(Received for publication, November 6, 1995; and in revised form, January 24, 1996)
From the
We have mutated two residues, Ala and
Leu
, in the C-terminal portion of the third intracellular
loop of the human platelet-activating factor (PAF) receptor into
Glu
and Arg
, respectively. The Leu
Arg
substitution led to two major
modifications: 1) increased constitutive activity of the PAF receptor
resulting in agonist-independent production of inositol phosphates and
2) increased affinity of the receptor for binding PAF (agonist) but not
WEB2086 (antagonist). The L231R mutant was able to adopt at least two
conformations: (i) a higher affinity state than the corresponding state
of the wild-type receptor (WT), dependent on G protein coupling, and
(ii) a low affinity state, higher than the one for the uncoupled WT
receptor. The Ala
Glu
substitution
also resulted in two major modifications: 1) unresponsiveness in terms
of phosphatidylinositol hydrolysis in response to PAF and 2) a marked
decrease in affinity of the receptor for binding the agonist but not
the antagonist. Competition binding studies of transient receptor
expression in COS-7 cells and the inability of guanosine
5`-O-(3-thiotriphosphate) to modulate the decrease in affinity
of a stable A230E mutant in Chinese hamster ovary cells suggest an
inherent low affinity conformation for this mutant. Alternatively,
mutation of Ala
to Gln
suggested that the
residue 230 has a fundamental effect on receptor affinity and its
charge is determinant in G protein coupling of the PAF receptor. In
this report, we show that substitution of two immediately adjacent
residues of the PAF receptor, Ala
and Leu
,
surprisingly leads to an inactive and a constitutively active
phenotype, respectively. These results further support the concept of
constitutively active G protein-coupled receptors as adopting
``active'' state conformations similar to those induced by
agonist binding to WT receptors.
Platelet-activating factor (PAF), ()identified as
1-O-alkyl-sn-glycero-3-phosphocholine, is a
phospholipid released from stimulated basophils, platelets,
macrophages, polymorphonuclear neutrophils, and other cell
types(1) . PAF is a potent mediator with numerous biological
activities related to inflammatory and immune responses, as well as
cardiovascular, reproductive, respiratory, and nervous system
physiology(1, 2) . PAF exerts its action by binding to
a specific, high affinity receptor on the target cell surface. This
receptor is stereospecific and PAF-dependent cellular responses can be
inhibited by a variety of structurally distinct PAF
antagonists(2) . PAF binding has been found on several cell
types and cDNA cloning from various sources revealed that the PAF
receptor belongs to the G protein-coupled receptor
superfamily(3, 4, 5, 6, 7) .
The PAF receptor couples with various second messenger systems,
including phospholipases A, C, and D activation (8, 9, 10) and activation of the
mitogen-activated protein kinase
cascade(9, 11, 12, 13) .
For the
majority of the G protein-coupled receptors studied so far, the
ligand-binding pocket appears to be formed by the transmembrane domains
of the peptide chain, whereas the coupling to specific G proteins seems
to be mediated by the intracellular loops, predominantly by the third
intracellular loop, and especially those regions in close proximity to
the inner surface of the plasma membrane ((14, 15, 16) , and references therein).
Recently, it has been reported that site-directed mutagenesis of
specific residues in the C-terminal portion of the third intracellular
loop of the ,
, and
-adrenergic receptors constitutively activate these
receptors(14, 15, 16) . We were interested in
defining a region involved in G protein interaction with the PAF
receptor. In addition, we wished to ascertain whether the
constitutively active receptor forms induced by mutations in the third
intracellular loop of the adrenergic receptor family could also be
generated in a G protein-coupled receptor for a lipid ligand. Finally,
the aim of this work was to develop a tool for screening inverse
agonists for this receptor as potential therapeutic agents in PAF
pathophysiology. We report that the Ala
Glu
and Leu
Arg
substitutions result, respectively, in an inactive and a
constitutively active PAF receptor.
We were interested in defining a region in the third
intracellular loop of the PAF receptor that was involved in G protein
interaction or whose modification might lead to the constitutive
activation of the receptor. Substitutions of amino acids were made with
residues which had a high probability of causing a change in the
structure-function of the PAF receptor, based on the work of Kjelsberg
and co-workers(15) . This group reported the constitutive
activation of the -adrenergic receptor by all amino
acid substitutions at the Ala
position (the residues with
the most pronounced effects being Arg, Lys, and Glu) which is
positioned similarly to Ala
and Leu
of the
PAF receptor with respect to the inner surface of the plasma membrane. Fig. 1shows a representation of the putative seven
membrane-spanning domain topography of the PAF receptor and indicates
the two amino acids which were replaced in the mutant receptors.
