From the Department of Biological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
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Agonist-induced phosphorylation of
G-protein-coupled receptors has been shown to facilitate the
desensitization processes, such as receptor internalization, decreased
efficiency of coupling to G-proteins, or decreased ligand affinity. The
lowered affinity may be an intrinsic property of the phosphorylated
receptor or it may be the result of altered interactions between the
modified receptor and downstream components such as G-proteins or
arrestins. To address this issue, we purified cAR1, the major
chemoattractant receptor of Dictyostelium discoideum by a
strategy that is independent of the ligand binding capacity of the
receptor. To our knowledge, this represents the first successful
purification of a chemoattractant receptor. The hexyl-histidine-tagged
receptor was solubilized from a highly enriched plasma membrane
preparation and purified by Ni2+-chelating chromatography.
The protocol offers a simple way to purify 100-500 µg of a G-protein
coupled receptor that can be targeted to the plasma membrane of
D. discoideum. The Kd value for the
purified cAR1 was about 200 nM, consistent with that of
receptors that are not coupled to G-proteins in intact cells. In
contrast, the affinity of phosphorylated cAR1, purified from
desensitized cells, was about three times lower. Treatment of the
phosphorylated receptor with protein phosphatases caused dephosphorylation and parallel restoration of higher affinity. We
propose that ligand-induced phosphorylation of G-protein-coupled receptors causes a decrease in intrinsic affinity and may be useful in
maintaining the receptor's sensitivity at high agonist levels. This
affinity decrease may precede other processes such as receptor internalization or uncoupling from G-proteins.
G-protein-coupled receptors
(GPCRs)1 are involved in a
wide variety of important biological processes including vision,
olfaction, chemotaxis, and immune response. It is remarkable that the
receptors for such diverse stimuli all share the same topological
feature of seven-membrane spanning segments. It is believed that these segments cluster to form the binding pocket. Upon agonist binding, the
receptors undergo conformational changes, activating intracellularly coupled G-proteins, which proceed to interact with the downstream effectors (1). Despite extensive pharmacological studies on certain
representative GPCRs, detailed biochemical and biophysical characterization of most of these receptors is still lacking. With the
exception of rhodopsin, the extreme difficulties in purifying most
GPCRs have hindered studies of the structures and functions of these proteins.
A physiologically important property of GPCRs is their tendency to
desensitize during exposure to agonist. Desensitization mechanisms
include "down-regulation" or reduction of receptor number,
"sequestration" or apparent shielding of the receptors from
interacting ligands, and "uncoupling" from G-proteins.
Agonist-induced receptor phosphorylation, usually carried out by
G-protein-coupled receptor kinases, may contribute to each of these
processes. In the case of the To investigate the intrinsic properties of a phosphorylated GPCR, we
purified cAR1, the major chemoattractant receptor of the social amebae,
Dictyostelium discoideum. cAR1 is coupled to the
heterotrimeric G-protein G2, which transmits the activation signal
downstream to mediate actin polymerization, chemotaxis, calcium uptake,
cell-cell signaling, and differentiation (9). We developed a
purification protocol which, unlike previous GPCR purification schemes,
does not rely on the ligand binding capacity of the receptor. In this
procedure, a specialized plasma membrane subdomain highly enriched in
receptor was isolated (10). After detergent extraction, the solubilized
receptor was applied to a Ni2+ column and purified in a
single step. Tens of micrograms of purified, active cAR1 are obtained
from a liter of cell culture. The agonist affinity of the final
purified receptor is similar to that of the binding sites on cells
lacking G-proteins. To our knowledge, this represents the first
successful attempt to purify a chemoattractant receptor to near
homogeneity. In principle, this protocol can be extended to the
purification of any GPCR that can be targeted to the plasma membrane of
D. discoideum cells.
Using this purification procedure, we have found that the lower
affinity displayed by phosphorylated receptors after agonist pretreatment in vivo is an intrinsic property of the
modified proteins. When this phosphorylation was blocked by
substituting serines 303 and 304, the major phosphorylation sites of
cAR1, with alanine and glycine, the mutant receptor failed to display lowered affinity after similar agonist pretreatment (5). Additionally, protein phosphatase treatment of the phosphorylated receptor led to its
dephosphorylation and a corresponding enhanced ligand affinity. This
suggests that receptor phosphorylation itself, independent of other
interacting components or downstream processes, may directly contribute
to the desensitization.
Vectors and Constructs--
C-terminal hexyl-histidine-tagged
cAR1 constructs were created by polymerase chain reaction. The
N-terminal primer,
GCCGGAAGATCTTATTAAAAAATGGGTCTTTTAGATGGAAATC, contains a BglII site (first underline) at the 5' end,
followed by the Dictyostelium consensus ribosomal binding
site (italicized) and the N-terminal residues of cAR1 (second
underline). The C-terminal primer,
CGAGGCGTAGCTAGCTGGTGGATTATTTCCTTGACCATTTGTTGCA,
contains the last six residues of cAR1 sequence (italicized),
followed by two prolines (underlined) and a NheI site. Two
constructs were made to create the hexyl-histidine-tagged wild-type
cAR1 (abbreviated as cAR1-H6), the wild-type cDNA sequence of cAR1
was used as template. To create hexyl-histidine-tagged
non-phosphorylatable form of cAR1 (abbreviated as cm1234-H6), a mutant
cAR1 sequence in which all C-terminal serine and threonine residues
were substituted (cm1234, Ref. 12) was used. A modified pBluescript
(Stratagene) containing a hexyl-histidine tag was created as follows:
the EcoRV site was replaced with a BglII site.
