(Received for publication, September 12, 1994; and in revised form, December 20, 1994)
From the
Surface cAMP receptors (cARs) in Dictyostelium transmit
a variety of signals across the plasma membrane. The best characterized
cAR, cAR1, couples to the heterotrimeric guanine nucleotide-binding
protein (G protein) -subunit G
2 to mediate activation of
adenylyl and guanylyl cyclases and cell aggregation. cAR1 also elicits
other cAMP-dependent responses including receptor phosphorylation, loss
of ligand binding (LLB), and Ca
influx through a
G
2-independent pathway that may not involve G proteins. Here, we
have expressed cAR1 and a related receptor, cAR3, in a g
strain (Lilly, P., Wu. L., Welker, D.
L., and Devreotes, P. N.(1993) Genes & Dev. 7,
986-995), which lacks G protein activity. Both cell lines failed
to aggregate, a process requiring the G
2 and G
-subunits. In
contrast, cAR1 phosphorylation in cAR1/g
cells showed a time course and cAMP dose dependence
indistinguishable from those of cAR1/G
controls.
cAMP-induced LLB was also normal in the
cAR1/g
cells. Finally,
cAR1/g
cells and
cAR3/g
cells showed a
Ca
response with kinetics, agonist dependence, ion
specificity, and sensitivity to depolarization agents that were like
those of G
controls, although they accumulated
fewer Ca
ions per cAMP receptor than the control
strains. Together, these results suggest that the G
-subunit is not
required for the activation or attenuation of cAR1 phosphorylation,
LLB, or Ca
influx. It may, however, serve to amplify
the Ca
response, possibly by modulating other
intracellular Ca
signal transduction pathways.
External stimuli trigger a wide variety of physiological
responses by altering the activity of ion channels and intracellular
enzymes. Seven membrane span receptors modulate effectors through a
family of heterotrimeric guanine nucleotide-binding proteins (G
proteins), comprised of -,
-, and
-subunits (reviewed in (1) ). Receptor activation triggers a GTP-GDP exchange reaction
on the G
-subunit and dissociation of G
from the
G
-subunit complex. The G
-subunits activate a broad range
of effectors in eukaryotic cells(1) . Although free
G
-subunit complexes were thought only to attenuate signaling
by reassociating with activated G
-subunits(1) , emerging
evidence indicates that they also directly regulate adenylyl
cyclase(2) , phospholipases(3, 4) , and ion
channels(5) .
The cellular slime mold Dictyostelium
discoideum provides an excellent system for molecular genetic
analysis of G protein-linked signaling pathways. Upon nutrient
depletion, these amoeboid cells aggregate into a multicellular
structure, which undergoes morphogenesis and differentiation to form a
fruiting body. This program is regulated by cAMP, which is synthesized
and secreted by centrally located cells. cAMP binding to a cell surface
cAMP receptor 1 (cAR1) ()directs adjacent cells to move
toward aggregation centers and to relay the signal outwardly to more
distal cells. The periodic stimulus also acts as a developmental timer,
accelerating gene expression in the early stages of the program
(reviewed in (6) ).
