(Received for publication, May 5, 1994; and in revised form, November 28, 1994)
From the
Many G-protein-coupled receptors display a rapid decrease in
ligand binding following pretreatment with agonist. cAR1, a cAMP
receptor expressed early in the developmental program of Dictyostelium, mediates chemotaxis, activation of adenylyl
cyclase, and gene expression changes that bring about the aggregation
of 10 amoebae to form a multicellular structure. Occupancy
of cAR1 by cAMP initiates multiple desensitization processes, one of
which is an apparent reduction in binding sites. In transformed cells
expressing cAR1 constitutively, Scatchard analyses revealed that this
apparent loss of ligand binding is largely due to a significant
reduction in the affinity of cAR1 for cAMP. A parallel increase in the
dose dependence of cAR1-mediated cAMP uptake was observed. Consistent
with these findings, proteolysis of intact cells and immunofluorescence
suggested that cAR1 remains on the cell-surface following cAMP
treatment. Finally, agonist-induced loss of ligand binding is impaired
in cAR1 mutants lacking a cluster of cytoplasmic serine residues, which
are targets of cAMP-induced phosphorylation.
Seven-transmembrane domain, G-protein()-coupled
receptors constitute a diverse molecular superfamily with
representatives in many eukaryotic species. In mammals, these receptors
regulate processes ranging from chemotaxis to synaptic signaling and
vasoconstriction. Their ligands likewise range from glycoproteins to
biogenic amines and lipid molecules (reviewed in (1) ).
Despite this heterogeneity of function and ligand structure,
occupancy of these receptors elicits an apparently conserved series of
activation and desensitization processes. Initially, occupancy causes
the activation of receptor-associated G-proteins, which, in turn,
stimulate such effectors as adenylyl cyclase, phospholipase C, and ion
channels (reviewed in (2) ). Simultaneously, at least two
different desensitization events, adaptation and ``loss'' of
ligand binding, are observed. While most studies of these processes
focused on the -adrenergic receptor (reviewed in (3) ),
similar observations have been made for other G-protein-coupled
receptors. Adaptation, a rapid reduction in agonist-induced effector
activation, has been attributed to the uncoupling of receptor from
G-protein. This uncoupling is proposed to result from agonist-induced
receptor phosphorylation and the subsequent association of arrestin,
which appears to obstruct further receptor-G-protein interaction. The
second agonist-induced desensitization process is a rapid reduction in
the apparent number of surface binding sites. In some instances, this
loss of ligand binding, often referred to as sequestration, has been
attributed to receptor endocytosis.
Dictyostelium utilizes a family of four cell-surface G-protein-coupled cAMP receptors (cARs) to mediate a transition from a unicellular amoeboid phase to a multicellular sporogenous phase upon nutrient depletion. One of these, cAR1, is expressed shortly after the initiation of starvation and is required for aggregation(5, 6, 7, 47) . cAR1 occupancy has three consequences: (i) chemotaxis of cells toward the source of cAMP, (ii) altered gene expression, and (iii) synthesis and secretion of more cAMP, which serves to propagate the signal outwardly from aggregation centers (reviewed in (4) ). Like other G-protein-coupled receptors, cAR1 exhibits multiple responses to cAMP binding including adenylyl cyclase activation(8) , cAR1 phosphorylation (which is correlated with adenylyl cyclase adaptation)(9, 10, 11) , and loss of cAMP binding(12, 13) .
Constitutively expressed cAR1 undergoes phosphorylation (14, 15) and a ligand-induced reduction of ligand binding (14, 16) in the growth stage when the endogenous receptor gene is not expressed. This property has been exploited for the study of cAR1 mutants resulting in the identification of the major sites of cAMP-stimulated cAR1 phosphorylation(15) . In the present study, we used this system to examine the mechanism of cAMP-induced loss of ligand binding. Our findings lead us to conclude that, following cAMP pretreatment, growth-stage cells overexpressing cAR1 exhibit reduced binding, not because of receptor internalization, but because of a reduction in the affinity of cAR1 for cAMP. We also demonstrate that this transition is defective in cAR1 mutants that lack targets of ligand-stimulated phosphorylation, suggesting a possible link between these processes.