After digestion with BglII and BamHI, a
double-stranded filler fragment containing sequentially BglII and NheI sites followed by a six-histidine
sequence and an in-frame stop codon and a BamHI site was
digested with BglII and BamHI and then cloned
into the modified vector through BamHI and BglII
sites. The sequence for the top strand of the filler (from 5' to 3') is
as follows: GATCTCGCTCTGCTAGCCACCATCACCATCACCACTAATAAG. The polymerase
chain reaction products were gel-purified, digested with
BglII and NheI, and then cloned into compatible
sites of the above mentioned pBluescript. The final fragments
containing the assembled cAR1 followed by a six-histidine tag and stop
codon was released by BglII/BamHI digestion and
cloned into pB18, a D. discoideum integration expression
vector; or released by BglII/NotI digestion and
cloned into pMC34, an extra-chromosomal expression vector. Under both
conditions the tagged sequence is downstream of the D. discoideum actin 15 promotor and expressed throughout the growth
and developmental stages.
Cell Lines and Growth Conditions--
The expression vectors
encoding His-tagged cAR1 were transformed into
car1 Intact Cell Binding Assays--
cAMP binding of intact cells in
either phosphate buffer or in saturated ammonium sulfate was carried
out essentially as described (12). Briefly, for assay in phosphate
buffer, 100 µl of cells at 108 cells/ml were incubated
with various concentrations of [3H]cAMP (NEN Life Science
Products Inc.) at 4 oC for 3 min and centrifuged at
12,000 × g for 2 min to remove unbound ligands. For
ammonium sulfate assay, cells were suspended in saturated ammonium
sulfate and [3H]cAMP was added. One wash with saturated
ammonium sulfate was carried out. Background binding was obtained by
adding unlabeled cAMP.
CHIFF Preparation--
CHIFF was prepared as described
previously (10). Briefly, cells were harvested and washed once in DB
(10). The cell pellet was resuspended in DB to 5 × 107/ml and shaken at 120 rpm at 22 oC for 4-6
h. Cells were harvested again and washed with TEB (40 mM
Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM EDTA). They
were finally resuspended in TEBP (TEB plus various protease inhibitor)
to 2 × 108/ml. CHAPS powder was added at 20 mg/ml.
After gentle mixing, the lysed cells were kept on ice for 3-5 min and
then sucrose crystals were added to 55% final concentration. They were
loaded under a 20-45% step sucrose density gradient and centrifuged
at 150,000 × g for 12 h. The CHIFF band was
collected and washed once, then resuspended to 5 × 108 cell equivalent/ml in TBP (TEBP without the EDTA) with
30% sucrose and then stored frozen at Purification of Hexyl-histidine-tagged cAR1 Over
Ni2+-chelating Sepharose Column--
CHIFF was thawed and
solubilized in 1-2% Lubrol PX for 2-4 h with mixing at
4 oC at a cell equivalent density of 3-4 × 108/ml. After centrifugation at 100,000 × g for 30 min, the supernatant was recovered. NaCl and
immidazole were added at final concentrations of 200 and 2 mM, respectively. About 40 ml of this supernatant was first
batch incubated with 0.6 ml (sedimented volume) of metal-chelating Sepharose resin (Pharmacia) precharged with 50 mM
Ni2+ according to the manufacturer's suggestion. After
2-4 h incubation, the resin was spun down and loaded into a 5-ml
Bio-Rad econocolumn. The supernatant was further absorbed over the
column by gravity flow (for about 1 h). After washing the column
with more than 20 ml of wash buffer (40 mM Tris-HCl, pH
7.5, 200 mM NaCl, 2 mM imidazole, and 0.1%
Lubrol-PX), the column was further washed with buffer containing 15 mM nonylglucoside (40 mM Tris, 200 mM NaCl, 2 mM imidazole, and 15 mM
nonylglucoside) to exchange the original Lubrol. This was found to be
necessary since our result showed that nonylglucoside preserved
receptor binding activity more consistently than Lubrol-PX. cAR1 was
eluted in four steps of increasing concentrations of imidazole: 25, 50, 100, and 250 mM. Each step consisted of 4 column volumes of
elution solution.
Preparation of Basalated and Maximally Phosphorylated
Hexyl-histidine-tagged cAR1--
The procedure was essentially as
described (5). cAR1-H6 cells suspended in DB were shaken at 200 rpm for
20 min with either 4 mM caffeine or 10 mM
dithiothreitol plus 10 cAMP-binding Analysis of CHIFF and Purified cAR1--
The major
cAR1 fractions of Ni2+ column elutions were pooled and
further characterized for cAMP binding through equilibrium dialysis
(13). The binding reaction typically contained 200 ng of pure protein,
15 mM nonylglucoside, 40 mM Tris-HCl, 2 mg/ml bovine serum albumin, and 10% sucrose. For background controls, 1,000-fold excess unlabeled cAMP was added to both sides of the chamber, the differential between the two sides was taken as the background. For CHIFF binding, no detergent was present and 5-10 mM dithiothreitol was added to inhibit phosphodiesterase
activity. Usually the reaction was stopped after 6-8 h of mild rocking
as this time was tested to be sufficient for equilibrium establishment. Ten different [3H]cAMP concentrations in the range of
1-5,000 nM were used to generate the binding curve.