cAMP receptor occupancy induces a
transient entry of external Ca(7, 8, 9) and elevates cellular levels
of inositol 1,4,5- trisphosphate
(IP
)(10, 11) , cAMP(12) , and cGMP (13) . Many of these cAMP-dependent responses appear to require
G proteins. In fact, four related cAMP receptors with striking
similarity to rhodopsin and the
-adrenergic receptor (reviewed in (14) ), eight G protein
-subunits (reviewed in (15) ), and a single G protein
-subunit (16) have
been identified in Dictyostelium. cAR1 is the major surface
cAMP binding site of aggregating cells and appears to couple to the
G
-subunit G
2, which is also expressed maximally during
aggregation. Mutants lacking functional G
2 fail to differentiate,
exhibit chemotaxis toward cAMP, or show cAMP-stimulated production of
cAMP, cGMP, or IP
(reviewed in (17) ). The other
cARs (cAR2, cAR3, and cAR4) are normally present during
post-aggregative development but are capable of eliciting some of these
responses when expressed in growth-stage wild-type cells or car1
cells(18, 19) . (
)
We have proposed that a subset of cAR-generated signals
may not require G proteins, based on the following observations. First,
cAR1/g2
and
cAR3/g
2
cells show essentially
wild-type levels of cAMP-dependent Ca
uptake, even
though they are defective in the G protein-linked pathways that control
chemotaxis, differentiation, and production of cGMP (18) or
cAMP(20) . Two additional responses of the receptors,
agonist-induced phosphorylation and reduction in cAMP binding affinity
(loss of ligand binding, LLB), also do not require
G
2(21) . Second, the complement of G
-subunits present
in growing cells is different from those expressed during aggregation
and post-aggregation when the cARs are present(15) . Yet, cAR1,
cAR2, and cAR3 elicit wild-type Ca
responses when
they are expressed in growing cells, and they appear to activate the
same pathway of ion entry(18) . Third, analysis of
G
-subunit null mutants indicates that G
1, G
2, G
3,
G
4, G
7, and G
8 are not essential for cAMP-induced
Ca
uptake(18) . Moreover, the developmental
regulation of G
5 and G
6 suggests that these subunits do not
couple to the three cARs(22, 23) . While these
observations imply a G protein-independent pathway, several caveats can
be raised. Although the eight G
-subunits cannot be classified into
subgroups(15, 24) , some of them could be functionally
redundant. Alternatively, G
-subunit complexes could regulate
the G
-subunit-independent responses.
We took advantage of the
observation that g cells possess a
single G
-subunit, which is expressed at a constant level during
growth and development(16) , to further explore this issue.
Here, we have characterized cAMP-stimulated Ca
uptake, cAR1 phosphorylation, and LLB in g
strains, which should not generate
activated G
-subunits or free G
-subunit complexes. Our
results support the conclusion that G protein-coupled cARs function in
the absence of G proteins to activate certain cellular responses.
Ca uptake into 5
10
cells in
the presence and absence of 100 µM cAMP was performed in a
medium containing 500 µM CoCl
as
described(18) . cAMP-induced Ca
uptake is
equal to the amount of Ca
taken up by cAMP-treated
cells minus the amount of Ca
taken up by
non-stimulated cells.
[H]cAMP binding to the
cells was performed in triplicate using the ammonium sulfate assay as
indicated (33) except that the stimulus concentration was 1
µM.
Protein was measured as described (34) using bovine serum albumin as standard.
When g cells were repeatedly
stimulated with exogenous cAMP, the cells showed weak levels of surface
cAMP binding and low levels of cAMP-induced Ca
uptake
(data not shown). The expression level of cAR1 in g
cells varies with the growth
conditions and the age of the culture. (
)To obtain more
reproducible levels of cAR expression and to permit analysis of
growth-stage cells, g
cells were
transformed with vectors containing the cAR1 or cAR3 cDNA under the
control of a promoter that is constitutively active during growth. The
expression of cAR1 and cAR3 in growth-stage g
cells is shown in Fig. 1A. Lysates of
cAR1/g
cells fractionated using
SDS-polyacrylamide gel electrophoresis showed a 40-kDa protein band
immunoreactive with cAR1-specific antiserum (lane2).
This protein band, indicative of cAR1(30) , was also present in
cAR1/G
lysates (lane3) but not
in g
lysates (lane1). When lysates of cAR3/g
cells were immunoblotted with a cAR3-specific antiserum, a 65-kDa
protein band was evident (lane5) characteristic of
cAR3(31) . The same protein band was also present in control
cAR3/G
cells (lane6) but not
in g
cells (lane4).