Figure 1:
Kinetics
and concentration dependence of cAMP-induced loss of cAMP binding in
cells overexpressing cAR1. A, washed, growth-stage cells were
incubated without (0 min) or with 10M cAMP
for 1-30 min in the presence of 10 mM dithiothreitol,
washed extensively, and cAMP binding measured at 10 nM by
centrifugation through silicone oil as described under
``Materials and Methods.'' B, washed,
growth-stage cells were incubated with various concentrations of cAMP
(0-10
M) for 15 min in the presence
of 10 mM dithiothreitol and 5 mM caffeine, washed
thoroughly, and [
H]cAMP binding measured as in panel A. The means of triplicate determinations from each of
two independent experiments are shown.
Figure 2:
Scatchard analysis of
[H]cAMP binding to cAR1 with and without cAMP
pretreatment. Growth-stage (A) or developed (B) cells
overexpressing cAR1 were treated without (filledsquares) or with (open squares) cAMP
(10
M, 15 min, with 10 mM dithiothreitol), washed, and [
H]cAMP binding
measured at cAMP concentrations from 10
M to 2
10
M by centrifugation
through silicone oil as described under ``Materials and
Methods.'' Data shown are the means from one of four (A)
or two (B) independent experiments performed in triplicate.
The lines represent the computer-generated fit for each set of data (Table 1, experiments 1 and 3).
To assess whether this pattern
of binding changes is a property unique to growth-stage cells, an
identical analysis was performed using developed cells (Fig. 2B). As with growth-stage cells, the Scatchard
plot of the untreated developed cells is curvilinear and cAMP
pretreatment causes a shallowing of the plot. Again, much of the
observed change results from a reduction in the affinity of the low
affinity receptor form (Table 1, experiments 3 and 4). The total
loss of cAMP binding sites may be slightly greater than in growth stage
cells (32 ± 6% and 15 ± 11% in experiments 3 and 4,
respectively) but does not fully explain the loss of binding. It is
unclear whether, in developed cells, there is a reduction in the number
of high affinity sites analogous to that observed in growth-stage
cells. An overall difference between growth-stage and developed cells
is an apparent trend toward increased affinities of all receptor
classes in the latter. This increase may reflect the expression of the
G-protein subunit, G2, which mediates cAR1 signaling during
aggregation(49) .
Figure 3:
Effect of cAMP pretreatment on the dose
dependence of [H]cAMP uptake. Cells were
incubated without or with cAMP (10
M, 15
min, with 10 mM dithiothreitol), washed, and
[
H]cAMP uptake determined at the various
concentrations shown on the abscissa (10
to
10
6 M) as described under
``Materials and Methods.'' Plotted for each
concentration is the percentage of control uptake exhibited by control
cells (filled squares, 100 by definition) and cAMP-pretreated
cells (opensquares). Values shown are the means
± standard deviations of nine (10
M and 2
10
M points) or two
(remaining points) determinations pooled from three independent
experiments. The mean absolute uptake exhibited by control cells (in
units of molecules per cell) is indicated above each data point.
Because the absolute uptake varied by as much as 2-fold from day to
day, all uptake values were normalized to the average level of uptake
exhibited by control cells for each experiment prior to the calculation
of standard deviation.
Figure 4:
Effect of cAMP on protease accessibility
of epitope-tagged cAR1. Washed, growth-stage cells expressing Myc-cAR1
were shaken without(-) or with (+) cAMP (10M, 15 min, 22 °C, with 10 mM dithiothreitol) and washed four times in ice-cold PB. They were
then incubated in the absence (0 min) or presence of trypsin (50
µg/ml, 0 °C) for the indicated times. After addition of trypsin
inhibitor (100 µg/ml) and extensive washing, a CHAPS-insoluble
fraction was prepared, subjected to SDS-PAGE and immunoblotted with
cAR1 COOH terminus-specific antiserum (A) or Myc-specific
antiserum as described under ``Materials and
Methods'' (B). The electrophoretic positions of wild-type
cAR1 (openarrowhead, not shown), Myc-cAR1 (asterisk), and phosphorylated Myc-cAR1 (filledarrowhead) are indicated.