Computer modeling program ORIGIN (Microcal Software, Inc., Northampton,
MA) was employed to fit the binding data and obtain the number of
affinity states and value of dissociation constant
(Kd). Other binding assays, such as spin column assay and a polyethylene glycol precipitation assay, also confirmed the
activity of the receptor preparations. But these methods were not
satisfactory for quantitative analysis. The equilibrium dialysis method
proved to be the most reproducible method. We confirmed the integrity
of cAMP throughout the incubation process by monitoring its
chromatographic behavior. Little phosphodiesterase activity was
detected in the purified preparations of receptor.
Dephosphorylation of Phosphorylated cAR1--
CHIFF prepared
from desensitized cells were resuspended in 1 × NEB III buffer at
a cell equivalent density of 1-2 × 109/ml. 2 units
of alkaline phosphatase (New England Biolabs) was added and
dephosphorylation carried out at 37 °C for 20-30 min. Gel Shifting
assay indicated that this treatment was sufficient to dephosphorylate
80-90% of cAR1. The CHIFF membrane was recovered by centrifugation
and resuspended in TEB for cAMP binding assay.
C-terminal Hexyl-histidine-tagged cAR1s Are Fully
Functional--
Several lines of evidence indicated that the
C-terminal hexyl-histidine fusion did not interfere with the functional
properties of the receptor. To avoid any potential receptor
heterogeneity caused by variable extents of phosphorylation, we
initially used a hexyl-histidine-tagged cAR1 in which all serines and
threonines in the C-terminal domain were substituted (cm1234-H6). After
transformation into
car1-/car3
We have previously shown that wild-type cAR1 is quantitatively
localized to a specialized plasma membrane fraction designated CHIFF
(10). Our strategy was to use CHIFF as an intermediate purification
step, providing a 500-fold enrichment, and then to solubilize and
purify the hexyl-histidine-tagged receptor by metal affinity column
chromatography. We first determined that the tagged receptor was also
enriched in CHIFF and its activity remained intact. Our results
indicated that cm1234-H6 displayed the same localization in CHIFF as
wild-type cAR1 (data not shown), showing that the histidine tag does
not interfere with the normal targeting of receptor to the specialized
plasma membrane domains.
The ligand binding activity of CHIFF membranes was characterized by
equilibrium dialysis. Computer analysis indicated that a single
affinity form was present, with a Kd of 250 ± 65 nM (Fig. 1C). This value was close to the
Kd of the predominant receptor population at the
cell surface (Kd = 235 nM, Fig.
1A) and indicated that the agonist binding sites remain
essentially unchanged during the CHIFF preparation process.
Solubilization and Purification of Hexyl-histidine-tagged cAR1 by
Ni2+ Column Chromatography--
cm1234-H6 cAR1 was
solubilized and purified to near homogeneity by chromatography on a
Ni2+-chelating column. We have previously shown that CHIFF
proteins can be efficiently extracted by the non-ionic detergent
Lubrol-PX (9). We resuspended the CHIFF sample to a density of
2-4 × 108 cell equivalents/ml and added 1%
Lubrol-PX. It is crucial to carry out solubilization at this density;
at higher densities, CHIFF fragments tend to aggregate, preventing
efficient solubilization. Under these conditions, more than 90% of the
receptor was typically extracted. The solubilized fraction was
supplemented with NaCl and imidazole to prevent nonspecific absorption
and then chromatographed over the Ni2+ column. After
extensive washes, the bound receptor was eluted with four steps of
increasing imidazole concentrations: 25, 50, 100, and 250 mM (Fig. 2). cAR1 emerged
from the column in two peaks, about 25% eluted at 100 mM
and the remainder eluted at 250 mM. This ratio varied
slightly between experiments. The purified receptor displayed two
major molecular weight forms according to gel migration positions,
corresponding to a monomeric (indicated by "M") and a dimeric form
(indicated by "D"). The second peak, emerging from the column at
250 mM imidazole, was enriched in the dimer form.
Samples taken from the three stages of the purification process were
examined by SDS-PAGE followed by silver staining (Fig. 2C).
Comparisons of protein profiles between intact cell, CHIFF, and final
purified samples revealed a significant purification. Gel scanning
showed that in both peaks eluted from the column the purity of receptor
was over 80%. Latter experiments suggested that the purity could be
further enhanced by prolonging the 25 mM wash step (data
not shown).
cAMP Binding Activity and Receptor Protein Co-fractionates on
Ni2+ Column--
Next we determined whether the
column-purified receptor retained cAMP binding activity. We assayed the
cAMP binding activity of each column fraction by equilibrium dialysis.
Specific bindings were obtained from these fractions (Fig.
3A, hatched bars). Comparison between this profile and the previous cAR1 protein elution profile (Fig. 3A, inset) clearly demonstrated a direct
correspondence between the levels of binding activity and receptor
protein, suggesting that the purified cm1234-H6 is responsible for the
observed binding.
Purified Receptors in Detergent Solution Display a Similar cAMP
Affinity as Receptors on Cell Surface--
Having established that the
purified receptor is still active, we quantitatively characterized its
affinity by equilibrium dialysis. For these studies, we pooled both
elution peaks containing cAR1, representing over 90% of the eluted
receptors (Fig. 2A, fractions 9-15). Specific binding data
at 10 different cAMP concentrations were obtained. The curve was best
fit by a single affinity state with a dissociation constant of 188 ± 39 nM (Fig. 3B). This value is similar to
that of cAR1 on CHIFF membranes and on the surface of cells lacking
G-proteins (22). These data suggest that the purified preparation
consists of a homogeneous population of receptors not coupled to
G-proteins.
