.81,
< 0.001. Separate analyses of the third
loop mutants ( r
= 0.78,
< 0.01)
and cluster 1 mutants ( r
= 0.81,
<
0.01) reflected similarly strong correlations (not
depicted).
These observations prompted us to re-examine the
mobility shift of cAR1 third intracellular loop mutants which we had
previously found to be impaired in LLB
(16) . As with the
cluster 1 mutants, we found that the lower mobility forms of some of
these mutants were less stable than that of wild-type cAR1 ( e.g.
A53 and A62, Fig. 3). Inclusion of data from these
mutants in Fig. 4further strengthened the correlation between
LLB and the electrophoretic shift.
cAMP Concentration-dependent Stabilization of the Shifted
Form of Wild-type cAR1
To further evaluate this correlation and
establish its relevance to the wild-type receptor, we performed a
detailed analysis of the stability of the mobility shift in wild-type
cAR1 and its relationship to LLB. We first sought to assess whether the
stabilization of the lower mobility form of wild-type cAR1 depended on
cAMP concentration. We therefore treated cells expressing wild-type
cAR1 with either 10
M or 10
M cAMP, removed the cAMP, and monitored the stability of
the lower mobility receptor form. As illustrated in Fig. 5 A,
each concentration elicited a complete conversion from unshifted to
shifted forms. Upon removal of cAMP and incubation at 4 °C,
however, the stabilities of the shifted forms differed markedly. While
receptors treated with the higher cAMP concentration remained
completely shifted for at least 2 h, those treated at the lower
concentration exhibited a time-dependent decay to the higher mobility
form ( t
60 min). Thus, as we found for the
mutants, electrophoretically shifted wild-type receptors can exhibit
markedly different stabilities. Their relative stabilities depend upon
the cAMP concentration with which they were stimulated.
M cAMP
and then exposed the cells for various times to 10
M cAMP (Fig. 5 B). As before, receptors
exposed to only the lower cAMP concentration exhibited an unstable
mobility shift. Those exposed to the higher concentration acquired
stability with a t
of approximately 4 min.
Figure 5:
Concentration-dependent stability of the
mobility shifted form of wild-type cAR1. A, cells expressing
wild-type cAR1 were incubated without (0
M) or with
10
M or 10
M cAMP (15 min, 22 °C), washed twice with ice-cold PB containing
10 m
M DTT (for the unstimulated sample) or ice-cold PB
containing 10
M cAMP and 10 m
M DTT, then twice with ice-cold PB alone, resuspended to 5
10
/ml, and incubated on ice. Cells were combined with
sample buffer at the indicated times after the initiation of the third
wash and analyzed by SDS-PAGE on 10% low bisacrylamide gels and
immunoblotting with cAR1-specific antiserum as described under
``Materials and Methods.'' B, time-dependent
stabilization of the lower mobility receptor form. Cells expressing
wild-type cAR1 were incubated with 10
M cAMP (15 min, 22 °C) after which the cAMP concentration was
adjusted to 10
M cAMP for the indicated
period of time. ( 0 indicates no further cAMP addition.) Cells
were then washed twice with ice-cold PB containing cAMP
(10
M) and 10 m
M DTT, then twice
with ice-cold PB alone, resuspended to 5
10
/ml, and
incubated on ice for 170 min. Cells were combined with sample buffer
prior to the addition of cAMP (no cAMP), just prior to washing (0 min)
or at 170 min after the initiation of the third wash. Data shown are
representative of two independent
experiments.
Correlation between Shifting and LLB of the Wild-type
cAR1
Previously reported ECvalues for the cAR1
mobility shift are up to 10-fold lower than those for LLB
(11, 13, 25, 32) . To assess whether
this apparent disparity could be explained by instability of shifted
receptors, we undertook a detailed analysis of the cAMP concentration
dependence of cAR1 mobility shifting prior to and at times following
cAMP removal. As shown in Fig. 6 A, the concentration of cAMP
required to convert half of the cAR1 molecules to the shifted form, or
EC
, is 7 n
M. Due to instability at low cAMP
concentrations, the apparent EC
values observed 20 and 150
min after cAMP removal and incubation at 4 °C are 30 and 140
n
M, respectively. We also assessed the
concentration-dependence of LLB at the 20- and 150-min time points. The
EC
values for LLB increased with time and closely
paralleled those of the mobility shift (Fig. 6 B).
Figure 6:
Concentration-dependence of wild-type cAR1
mobility shift and LLB. A, cells expressing wild-type cAR1
were treated 15 min with a range of cAMP concentrations (0 to
10
M) in the presence of caffeine and DTT.
Cells treated at concentrations below 10
M were washed twice with ice-cold PB containing DTT and the same
concentration of cAMP. Cells treated with 10
M cAMP and above were washed twice with ice-cold PB containing
10
M cAMP and DTT. All cells were then
washed twice with ice-cold PB alone, resuspended to 10
/ml,
and incubated at 4 °C with shaking (200 rpm) for an additional 130
min. Cells were combined with sample buffer immediately after the
incubation with cAMP ( filled triangles) and at 20 min
( open circles) or 150 min ( filled circles) after the
initiation of the third wash. Samples were subjected to SDS-PAGE on 10%
low bisacrylamide gels, immunoblotted with cAR1-specific antiserum, and
the fraction of receptor in the lower mobility form determined as
described under ``Materials and Methods.'' These values were
then normalized to the percentage of shifting observed at 20 min in
cells treated with 10
M cAMP (90%). Data
shown represent the mean ± range of two independent experiments.