Unlike the wild-type cAR1, the
Myc-tagged cAR1 is sensitive to surface trypsinization. Within 15 min,
more than 87% of the molecules are cleaved to yield slightly smaller
products which have lost the Myc epitope (Fig. 4B, left). The time dependence of the cleavage strongly suggests
that trypsinization is occurring on intact cells, and not during
subsequent sample preparation. Co-incubation of samples from
trypsin-treated and untreated cells results in no further loss of
Myc-tagged cAR1 (data not shown), further supporting this conclusion.
When the cells were pretreated with cAMP, the mobility of the
Myc-tagged cAR1 on SDS-PAGE was observed to further decrease (Fig. 4, right). This mobility alteration, previously
reported for wild-type cAR1, is due to serine phosphorylation on the
cytoplasmic COOH-terminal domain(15) . When the pretreated
cells were subjected to proteolysis, removal of the Myc epitope
occurred as extensively as it did in untreated cells (Fig. 4B, right). The slight decrease in the
amount of Myc-cAR1 detectable with anti-cAR1 serum (approximately 30%
at 15 min, Fig. 4A) might reflect cleavage at basic
residues within the cAR1 sequence or an inherent instability in the
NH-terminally cleaved Myc-cAR1. The failure of cAMP
pretreatment to protect Myc-cAR1 from proteolysis is consistent with
the notion that the reduction in cAMP binding observed upon
pretreatment can occur without significant receptor internalization.
This interpretation was corroborated by immunofluorescence studies.
When untreated cells expressing Myc-cAR1 were rapidly fixed in
suspension, they exhibited a predominantly peripheral pattern of
immunofluorescent staining with anti-cAR1 antibodies (Fig. 5A). This pattern is similar to that described
previously for endogenous cAR1 in developed cells and suggests that
most receptor molecules are within the plasma membrane. Despite a 74%
reduction of binding at 16 nM [H]cAMP,
cAMP-pretreated cells revealed the same pattern of cAR1 distribution as
untreated cells (Fig. 5B), suggesting that little if
any relocalization had occurred. Vector control cells expressing no
cAR1 displayed no peripheral staining under these conditions (data not
shown).
Figure 5:
Effect of cAMP pretreatment on cAR1
immunofluorescence. Washed, growth-stage cells expressing Myc-cAR1 were
shaken without (A) or with (B) cAMP (10M, 12 min, 22 °C) and washed four times with
ice-cold PB. They were then fixed in suspension with methanol, 1%
formaldehyde, stained with cAR1 antiserum (29) and fluorescein
isothiocyanate-conjugated goat anti-rabbit antibodies as described
under ``Materials and Methods.'' Pretreated cells in
this representative experiment displayed 74% less binding to 16 nM [
H]cAMP than control cells, when assessed at
the time of fixation. The cells shown are approximately 12 µm in
diameter.
Figure 6:
Loss of ligand binding in cAR1 mutants
lacking cytoplasmic serine residues. Washed growth-stage cells
expressing wild-type cAR1 (opensquares), cm234 (filledcircles), cm1 (filledsquares), or cm1234 (opencircles) were
shaken without (0 min) or with cAMP (10M,
22 °C) for 5, 15, or 30 min and washed extensively with PB.
[
H]cAMP binding was then measured at 16 nM by the sedimentation assay, as described under
``Materials and Methods.'' Data shown represent the
means ± S.E. of three experiments performed in
triplicate.
The nature of loss of ligand binding by G-protein-coupled
receptors has been difficult to ascertain. Often, this process has been
equated with receptor internalization. Studies using immunofluorescence (31, 32) and cell fractionation (33, 34) have revealed a strong correlation between
the loss of ligand binding and the ``sequestration'' or
movement of receptors to a new, apparently intracellular compartment.