When cAR1/g
or
cAR3/g
cells were plated for
development on non-nutrient agar, the amoebae remained as a flat
monolayer. Control cAR1/G
cells (Fig. 1B) differentiated to form fruiting bodies, as
has been shown for cAR3/G
cells. (
)
Figure 1:
Expression
of cAMP receptors in cAR1/g and
cAR3/g
strains and their development on
phosphate-buffered agar. A, SDS-solubilized growth-stage g
(lanes1 and 4), cAR1/g
(lane2), cAR1/G
(lane3), cAR3/g
(lane5), and cAR3/G
(lane6) cells (1
10
) were
electrophoretically separated and immunoblotted using antibodies to
cAR1 (lanes1-3) and cAR3 (lanes4-6) as described under ``Experimental
Procedures.'' Numbers at the margin of the
figure indicate the migration pattern of molecular mass standards
expressed in kDa. B, development of
cAR1/g
,
cAR3/g
, and cAR1/G
cells. Growth-stage amoebae were starved (22 °C) on
non-nutrient agar for 48 h as described(58) . Immunoblot
analysis of 0-h cells indicated that each line had comparable levels of
cAR1 or cAR3, respectively, to levels shown in A.
We assessed cAMP-induced cAR1 phosphorylation in
cAR1/g and cAR1/G
cells by monitoring a parallel change in the apparent molecular
mass of the receptor from 40 to 43 kDa. This electrophoretic mobility
shift is due to serine phosphorylation within the C-terminal domain of
cAR1(46) . Extracts of non-stimulated g
cells contained cAR1, which primarily
migrated at 40 kDa, although there was also a detectable 43-kDa band (Fig. 2A, lane1), which is the
phosphorylated form of cAR1 (30) . Upon addition of a
saturating cAMP stimulus, the proportion of cAR1 in the 43-kDa form
increased by 15 s and was almost entirely shifted to this form by 2 min (lanes2-9). Comparable results were obtained
in the cAR1/G
cells. In the presence of
increasing concentrations of cAMP, both
cAR1/g
and cAR1/G
cells required 5 nM cAMP to induce the shift in mobility (Fig. 2B, lane3) and showed maximal
responses at 100-500 nM (lanes6 and 7). In both instances, the concentration of cAMP required for
half-maximal response (EC
) was between 10 and 50
nM. Thus, the time course and dose dependence of cAMP-induced
cAR1 phosphorylation are identical in the presence or absence of
G
. These values are similar to those of cAR1 expressed in
aggregating wild-type cells(32) .
Figure 2:
cAMP-induced phosphorylation of cAR1 in
cAR1/g cells. Growth-phase
cAR1/G
or cAR1/g
cells (1.5
10
) were resuspended in phosphate
buffer containing 5 mM caffeine and 10 mM dithiothreitol and stimulated with 100 µM cAMP for
the indicated times (A) or with various concentrations of cAMP
for 15 min (B), solubilized, separated electrophoretically,
and immunoblotted with cAR1 antiserum as described under
``Experimental Procedures.'' A, time course of cAR1
phosphorylation. Lanes 1-9 represent the following times
of treatment with cAMP: 0 s, 7 s, 15 s, 30 s, 1 min, 2 min, 5 min, 10
min, and 20 min. B, cAMP dose dependence of cAR1
phosphorylation. Lanes 1-9 represent the following doses
of cAMP: 0, 1 nM, 5 nM, 10 nM, 50
nM, 100 nM, 500 nM, 10 µM, and
100 µM. This experiment was repeated once with similar
results.
Pretreatment of Dictyostelium amoebae with cAMP induces a rapid reduction in
subsequent cAMP binding within 7 min(35, 36) . This
response appears to reflect a reduction in the affinity of cAR1 for
cAMP and likely depends upon receptor phosphorylation. ()As
shown in Fig. 3, cAR1/g
cells
pretreated with 10 µM cAMP exhibited a 75% reduction in
the level of binding to a subsaturating concentration of cAMP relative
to control cells treated with buffer. cAR1/g
cells also showed a pronounced response, with a 90% reduction in
cAMP binding upon cAMP pretreatment.