The data from a representative purification were tabulated (Table
I) to illustrate the recoveries of
protein, [3H]cAMP binding activity and changes in
specific activity in this experiment. Step 1, which involves purifying
CHIFF from cells, yielded approximately 50% of the receptor protein
and 60% of cAMP binding activity. Since the CHIFF fraction contains
less than 0.2% of the cellular protein, the specific activity of
receptors increased nearly 500-fold during this step. Step 2, the
purification of receptors from solubilized CHIFF, yielded 40%
recoveries of both receptor protein and cAMP binding activity. The
cumulative recoveries for the whole procedure were about 18% for the
protein and 25% for binding activity. The overall fold of purification was about 7,000. The specific activity of the final purified sample was
about 3 × 104 pmol/mg, corresponding to 1.8 × 1016 sites/mg. The theoretical specific activity of pure
cAR1 is calculated to be 1.6 × 1016 sites/mg,
assuming one binding site per cAR1 molecule. These data suggest that
the purified receptors in detergent solution are fully active, although
these calculations were based on several measurements which all have
margins of error. We routinely purify about 10-20 µg of pure active
receptor from 1010 cells, which corresponds to 1 liter of
axenic culture. We can conveniently grow and harvest 30 liters of
cells, which would correspond to about half a milligram of purified
cAR1. The current limitation in the procedure is the requirement of a
separate ultracentrifugation run to purify CHIFF from each 5 liters of
culture.
The cAMP Binding as Measured by Equilibrium Dialysis Depends on
Native Receptors--
To control for the specificity of the
equilibrium dialysis binding assay, we performed several experiments.
Purified receptors were divided into three equal sets: the first was
not treated, the second was heat-denatured (95 °C for 5 min), and
the third was treated with 1% SDS at room temperature for 10 min. The
three sets were then assayed in parallel for [3H]cAMP
binding. As shown in Fig. 3C, only the first set displayed binding activity. The other two sets showed only very low activity, indicating that the observed binding was due to a native protein.
We carried out a further test to confirm that the binding activity was
specifically due to the receptor. We used parallel sets of cells,
overexpressing two versions of cAR1 which did or did not contain the
hexyl-histidine tag. CHIFF were prepared from both cell lines and then
both samples were solubilized and chromatographed on Ni2+
columns. Presumably both receptors would fractionate with CHIFF, but
only the hexyl-histidine-tagged receptor will be retained and purified
by the column. At each stage, cAMP binding was assayed by equilibrium
dialysis (Fig. 3D). At the CHIFF stage (1 and 2) both
cell lines displayed comparable specific binding; while at the purified
stage (3 and 4), only the His-tagged receptor preparation displayed
binding. The immunoblot showed that CHIFFs from both cell lines
contained cAR1 (inset, lanes 1 and 2), but only
the hexyl-histidine-tagged receptor was purified (inset, lanes
3 and 4), demonstrating that cAMP binding was
specifically due to purified receptor.
We also carried out an additional experiment to further quantitate the
step from CHIFF to purified receptor. We prepared CHIFF from cm1234-H6
cells and divided it into two equal aliquots. The first was saved
(CHIFF) while the second was subjected to extraction and purification
(purified). Equal fractions of each sample were assayed both for cAMP
binding by equilibrium dialysis and by immunoblotting to quantitate the
recovery of binding activity and receptor protein, respectively. For
immunoblotting, a dilution series of four decreasing concentrations of
samples were applied to better assess the recovery. As shown in
Fig. 3E, the recoveries of binding activity and receptor protein were about 78 and 69%, respectively. This observation indicates that there was no significant inactivation of the receptor during the solubilization and purification process.
Phosphorylated Receptors Have an Intrinsically Lower Affinity for
[3H]cAMP--
To address whether the lowered affinity of
phosphorylated receptors on the cell surface (5) was intrinsic to the
modified protein or due to interactions with other proteins, we
purified the unphosphorylated and phosphorylated forms of receptors in parallel. We used the cell line expressing hexyl-histidine-tagged wild-type receptor (cAR1-H6). The cells were pretreated with excess cAMP to induce maximal phosphorylation or with caffeine to ensure complete dephosphorylation of the receptor ("Experimental
Procedures"). Binding assays were performed on these two sets of
cells to determine their respective affinities. As shown in Fig.
4A, consistent with previous
studies, the cAMP pretreated set displayed a decreased affinity
compared with the untreated control set (Kd for
pretreated cells: 728 ± 76 nM; Kd
for untreated cells: 220 ± 60 nM). The total number
of binding sites was the same for both sets of cells. These
observations also demonstrated that hexyl-histidine tagging did not
interfere with the normal desensitization properties of receptor.
It has been previously shown that upon detergent lysis, cAR1 from
cAMP-pretreated cells was stably maintained in its phosphorylated state, and phosphorylation did not alter the localization of receptor to the CHIFF fraction (10). Thus we were able to use CHIFF to purify
both forms of receptors. CHIFF preparations were obtained for both
untreated and cAMP-pretreated cells and tested for ligand binding by
equilibrium dialysis (Fig. 4B). The two forms of cAR1-H6 essentially retained the same affinities as in intact cells. The 3-fold
difference in affinities between the two forms persisted; the CHIFF
preparation from cAMP-pretreated or resting cells displayed Kd values of 812 ± 69 and 279 ± 50 nM, respectively. We have shown previously that G-proteins
are absent from CHIFF (10). This indicates that the lowered affinity of
phosphorylated receptors could not be due to their
uncoupling from G-proteins.
Subsequently, the two forms of receptors were extracted from CHIFFs and
purified in parallel over Ni2+ columns. Similar
fractionation profiles were obtained for both forms and were
essentially the same as for the cm1234-H6 receptors except that the
dimer form was less abundant. The pure fractions were pooled, analyzed
by SDS-PAGE, and silver stained (Fig.