B, binding to 10 n
M [
H]cAMP was
assessed for the cells described in Panel A, 20 min ( open
squares) or 150 min ( filled squares) after the initiation
of the third wash. Binding, measured by the silicone oil method, was
normalized to that of untreated cells. In order to allow a comparison
between LLB and the mobility shift ( dashed lines, reproduced
from Panel A), binding data were expressed as a percentage of
LLB observed at the 20 min time point in cells treated with
10
M cAMP (86%). Data shown represent the
mean ± range from two independent experiments performed in
triplicate. The 20 min LLB data for these two experiments have
previously been presented elsewhere (32).
Lastly, we examined the relationship between the electrophoretic
mobility shift and LLB of wild-type cAR1, by evaluating the kinetics of
both processes. As shown in Fig. 7, the kinetics of the
electrophoretic mobility shift and LLB, induced by 10
M cAMP and assessed immediately after cAMP removal, were
nearly identical. These findings, taken together with the results of
the concentration-dependence experiments, extend the correlation
observed between LLB and phosphorylation in cAR1 mutants to the
wild-type receptor.
Figure 7:
Kinetics of wild-type cAR1 mobility shift
and LLB. Kinetics of LLB ( open circles) and the mobility shift
( filled circles). Washed, transformed cells expressing
wild-type cAR1 were incubated without or with cAMP (10
M, 15 min) washed four times with PB, and resuspended to
10
/ml. The percentage of receptor in the lower mobility
form and the percentage of control binding lost upon cAMP pretreatment
were simultaneously determined as in Fig. 6. To allow a direct
comparison of kinetics, data were normalized to those values obtained
for cells exposed to a 15 min cAMP stimulus (81% LLB, 92% mobility
shift). Data shown represent the means ± range from two
experiments performed in triplicate. The LLB data from these
experiments have previously been reported elsewhere
(32).
, Ser
, Ser
,
Ser
, and Ser
, accounts for two-thirds of
the total and causes a marked decrease in cAR1's electrophoretic
mobility
(15) . In this report, we show that the lower
electrophoretic mobility form can result from the phosphorylation of
either Ser
or Ser
. Presumably, one or both
of these two serine residues are targets of phosphorylation in the
wild-type receptor.
yielded
a mutant, mut15, which exhibited the electrophoretic mobility shift but
virtually no LLB. Careful examination, however, revealed that the lower
mobility form of this mutant is abnormally unstable. When cells were
washed free of ligand and incubated on ice, this receptor returned to
the higher mobility form with a half-life of several minutes or less
compared with hours for the wild-type receptor. Consequently, this and
other similar mutant receptors are found predominantly in the higher
mobility form at the time of LLB assay. The discovery of this
instability prompted us to re-examine receptors with mutations in the
third cytoplasmic loop that exhibit impaired LLB
(16) . Some of
these mutants also displayed unstable mobility-shifted forms ( e.g. A53). When these instabilities are taken into account, the extent
of LLB is significantly correlated with the fraction of receptors in
the lower mobility form at the time of LLB assay for all mutants
examined. Dose-dependence and kinetics studies, presented in this
report, revealed a similar correlation for the wild-type receptor.
)-cAMPS induces LLB without
causing the electrophoretic shift
(26) . Still, the LLB induced
by ( R
)-cAMPS is unstable (reversing within minutes
at 4 °C),
(
)
suggesting that phosphorylation
might stabilize the low affinity state. Interestingly, the G
protein-coupled receptor kinase GRK2 has been shown to facilitate the
sequestration of mammalian m2 muscarinic receptors
(3) ,
suggesting that similar relationships between receptor phosphorylation
and agonist binding alterations may exist in other systems.
10
M) induce the electrophoretic mobility shift. As
described above, phosphorylation of either Ser
or
Ser
could be sufficient to produce the shift. Upon cAMP
removal, however, receptors phosphorylated under these conditions are
rapidly dephosphorylated and return to the unshifted state with a
t
of
20 min at 4 °C. Saturating cAMP
concentrations (>10
M), in addition to
causing the shift, trigger a process which dramatically increases the
stability of the shifted form ( t
of several
hours at 4 °C). Assuming that the involved kinase ceases to
phosphorylate the receptor after cAMP is removed, this stabilizing
process must reflect either inactivation of the involved phosphatase or
a change in the shifted receptor which impedes its dephosphorylation.
We suspect the latter possibility is the case and that stabilization of
shifted receptors might be the consequence of further phosphorylation
of the receptor, beyond that required to cause the shift. Consistent
with this possibility,
P-labeling experiments have shown
that the specific radioactivity of shifted receptors increases
2-fold during a response to saturating cAMP
(11) .
Furthermore, we have previously shown that cAMP occupancy triggers the
addition of up to 2 or 3 phosphates to cluster 1 serines
(15) .
-adrenergic receptor kinase and rhodopsin kinase, the kinase
responsible for cAR1 phosphorylation is activated by interaction with
the receptor's intracellular loop domains
(27, 28, 29) .
-adrenergic receptors must be internalized in order to
be dephosphorylated
(7, 30) . This appears not to be the
case for cAR1 since we observed dephosphorylation at 4 °C, a
temperature which should preclude vesicular traffic. Furthermore, under
the conditions of our experiments, prior internalization is unlikely as
nearly all cAR1 molecules remain on the cell surface following cAMP
treatment
(32) .
Table:
Definition of cAR1 cluster 1 mutants
)-cAMPS, cyclic adenosine
3`,5`-monophosphorothioate, ( R
) - isomer; PAGE,
polyacrylamide gel electrophoresis; LLB, loss of ligand binding.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.