Figure 3:
Loss of ligand binding in
cAR1/g or cAR1/G
cells. Growth-stage amoebae were resuspended to 3
10
cells/ml in phosphate buffer, shaken (20 min, 22 °C,
200 rpm) in the presence of 10 mM dithiothreitol, and then
treated for 15 min with buffer (shaded bars) or with 10
µM cAMP (open bars). Cells were diluted 15-fold
in ice-cold phosphate buffer, washed extensively in the same buffer,
and placed on ice. Binding assays of each sample were performed in
triplicate using 16 nM [
H]cAMP and 10
mM dithiothreitol. Results shown are the average of two
experiments. Absolute [
H]cAMP binding to
buffer-treated cAR1/G
cells and
cAR1/g
cells was
1500 cpm/8
10
cells and 3400 cpm/8
10
cells, respectively.
Next, agonist-induced
Ca uptake in the g
strains was examined. Growth-stage
cAR1/g
cells treated with cAMP
initially accumulated Ca
at the same rate as
non-stimulated cells. After a delay of 10-15 s, however, the rate
of Ca
uptake into these cells increased until
30
s after receptor activation, when it returned to prestimulus rates (Fig. 4A). The agonist induced the accumulation of
25 pmol of Ca
/mg of protein. Growth-stage
cAR3/g
cells showed similar profiles of
cAMP-induced Ca
uptake except that the delay
preceding stimulated uptake was only 5-10 s, and the agonist
induced the accumulation
50 pmol of Ca
/mg of
protein (Fig. 4B). Presumably, there are few endogenous
receptors during growth since g
cells
showed no cAMP-dependent Ca
response (Fig. 4C). Each of the three strains showed similar
levels of basal Ca
entry.
Figure 4:
Time course of Ca uptake
into cAR1/g
cells (A),
cAR3/g
cells (B), and g
cells (C). Growth-stage
amoebae were assayed for Ca
uptake as described under
``Experimental Procedures.'' Values are shown for
Ca
into resting (
) and cAMP-stimulated cells
(
) and for cAMP-induced Ca
uptake (
).
Results shown represent the means of data from five (A), four (B), and two (C) independent experiments. In A and B, bars represent S.E. For clarity, half-error bars are shown.
The time courses of
cAMP-dependent Ca uptake into
cAR1/g
and
cAR3/g
cells (Fig. 4) are like
those of cAR1/G
and cAR3/G
cells(18) . However, the magnitude of cAMP-dependent
Ca
uptake into the g
strains was less than that seen earlier in G
lines(18) . To assess the ability of cAR1 and cAR3 to
promote Ca
influx in g
strains, levels of cAMP-induced Ca
uptake in
cAR1/g
and
cAR3/g
cells were standardized to the
levels of surface cAMP binding sites in these cells.
cAR1/g
cells transported 1.8
Ca
ions/cAMP binding site, while
cAR3/g
cells accumulated 4.2
Ca
ions/binding site (Table 1). These results
are consistent with the finding that cAR3/G
cells
transport higher levels of Ca
/receptor than
cAR1/G
cells (Table 1). However, both g
strains transported Ca
4-fold less effectively than the
208-derived
G
strains. To determine if this was due to a
defective cell line, fresh g
cells were
retransformed with the cAR1 expression vector, and 10 clones expressing
high levels of cAR1 were screened for cAMP-dependent Ca
uptake. All lines displayed a similar agonist-induced
Ca
response (data not shown). Since the
208
cells were constructed in a HPS400 background, while g
cells were constructed using an
AX3-derived strain, the experiment was repeated using
cAR1/g
cells and cAR1/G
cells (JB4 line) constructed in the same parental background.
Comparable results were obtained (Table 1).
The cAMP
requirements of Ca uptake into
cAR1/g
and
cAR3/g
cells are like those of
cAR1/G
and cAR3/G
cells,
respectively. In both cAR1/g
and
cAR1/G
cells, the Ca
response
was evident at 100 nM cAMP, reached a maximum at 3 µM cAMP, and displayed an EC
value of
250 nM (Fig. 5A). Moreover, both
cAR3/g
and cAR3/G
cells showed similar cAMP dose-response curves. Stimulated
Ca
uptake was detectable at 300 nM cAMP, was
maximal at 10 µM, and had an EC
value of
780 nM (Fig. 5B). The EC
values of cAR1- and cAR3-dependent Ca
uptake
are similar to the corresponding affinities of cAMP binding to cAR1 and
cAR3, which have K
values of
230 and
500-700 nM, respectively(26) .