5A). The phosphorylated form
displayed a lower mobility, consistent with previous results (12),
indicating that the extent of phosphorylation was fully preserved
during the purification. The ligand binding properties of these
preparations were characterized by equilibrium dialysis (Fig.
5B). Computer fitting yielded predominantly a single site
for both forms. The Kd values were essentially the same as those in intact cells and CHIFF. The purified phosphorylated receptors displayed a Kd of 882 ± 70 nM, three times higher than the unmodified receptors which
had a Kd of 249 ± 63 nM. The
Kd values for both forms of purified receptors
closely reflect their original cell surface values, suggesting that the
decreased ligand affinity of phosphorylated cAR1 is an intrinsic
property of the receptor itself and does not require associated
proteins. The receptor affinities during the purification process are
summarized in Table II.
Receptor Dephosphorylation Enhanced Its Ligand Affinity--
To
demonstrate that phosphorylation, not other possible concurring
modifications, directly accounts for the lowered affinity of the
phosphorylated receptor, we dephosphorylated the receptor with
different protein phosphatases and then determined whether removal of
phosphates lead to enhanced receptor affinity. Various phosphatases,
including protein phosphatase 1,
[3H]cAMP binding assays were performed on the alkaline
phosphatase-treated samples in comparison with untreated control
samples (Fig. 6B). In one experiment, untreated controls
showed a ligand binding of 17,000 cpm, the mock-treated controls showed
somewhat lowered binding at 8,700 cpm, indicating that incubation of
CHIFF at 37 °C for 20 min lead to partial reduction in cAMP binding. The samples treated with alkaline phosphatase displayed an enhanced binding of 42,000 cpm, 2-3-fold higher than the untreated control. The
real increase may be even higher since 37 °C incubation leads to
partial reduction of receptor ligand binding.
Our current study demonstrates that lowered affinity of receptor
induced by agonist pretreatment is an intrinsic property of the
phosphorylated receptor. That is, phosphorylation within the C-terminal
domain of the receptor (at serines 303 and 304) imposes a lower
affinity. Removal of these phosphate groups caused a 2-fold increase in
cAMP binding capacity. This in vitro enhancement is
consistent with the observed affinity difference between
unphosphorylated and phosphorylated receptor. These results argue that
phosphorylation, rather than other putative accompanying modifications,
directly causes the decreased affinity. This suggests that
phosphorylation per se could directly lead to receptor
desensitization through reduced ligand occupancy.
It has been proposed for other GPCRs that the ligand-binding site
resides within a pocket formed by the seven hydrophobic transmembrane
segments (1). To explain the relatively higher affinity of the basal
form of receptor and the lower affinity of the phosphorylated receptor,
we propose that under resting conditions, there is minimal contact
between the C-terminal domain and intracellular loops of the receptor
(which underline the ligand binding pocket). After agonist binding and
phosphorylation, the C-terminal domain may undergo a conformational
change and interact with the loop regions, thus imposing the lowered
affinity. Although other models are possible, two lines of observation
favor this proposal. First, truncation of the C-terminal domain yields
a receptor with high affinity that is resistant to agonist-induced affinity reduction (14), suggesting that the higher affinity of
unphosphorylated receptor does not require the C-terminal domain. Second, the current study demonstrates that completely purified phosphorylated receptors display the lowered affinity, ruling out the
need for interacting proteins in this process.
Lowered affinity displayed by phosphorylated GPCRs has not been widely
recognized as a major cause of desensitization. In in vitro
reconstitution studies with unphosphorylated or phosphorylated receptors, the phosphorylated receptors were found to have reduced coupling to G-protein (15), but comparisons between the affinities of
two forms of receptors were typically not carried out. In in vivo studies, receptor internalization, sequestration, or
down-regulation often occur during or immediately after receptor
phosphorylation, thus making direct assessment of receptor affinity
after phosphorylation impractical. Hence, the processes such as
internalization or sequestration are usually considered the major
contributing factors to desensitization while altered affinity is not.
Possible decreases in receptor affinity can only be observed in
situations where internalization/sequestration is delayed or does not
occur. Alternatively, the phosphorylated and unphosphorylated receptors
can be purified so their affinities can be directly compared. We
propose that such unrecognized affinity decreases may be a typical
consequence of GPCR phosphorylation and could directly contribute to
receptor desensitization.
The purification scheme we have developed is in principle applicable to
any GPCR or membrane protein that can be expressed in D. discoideum as long as that protein is quantitatively enriched in
the minor detergent-resistant plasma membrane fraction, CHIFF. The
CHIFF purification step provides a 500-fold purification, which makes
the the subsequent fractionation on the Ni2+ column very
effective. Direct loading of whole cell extracts on the column yielded
little purification (data not shown). Purification schemes for other
GPCRs have often included ligand affinity chromatography as a major
enrichment step (16-18). In contrast, our procedure is independent of
ligand binding. We required such a scheme since we wished to purify
various forms of receptors, including those with decreased binding
activities. In the current study we have used it to purify the
phosphorylated and unphosphorylated forms of cAR1 even though they have
different affinities. Furthermore, we have constructed many mutant
forms of cAR1 which are specifically deficient in ligand binding,
G-protein coupling, signal transduction, or desensitization and they
have been analyzed in vivo (12). This purification scheme
which is applicable to both wild-type receptor and all the mutant forms
will enable us to dissect the structural differences between these
mutants and identify the structural determinants for each property of
the receptor.