Consistent with this interpretation, cells expressing -adrenergic
and muscarinic receptors, upon pretreatment, lose their ability to bind
hydrophilic but not hydrophobic ligands (35) . While the
reduction in binding and internalization may be tightly correlated,
however, they might be separate processes. Several studies, in fact,
have suggested that loss of binding can occur without receptor
internalization(36, 37) . Previous studies of cAR1 (38, 39) and our present results support this
hypothesis.
We demonstrate here that in response to cAMP pretreatment, growth-phase Dictyostelium cells overexpressing cAR1 exhibit a reduction of cAMP binding similar in rate, extent, and concentration dependence to that displayed in developed cells expressing endogenous cAR1. Scatchard analysis and computer fitting of cAMP binding data suggest that this binding alteration is due predominantly to a reduction in the affinity of the lower affinity receptor form, which represents the bulk of cell surface cAR1, with a concomitant small change in the number (but not the affinity) of high affinity binding sites. It is unclear whether this latter change represents an interconversion of high to low affinity sites or a selective removal of high affinity sites from the cell surface. Nevertheless, there is a minimal reduction in the total number of binding sites under these conditions, supporting the hypothesis that even at saturating cAMP concentrations, at least 80% of cAR1 molecules are neither internalized(38, 39) , ``irreversibly'' occupied with ligand (25) or incapable of binding for other reasons. Developed cells overexpressing cAR1 exhibit a similar pattern of affinity changes, demonstrating that this phenomenon is not a function of the developmental stage. There is some suggestion from the present data, however, that in these cells, cAMP might induce a slightly higher fractional receptor internalization than in growth-stage cells.
The binding affinity changes described above are mirrored functionally by changes in cell sensitivity to cAMP. The concentration dependence of cAR1-mediated cAMP uptake, a process distinct from binding, is altered by pretreatment. As with binding, pretreatment causes an apparent transition from high to low sensitivity of uptake, with little or no change in maximal uptake.
A different conclusion was drawn in previous studies involving endogenous cAR1 in developed cells. There, Scatchard analysis suggested that loss of ligand binding was due to a reduction in total cAMP binding sites, with little change in affinity(12, 13, 25) . We attribute these differences to our use of cells overexpressing cAR1, which enhances the detection of low affinity binding sites. In cAMP binding experiments involving endogenous cAR1, the expression levels are lower than those presented here, resulting in a lower signal-to-noise ratio at higher cAMP concentrations, and thereby precluding the detection of low affinity binding sites. In addition, our initial analysis of growth-stage cells, which exhibit less curvilinear Scatchard plots and less reduction in total binding, also facilitated the detection of changes among the low affinity sites.
The protease accessibility and immunofluorescence experiments described here provide physical evidence that nearly all cAR1 molecules remain on the cell surface following cAMP pretreatment. The immunolocalization of cAR1 in cAMP-treated and untreated cells is virtually indistinguishable when these cells are rapidly fixed in suspension, suggesting there is not significant internalization. Furthermore, even after maximal induction of the response, nearly all epitope-tagged cAR1 molecules remain susceptible to surface trypsinization. Consistent with these results, sustained chymotrypsin sensitivity of endogenous cAR1 following cAMP pretreatment has been previously reported(39) . Our results with epitope-tagged cAR1 strengthen the proposal put forth by these authors that loss of ligand binding can occur without cAR1 internalization.
Our immunofluorescence results appear to contradict the previously reported observation that cAR1 localization changes upon cAMP pretreatment from a peripheral pattern to a more punctate, possibly vesicular, one(32, 38) . We believe that this difference arises from the distinct treatment of the cells used in each case. In the previous studies, cells were allowed to adhere to glass slides, flattened with a sheet of agar, and then methanol-fixed. In the present study, in contrast, shaking cells were rapidly fixed in suspension. We too have found that adherent cells exhibit an apparent cAMP-induced redistribution of cAR1 immunofluorescence (data not shown), unlike cells fixed in suspension. Perhaps cAMP treatment normally induces cAR1 relocalization in the physiological context of a cell attached to a solid surface, while in suspension, the internalization process is uncoupled. Thus, the transition in cAR1 affinity appears not to depend upon internalization, though it might reflect an early step in this process, such as the binding of a component of the endocytic machinery to the receptor.