Figure 5:
Effect of cAMP concentration on the
magnitude of Ca uptake into (A)
cAR1/g
(
) and (B)
cAR3/g
(
) and
cAR3/G
(
) cells. Growth-stage amoebae were
assayed for cAMP-dependent Ca
uptake as described
under ``Experimental Procedures,'' except that uptake was
followed for 30 s in the presence of 10 nM - 100
µM cAMP. Ca
uptake values for each
strain are expressed relative to the maximum uptake value measured in
the presence of 3 µM (A) or 100 µM (B) cAMP. Each point is the mean ± S.E.
of results obtained in three (
), three (
), and four (
)
separate experiments. The maximum levels of stimulated Ca
uptake were 22 ± 2 (
), 48 ± 11 (
), and
58 ± 9 (
) pmol of Ca
/mg of protein
± S.E., respectively. Data showing the effect of cAMP on
receptor-induced Ca
uptake by cAR1/G
cells (A,
) were taken from (18) .
The
pharmacological properties of Ca influx into
cAR1/g
and
cAR3/g
cells are comparable with those
of G
strains expressing cAR1 or cAR3. In
G
strains, Ca
uptake is
inhibited effectively (95%) by 500 µM La
or Gd
, moderately (35%) by 500 µM Cd
, and not by 500 µM Co
. In these strains, 1-2 µM carbonylcyanide-m-chlorophenylhydrazone (CCCP) and 10
µM Ruthenium Red reduce the response by
50%(18) . The cAMP-dependent Ca
uptake
of cAR1/g
and
cAR3/g
cells show the same pattern of
inhibition by these compounds (Fig. 6). The Ca
responses were reduced considerably by 125 µM La
or Gd
, moderately by 500
µM Co
, and not by 500 µM Co
. CCCP (1 µM) reduced the
magnitude of stimulated Ca
uptake by
50%, while
Ruthenium Red (10 µM) almost completely blocked this
response.
Figure 6:
Effect of Ruthenium Red (RR),
CCCP, and different cations on receptor-stimulated Ca accumulation. cAMP-induced Ca
uptake into
cAR1/g
cells (closed bars) and
cAR3/g
cells (open bars) was
measured for 30 s as described under ``Experimental
Procedures'' except that the assay medium contained no CoCl
and was supplemented with 500 µM CoCl
,
500 µM CdCl
, 125 µM LaCl
, 125 µM GdCl
, 2
µM CCCP, or 10 µM Ruthenium Red. Results are
expressed relative to control samples not receiving test compounds and
represent the average of three (closed bars) or four (open
bars) independent experiments. Bars represent S.E. The
magnitude of stimulated Ca
uptake in the untreated
cells was 15 ± 5 (closed bars) and 64 ± 7 (open bars) pmol of Ca
/mg of protein
± S.E.
Our analysis of cAR1/g and
cAR3/g
cells suggests that the G
protein-coupled cAMP receptors of Dictyostelium induce certain
signal transduction events in the absence of G
-subunits.
cAR1/g
cells showed agonist-dependent
receptor phosphorylation, which had a time course and cAMP
dose-response curve comparable with that of cAR1/G
cells (Fig. 2). cAR1/g
cells pretreated with cAMP displayed a normal LLB response (Fig. 3). Finally, cAR1/g
and
cAR3/g
cells exhibited a
cAMP-stimulated Ca
uptake with kinetics and agonist
requirements ( Fig. 4and Fig. 5), which were markedly
similar to those of cAR1/G
or
cAR3/G
cells(18) .
Since these
responses are not activated or attenuated by G, it is also
unlikely that they are activated by the GTP-bound form of an
G
-subunit. Consistent with this idea, analysis of Ca
uptake in G
-subunit null cells (G
1, G
2, G
3,
G
4, G
7, and G
8) (18) or of the developmental
regulation of G
-subunits (G
5, G
6) (22, 23) suggested that none of these proteins
regulate Ca
responses triggered by the three cARs.