It has been reported that certain GPCRs display dimeric and higher
oligomeric forms when solubilized in non-ionic detergents and run on
SDS-PAGE (19). We often detected cAR1 dimer on SDS-PAGE in both whole
cell extracts and plasma membrane samples. The dimers are not formed
through disulfide bonds since they are resistant to 100 mM
dithiothreitol treatment (data not shown). Since the dimers may be only
partially resistant to SDS, we speculate that the apparent dimers on
SDS-PAGE represent a residual portion of the dimers originally present
in non-ionic detergents. Several lines of evidence further confirm the
dimerization of cAR1 in non-ionic detergents. First, cAR1 solubilized
in non-ionic detergents migrates as two forms on sucrose velocity
gradients (data not shown). The more rapidly sedimenting form contained
a higher proportion of apparent dimers on SDS-PAGE. Second, unlike the
monomers which eluted at both 100 and 250 mM imidazole
concentrations, the apparent dimers only eluted at the 250 mM imidazole range in the Ni2+ column profile
(Fig. 2), consistent with the anticipated tighter binding two histidine
tags would confer. Third, we performed the following
co-immunoprecipitation test to confirm the presence of receptor dimers
(data not shown). A cAR1-GFP (green fluorescence protein, Ref. 20)
fusion construct was transformed into wild-type cells such that the
cells would co-express both wild-type cAR1 and cAR1-GFP fusion
proteins. Detergent extracts from these cells were prepared and
immunoprecipitated with anti-GFP antibody. Wild-type cAR1 as well as
cAR1-GFP was found in the final immunoprecipitate, demonstrating the
existence of mixed dimers. It is difficult to determine the
physiological relevence of dimerization from biochemical analysis. Our
results suggest that the dimer and monomer forms do not differ
dramatically in ligand affinity (since only one affinity form was
present for the whole preparation). Further analysis will require a
complete purification of each of the two forms.
In D. discoideum, many cAR1-mediated responses desensitize,
and their time courses closely reflect that of receptor
phosphorylation. Conversely, the resensitization of these processes
closely follows the dephosphorylation of cAR1 (14). This suggests that
receptor phosphorylation is the central step in desensitization.
However, we have shown definitively that receptor phosphorylation is
not essential for termination of receptor-mediated
responses. Cells expressing only cm1234, the non-phosphorylatable form
of cAR1, display the normal kinetics in attenuation of adenylate
cyclase and other cAR1-mediated responses. This observation indicates that a mechanism(s) other than receptor phosphorylation operates to
terminate multiple receptor-mediated pathways. However, the cells
expressing cm1234 do recover more quickly from adaptation after removal
of the stimulus, suggesting that both
phosphorylation-dependent and independent mechanisms are
functioning redundantly in mediating adaptation.4
Although the phosphorylated receptors have a lower affinity for
agonist, they are not necessarily signaling deficient. On the contrary,
we speculate that this affinity decrease may be important for fine
tuning the responses that chemoattractant receptors mediate. As cells
initially move toward a source of attractant, the attractant
concentration is low, so the higher affinity of the unmodified
receptors is needed. As the cells approach the source, a higher ligand
concentration will be encountered. As the concentration nears
saturation level, cells carrying receptors that cannot be modified and
hence cannot assume a lower affinity will be unable to distinguish the
directionality of the gradient; whereas cells with receptors whose
affinity can be adjusted downwards by phosphorylation will be able to
detect a larger range of chemoattractant concentrations. In summary,
the agonist-induced phosphorylation of the chemoattractant receptor
which lowers affinity may be useful in maintaining the receptor's
sensitivity at high agonist levels.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
-adrenergic receptor and rhodopsin,
the two most extensively characterized GPCRs, phosphorylation is
proposed to promote the association of arrestins and subsequent
uncoupling from G-proteins and internalization (2-4). Desensitization
may also be accompanied by a lowered affinity of the phosphorylated receptor as in the cases of cAR1, angiotensin II receptor, D2 dopamine
receptor (5-7), and possibly the yeast phermone receptor (21). It has
been speculated that the lowered affinity is due to receptor
uncoupling from G-proteins and consequent coupling to arrestins, but
recent evidence suggests that receptor-arrestin complexes also display
high affinity (8). It is possible that agonist-induced
phosphorylation may lower the intrinsic affinity of the receptor.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
/car3
cells (12).
Clones were grown up and further characterized by cAMP binding assay.
The clone giving the highest binding was selected. Cells are maintained
in a Petri dish in HL-5 medium (12) plus 20 µg/ml G418. Cells are
washed off the plate into large axenic cultures and grown up by shaking
at 200 rpm at 22 oC to a density of 5-8 × 106/ml.
70o.
5 M cAMP to convert
cAR1 into either the basal (unphosphorylated) or desensitized
(phosphorylated) forms, respectively. The efficiency of this treatment
was monitored by analyzing the treated sample on SDS-PAGE and
visualizing positions of the cAR1 band on the gel by
immunoblotting. Basal state corresponded to a faster migrating form;
the phosphorylated state corresponded to a slower migrating form. The
cells were then lysed and processed for cAR1 purification through CHIFF
as detailed above.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
cells,
transformed clones that expressed 3-fold higher surface cAMP-binding
sites (3 × 105 sites/cell) than optimally developed
wild-type cells were isolated. As shown in Fig.