Several domains within G-protein-coupled receptors
have been described as important for agonist-induced reductions in
ligand binding. Mutant adrenergic, muscarinic, or cAMP receptors
bearing substitutions or deletions within the putative second and third
intracellular loops have shown drastic impairment in this
process(16, 40, 41, 42) .
Replacement of a human -adrenergic receptor tyrosine
(Tyr
) with an alanine has no influence on G-protein
activation or desensitization with respect to adenylyl cyclase
activation but blocks loss of ligand binding, receptor relocalization,
and resensitization of the cyclase response(43) . This tyrosine
occurs within a sequence motif near the COOH-terminal end of the
putative seventh transmembrane domain of many G-protein-coupled
receptors (NPX
Y, where X represents aliphatic residues). A similar phenotype is seen with
-adrenergic receptor mutants in which putative targets of protein
kinase A phosphorylation have been changed to
alanines(44, 45) . Nevertheless, removal of all
serines from the COOH terminus of this receptor does not prevent the
agonist-induced reduction of ligand binding(44) , suggesting
that
-adrenergic receptor phosphorylation is unnecessary for loss
of binding.
Our present studies with cAR1 suggest that a subset of COOH-terminal serines is required for loss of cAMP binding. A cAR1 mutant lacking all serines in the cytoplasmic COOH terminus cannot undergo this process. Substitution of the 5 serines that comprise cluster 1 (serines 299, 302, 303, 304, and 308) with alanine and glycine residues (mutant cm1) results in a severe reduction in both the rate and extent of the agonist-induced binding changes, while substitution of all other serines in the COOH-terminal tail has virtually no effect. We previously demonstrated (15) that approximately two-thirds of the cAMP-induced phosphorylation of cAR1 occurs within serine cluster 1 and the remainder occurs within cluster 2 (serines 324, 325, and 331). Which of these residues are modified has not yet been determined. The remaining 10 serines of the COOH-terminal domain, which comprise clusters 3 and 4 (as defined in (15) ) are not phosphorylated in response to cAMP binding. Phosphorylation occurring within cluster 1 is strongly correlated with the adaptation of cAMP-stimulated adenylyl cyclase(11, 29) . Thus, the affinity transition and phosphorylation undergone by cAR1 in response to cAMP appear to share either common or overlapping structural requirements within this domain.
cAR1 contains two
NPXY motifs similar to that implicated in
-adrenergic receptor loss of ligand
binding(5, 43) . The first of these
(NPLMWRY
) like those of other receptors is located at the
COOH-terminal portion of the seventh transmembrane domain. The second
(NPSPY
) includes Ser
of serine cluster 1 in
the COOH-terminal cytoplasmic domain. It is tempting to speculate that
the latter is important for the loss of ligand binding in light of its
proximity to cluster 1. Note, however, that in mutant cm1, which is
impaired in loss of ligand binding, this sequence (NPAPY
)
resembles the proposed consensus sequence even more closely than does
the wild-type sequence. The mechanisms by which serine replacement
affects cAR1 loss of ligand binding therefore remain to be clarified.
In summary, agonist-induced loss of cAMP binding sites appears to involve a 3-5-fold reduction in the affinity of the predominant low affinity receptor form and is not dependent upon receptor internalization. The reduction in affinity could, however, reflect an intermediate step in the internalization process. Furthermore, this affinity transition depends upon a domain in the cAR1 cytoplasmic carboxyl terminus, which is also a target of cAMP-induced phosphorylation. Continued analysis of these processes should allow a detailed molecular description of the various adaptive changes undergone by cAR1 upon agonist binding.