The non-involvement of G
2 is striking, since this subunit couples
to cAR1 to induce multiple G protein-dependent responses(17) .
Our results with the g
strains,
together with the finding that sequence comparison of the eight
G
-subunits reveals no evidence of
subgroups(15, 24) , suggests that G
-subunits do
not substitute for each other to activate cAR1 phosphorylation, LLB,
and Ca
influx.
It is not likely that G itself
is redundant since it is expressed at a constant level during the
entire developmental program and since low stringency Southern analysis
of genomic DNA preparations yields a single band(16) . g
cells lack an intact copy of the
unique G
gene, do not express proteins immunoreactive with
G
-specific antiserum, fail to aggregate(16) , and lack
functional heterotrimeric G protein activity.
Moreover,
expression of cAR1 or cAR3 in g
cells
did not rescue the aggregation-minus phenotype of g
cells (Fig. 1B),
which might be expected if the cARs interacted with unknown G proteins
to generate activated G
-subunits. Importantly,
cAR1/g
cells show neither
cAMP-dependent synthesis of cAMP or cGMP nor GTP inhibition of cAMP
binding, responses indicative of receptor/G protein coupling.
Recently, novel proteins containing a sequence motif conserved
in G-subunits have been discovered, including coronin, an
actin-binding protein from Dictyostelium(37) . While
the role of coronin in cAR-mediated signal transduction remains to be
defined, it is unlikely that it, or other proteins with regions of
homology to G
, substitutes for G
to activate G
protein-dependent signal transduction across the plasma membrane.
First, coronin exerts pleiotropic effects on cells, influencing growth,
chemotaxis, and cytokinesis(38) . Like coronin, other proteins
containing regions of homology to G
regulate diverse processes
occurring in a variety of intracellular compartments (see (39) and references therein). Second, G
-subunits are
highly conserved throughout evolution, likely reflecting the presence
of domains essential for interactions with other components of the
receptor-G protein complex. For example, except for the 58 N-terminal
amino acids of the Dictyostelium G
-subunit, the remaining
300 amino acids of this protein show 70% identity (90% homology with
conservative replacements) to other G
-subunits(16) . In
contrast, the Dictyostelium G
-subunits show lower
homology (35-55% identity) to each other and G
-subunits of
other organisms(15, 24) . Furthermore, at least two of
the Dictyostelium G
-subunits couple to distinct receptors (17, 40) .
cAR1, cAR2, and cAR3 appear to activate
the same voltage-independent transporter (or channel) to promote
Ca entry(18) . Stimulated Ca
uptake into cAR1/g
and
cAR3/g
cells likely occurs through the
same pathway since the time course, ion specificity, and
pharmacological properties of the responses are similar to each other ( Fig. 4and Fig. 6) and to cAR1/G
or
cAR3/G
cells(18) . Ion competition
experiments indicated that, like the G
strains,
the Ca
uptake response of cAR-containing g
strains was highly specific for the
transport of Ca
over other multivalent ions (Fig. 6). The relative effectiveness of inhibitors was
La
and Gd
> Cd
> Co
, which was ineffective at 500
µM. Moreover, like G
strains, each
system was inhibited
50% by CCCP. This agent does not alter the
time course of Ca
influx but lowers the magnitude of
the response(9) , probably by depolarizing the plasma membrane
to reduce the electrochemical potential for Ca
entry
across the plasma membrane(41) . 10 µM Ruthenium
Red, which reduces the Ca
response of
G
strains by
50%(18) , more potently
inhibited Ca
uptake of
cAR1/g
and
cAR3/g
cells. The reason for this
enhanced sensitivity is unclear, although this compound can induce
membrane depolarization, block Ca
channels, and
interact with Ca
-binding proteins(42) .
Although the properties of agonist-induced Ca influx are markedly similar in the presence or absence of G
,
cAR1/g
and
cAR3/g
cells transported fewer
Ca
ions/surface cAMP binding site than the
corresponding G
strains (Table 1).