1A, under physiological
conditions in phosphate buffer, most of the receptors displayed an
average affinity of 200-300 nM (Kd = 235 ± 40 nM), and in the presence of ammonium
sulfate,2 the affinity
increased about 50-fold (Kd = 3.5 ± 0.3 nM). These values are essentially the same as those
previously reported for untagged receptors (11). To assess the
functional properties of the tagged receptor, we plated the
transformants on non-nutrient agar and observed the developmental
properties of the cells.3 As
shown in Fig. 1B, cm1234-H6 rescued the development of
car1-/car3
cells as
efficiently as the untagged cm1234, indicating that the C-terminal
hexyl-histidine does not interfere with the functions of receptor. As
expected, the vector transformed car1 -/car3
cells showed no development.
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Fig. 1.
A, hexyl-histidine-tagged receptor
displays the same cAMP-binding profiles as wild-type receptor in both
phosphate buffer and ammonium sulfate. cm1234 cAR1, tagged at the C
terminus with hexyl-histidine (cm1234-H6), was overexpressed in
cAR1-null cell line (RI9). Kd values in both
phosphate buffer (open circles) and ammonium sulfate
(solid circles) were assessed as described under
"Experimental Procedures." Ten different ligand concentrations were
used. The dissociation constant (Kd) values are
derived by computer modeling method (ORIGIN). For ammonium sulfate,
Kd was 3.5 ± 0.2 nM, for phosphate
buffer, Kd for the predominant population (>90%)
of receptors is 235 ± 42 nM. B,
histidine-tagged cAR1 rescues development of cAR1-null cells.
Vegetative state cm1234-H6 cells were washed in DB once and then plated
on non-nutrient agar (1). Untagged cm1234 cAR1-rescued RI9 cells (2)
and vector transformed RI9 cells (3) were also plated to offer positive
and negative controls. After 24 h, pictures were taken for each
plate to assess their developmental conditions. C,
histidine-tagged receptor on CHIFF displays similar cAMP binding
affinity as receptors on cell membranes. Purified CHIFF from His-tagged
cAR1 cells was isolated and resuspended in TB buffer. cAMP binding was
assayed by equilibrium dialysis. Ten cAMP concentrations were used to
create the binding curve (1, 2, 5, 10, 20, 50, 100, 200, 500 nM, 1 µM, and 5 µM). For each
point, about 2-3 µg of CHIFF was used which contains about 100 ng of
cAR1 (5% of total CHIFF proteins).
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Fig. 2.
Solubilization and purification of cm1234-H6
cAR1 over Ni2+ column. cm1234-H6 CHIFF preparation
were solubilized with Lubrol-PX and the extracts were loaded onto
Ni2+ chelating column. After extensive washes with 20-40
column volumes of binding buffer, bound receptors were eluted off the
column by four wash steps of increasing imidazole concentration (25, 50, 100, and 250 mM in binding buffer). Each step consists
of four column fractions. The elution was shown in both: A,
silver staining profile; and B, cAR1-immunoblotting profile.
Lanes 1-4, 25 mM elution; 5-8, 50 mM elution; 9-12, 100 mM elution;
and 13-15, 250 mM elution. C, sample
purities at different stage of purification (silver staining).
Lane 1, whole cells; 2, CHIFF; 3 and
4, purified receptor (two peaks). For lanes 1 and
2, same total proteins were loaded. Monomer and dimer forms
of cAR1 are indicated by "M" and "D,"
respectively.
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Fig. 3.
A, cofractionation of cAMP binding
activity with His-cAR1 over Ni2+ column. To establish that
the solubilized cAR1 is active, equilibrium dialysis binding was
performed on each of the Ni2+ column elution fractions
(Fig. 2A; see "Experimental Procedures" for binding
conditions). The final ligand concentration is 100 nM
[3H]cAMP for all fractions. Since fraction 13 displays
the highest level of cAMP binding, this fraction was given 100% value.
All the other fractions were quantified as a relative percentage value
of this fraction. Bindings were plotted as hatched columns.
The upper inset shows the corresponding cAR1 immunoblot
profile. Each lane was aligned with its respective column of binding.
B, ligand affinity of purified cm1234-H6 receptor. Final
purified His-tagged cAR1 was assayed for cAMP binding through
equilibrium dialysis. Ten different cAMP concentrations were used: 1, 2, 5, 10, 20, 50, 100, 500 nM and 1 and 5 µM.
Computer modeling was used to assess the Kd value at
188 ± 39 nM. C, specific cAMP binding of
purified receptor is sensitive to heat and SDS denaturations. Equal
amounts of purified His-cAR1 was directly used in the equilibrium
dialysis binding assay (Native), or first treated with
heating at 95 °C for 5 min (Heat), or treated with 1%
SDS for 10 min (SDS), and then used for the binding assay. Assay was
performed as previously and specific bindings were plotted.
D, the observed cAMP bindings is specifically due to the
receptor. CHIFF samples were prepared from WT cAR1
(non-histidine-tagged) expressing cells or hexyl-histidine-tagged
cAR1-expressing cells. They were solubilized and purified over
Ni2+ column. The purified samples and the original CHIFFs
were assayed for cAMP binding through equilibrium dialysis (bar
graph). 1, CHIFF with WT cAR1; 2, CHIFF with
His-cAR1; 3, column-purified sample from 1; 4, column-purified sample from 2. These four samples were also
loaded on SDS-PAGE for cAR1 immunoblotting (upper inset).
E, recoveries of receptor protein and binding activity
through the last purification step (purification of receptor from CHIFF
through Ni2+ column). CHIFF was prepared from His-cAR1
cells and divided into two equal aliquotes: the first one was saved on
ice (sample 1) while the second one was subjected to
extraction and purification of receptor (sample 2). Equal
fractions of samples 1 and 2 were assayed for cAMP binding (bar
graph) and cAR1 immunoblotting (upper inset) to compare
the recovery rates of activity and protein, respectively. For
immunoblotting, a dilution series consisting of four decreasing
concentrations (a, b, c, and d for sample 1; a', b', c', and d' for
sample 2) were used to better assess the recovery.