Downstream components of the Ca
-uptake system are
probably not limiting, since developed g
cells possessed lower levels of cAR1 than growth-stage
cAR1/g
cells, but transported
comparable levels of Ca
/cAMP binding site (data not
shown). G
-subunits or activated G
-subunits may influence
other aspects of cellular Ca
signaling, which
indirectly modulate Ca
entry into the cells. For
example, in liver cells, G
-subunits regulate Ca
pumps that extrude the ion across the plasma
membrane(43) . Moreover, G protein-modulated changes in the
Ca
content of intracellular stores, such as those
released by IP
, influence the rate of Ca
influx into certain mammalian cells(44) . In this light,
it is interesting that g
2
strains,
which do not produce IP
(45) , also show less (50%)
Ca
uptake/cAMP binding site(18) .
Alternatively, g
cells might express a
cryptic population of surface cAMP receptors that can bind cAMP but
cannot activate Ca
influx. This idea is less
plausible since cAR1/g
cells exhibited
normal cAR1 phosphorylation and LLB.
The biochemical component(s)
and function(s) of G protein-independent signaling in Dictyostelium remain to be defined. cAMP-dependent cAR1 phosphorylation occurs
in a cluster of serines at the N terminus of the cytoplasmic C-terminal
domain of cAR1 (46) and may be required for LLB. cAR1 phosphorylation appears not to be regulated by stimulated
Ca
entry, since inclusion of 1 mM EGTA or 30
µM Ruthenium Red in the assay medium did not influence
this response (data not shown). Receptor phosphorylation may simply
occur when agonist-mediated changes in cAR1 conformation expose a
kinase-binding domain or the C-terminal phosphorylation domain.
Agonist-induced regulation of cAR1 binding sites is probably
physiologically significant since car1
cells
expressing a mutant cAR devoid of C-terminal domain serines exhibit
abnormal development(46) . An analogous situation occurs in the
yeast Saccharomyces cerevisiae, where the G protein-coupled
receptor for
-pheromone is phosphorylated and internalized through
a G protein-independent mechanism (47) that also promotes
mating discrimination through this receptor(48) . In contrast
to these systems, the rate and extent of agonist-dependent
phosphorylation of the
-adrenergic receptor is notably enhanced by
G
-subunits that recruit the
-adrenergic receptor kinase
to the membrane(49) .
While the Ca influx
response of Dictyostelium cells is less well understood than
the desensitization of cAR1 activity, agonist-generated Ca
fluxes across the plasma membrane and intracellular membranes
raise levels of cytosolic free Ca
(50, 51) and appear to be critical for motility,
differentiation, and morphogenesis in this organism (see Refs. 18 and
51 and references therein). The Ca
entry system of
this organism appears to be novel since, in contrast to those of other
cell types(52, 53, 54) , it is not activated
by voltage changes, second messengers (intracellular cAMP, cGMP,
IP
), G
-subunits (9, 18) , or
G
-subunit complexes (Fig. 4). Interestingly, the m3
muscarinic receptor also triggers a voltage-insensitive Ca
influx that is independent of inositol phosphates and probably of
G proteins(55) , although this response was not characterized
in g
or g
cell lines.
The determinants of cAR1 required for the
regulation of Ca influx are unclear. The
phosphorylation state of cAR1 appears not to influence this response
since receptor mutants, in which all of the C-terminal receptor
phosphorylation sites have been replaced, induce a Ca
uptake with normal kinetics of activation and attenuation. (
)Moreover, mutant receptors lacking most of the
intracellular C-terminal domain or with extensive alterations of the
third intracellular loop promote Ca
entry.
Whether additional proteins or the receptor itself mediate the
Ca
response must be assessed. In this regard,
bacteriorhodopsin and halorhodopsin, seven membrane span receptors that
do not interact with G proteins, transport ions across membrane
bilayers(56) . Our results, together with the discovery of an
angiotensin receptor that is required for growth and development but
does not appear to couple to G proteins(57) , reflect the
growing diversity among this family of seven helix receptors. It is
interesting to speculate that G protein-coupled receptors in mammalian
cells also signal through G protein-independent mechanisms.