Two-step purification of cAR1
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Fig. 4.
A, ligand affinities of basalated and
desensitized cAR1-H6 cells. cAR1-H6 cells (hexyl-histidine-tagged WT
cAR1) were pretreated with caffeine or excess cAMP to basalate or
desensitize the receptors as under "Experimental Procedures." cAMP
binding was carried out for both sets as described before. 10-point
binding assay was performed to determine the binding curve and
Kd values were derived by computer fitting program:
220 ± 60 nM for basalated cells (predominant
population, open circles), 728 ± 76 nM for
desensitized cells (solid circles). B, ligand
binding profiles of CHIFF from basal and desensitized cAR1-H6 cells.
CHIFF from both basalated and desensitized cAR1-H6 cells were prepared
and assayed for ligand binding as described in the legend to Fig.
1C. Ten-point bindings were performed to determine the
respective Kd values: 279 ± 50 nM
for unphosphorylated CHIFF (open circles), 812 ± 69 nM for phosphorylated CHIFF (solid
circles).
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Fig. 5.
A, purification of cAR1-H6 receptor in
both unphosphorylated and phosphorylated form. cAR1-H6 cells were
treated before detergent lysis to induce receptor phosphorylation or
dephosphorylation(as in Fig. 4). The two forms of cAR1 were purified in
similar fashion from CHIFF over the Ni2+ column. The pooled
major elution fractions were shown for each form. Lane 1, unphosphorylated receptor; 2, phosphorylated receptor;
3 and 4, corresponding cAR1 immunoblotting
profile of 1 and 2. B, phosphorylated receptor
has intrinsically lower ligand affinity. The phosphorylated and
unphosphorylated forms of purified cAR1 from A were assayed
for cAMP affinities through equilibrium dialysis. Kd
values were derived from computer modelings: 249 ± 63 nM (open circle) for unphosphorylated receptor,
882 ± 70 nM for phosphorylated form (solid
circle). This experiment was repeated twice with consistent
findings.
Comparisons of cAMP affinity values of receptor at different stages of
purification
-phosphatase, and alkaline
phosphatase (calf intestinal) dephosphorylated cAR1 efficiently (Fig.
6A). Alkaline phosphatase
displayed the highest efficiency (lanes 10-13): a 10-min
incubation led to about 80% dephosphorylation according to the
electrophoretic mobility shifting assay, while protein phosphatase 1 and
-phosphatase required several hours to achieve the same result.
Control samples (mock-treated with buffer) incubated at 37 °C for 0 and 18 h showed minimal self-dephosphorylation (lanes 9 and 14).
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Fig. 6.
A, dephosphorylation of cAR1 by
different protein phosphatases. CHIFF with phosphorylated WT-cAR1 was
resuspended in appropriate buffers and treated for increasing times
with protein phosphatase 1 (PP-1: lanes 1, 0.5 h; 2, 1 h; 3, 2 h;
4, 18 h), -phosphatase (LP: lanes
5, 0.5 h; 6, 1 h; 7, 2 h;
8, 18 h), and alkaline phosphatase (AP: lanes
10, 15 min; 11, 30 min; 12, 1 h;
13, 2 h). Lanes 9 and 14 are
control samples treated with buffer at 37 °C for 0 and 18 h,
respectively. cAR1-P and Dimer-P indicate
phosphorylated cAR1 monomer and dimer, respectively. B,
dephosphorylation of cAR1 enhances its cAMP affinity. Phosphorylated
WT-cAR1 was dephosphorylated by alkaline phosphatase and tested for
cAMP-binding by equilibrium dialysis. Column 1, untreated
phosphorylated cAR1 control; 2, phosphorylated cAR1 treated
with buffer at 37 °C for 20 min; 3, phosphorylated cAR1
treated with alkaline phosphatase at 37 °C for 20 min.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
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ACKNOWLEDGEMENT |
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We thank Rachelle Gaudet for making the cm1234-H6 construct and cell line and Dr. Dale Hereld for stimulating discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM34933 (to P. N. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom all correspondence should be addressed. Tel.:
410-955-4699; Fax: 410-955-5759.
The abbreviations used are: GPCR, G-protein-coupled receptor; cAR1-H6, hexyl-histidine-tagged wild-type cAR1; cm1234-H6, hexyl-histidine-tagged non-phosphorylatable cAR1; PAGE, polyacrylamide gel electrophoresis; GFP, green fluorescent protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
2 The binding in phosphate buffer corresponds to binding at physiological conditions. Ammonium sulfate has been shown to convert the receptors into a single high affinity state (Kd 3-5 nM), so bindings obtained at 20-30 nM cAMP in ammonium sulfate reflect the total binding of cells.
3 During nutrient deprivation, the D. discoideum cells start a developmental program whereby central cells secrete cAMP, attracting surrounding cells through chemotaxis. The surrounding cells in turn secrete cAMP to attract more distal cells. Eventually tens of thousands of cells will aggregate to form a mound. The four cAMP receptor proteins, cAR1-cAR4, are the cell surface receptors responsible for sensing the pulses of cAMP and activating downstream events. cAR1 is the major type expressed during the initial stage of aggregation. In its absence, cells fail to aggregate.
4 R. Valkema and P. van Haastert, personal communication.
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