Figure 1: Putative seven-transmembrane segment topography of the PAF receptor. Solid circles indicate the amino acids of the PAF receptor that were mutated; the amino acids replaced in the mutant PAF receptors are indicated on the right.
We first examined the agonist-independent IP production and agonist-specific increase of binding affinity in these mutants. For these initial experiments, COS-7 cells were chosen for their high expression levels, which maximized basal activity, and for determining ligand binding properties independent of G protein coupling(16) .
Figure 2:
Basal and stimulated IP levels in COS-7
cells expressing WT or mutant PAF receptors. The receptor densities in
transfected COS-7 cells are indicated in Table 1. Shown are the
IP levels per 30-mm dish above those of control cells which were
transfected with vector alone (A) in the absence of PAF and (B) after a 30-s stimulation with 10M PAF. The results are the means ± S.E. of three independent
experiments, each done in duplicate.
Figure 3: IP accumulation in response to graded concentrations of PAF. Total IPs were measured in COS-7 cells transfected with vector alone (control), the A230E mutant, the A230Q mutant, the L231R mutant or the WT receptors following a 30-s stimulation with the indicated PAF concentrations. The results are the means of three independent experiments, each done in duplicate.
Figure 4:
Competition binding isotherms of H-WEB2086 by WEB2086 (A) or PAF (B) in
COS-7 cells.
H-WEB2086 binding was measured as indicated
under ``Materials and Methods'' on membranes derived from
COS-7 cells transiently expressing the WT, A230E, A230Q, or the L231R
mutant receptors. The results are representative of three independent
experiments the mean values of which are reported in Table 1.
We examined the ligand binding
properties of the WT and mutant receptors in membranes derived from
transfected CHO cells in the presence or absence of 10 µM GTPS. Binding isotherms revealed that WT and mutated
receptors bound the antagonist
H-WEB2086 with the same
affinity (Table 1) which is not modulated by the presence of
GTP
S (data not shown). In contrast with the situation described
above for the COS-7 cells overexpressing the receptors, competition of
H-WEB2086 binding by PAF in membranes of CHO stable
transfectants resulted in curves that could be resolved into high and
low affinity components, except for the A230E mutant (Fig. 5).
Similar results were obtained in COS-7 cells transiently transfected
using conditions to allow for low levels of receptor expression (data
not shown). Binding properties of the CHO transfectants are summarized
in Table 1. As shown by the competition binding curves, the high
affinity state of the L231R mutant (K
= 0.2 nM) is much higher than the WT receptor (K
= 6.7 nM).
According to Fig. 5A, around 60% of total
H-WEB2086 binding loss by competition with PAF is
associated with high affinity receptors for the L231R mutant compared
to 40% for the WT receptors, respectively. These data suggest that
there could be a higher percentage of receptors coupled to a G protein
for the L231R mutant compared with the WT receptor. GTP
S mediated
the conversion of both WT and L231R mutated receptors from their high
affinity states into their respective low affinity states. In agreement
with our observations in COS-7 cells, these experiments indicated that
the low affinity state of the mutant receptor has higher affinity for
PAF than the corresponding state of the WT receptor (Fig. 4B).
Figure 5:
Competition binding isotherms of H-WEB2086 with PAF in the absence or presence of GTP
S
in CHO cells.
H-WEB2086 binding was determined as described
under ``Materials and Methods'' on membranes derived from CHO
cells expressing the L231R mutant (A), the A230E mutant (B), the A230Q mutant (C), or the WT receptor (A,
B, and C) in the absence or presence of 10 µM GTP
S. Results shown are representative of three independent
experiments the mean values of which are reported in Table 1.
The inactive A230E mutant shows no high
affinity component in its competition binding curve (Fig. 5B). In fact, the curve obtained is monophasic
and the PAF binding properties for the A230E mutant are unaltered by
GTPS. This mutant binds the agonist with at least 10 times less
affinity than the uncoupled WT receptor. Again, these results are in
agreement with the experiments carried out in COS-7 cells (Fig. 4B).
The A230Q mutant displayed a biphasic
binding isotherm with a high affinity state almost 100 times lower than
the corresponding state of the WT receptor (Fig. 5C).
The percentage of H-WEB2086 binding loss associated with
high affinity PAF receptors was essentially the same between the A230Q
and the WT receptors. The uncoupled A230Q receptor had an affinity for
PAF approximately 10 times lower than the uncoupled WT receptor.
Together, these results suggest that the A230Q mutation brings a severe
loss of affinity of the PAF receptor for its agonist and a decreased
responsiveness of the PAF receptor to agonist stimulation in terms of
IP accumulation without apparently altering the G protein interaction
with the receptor.
The PAF receptor activates multiple signaling pathways in response to its agonist, which has numerous biological activities(11) . The present study was undertaken to determine a region involved in G protein coupling of the PAF receptor.
Constitutively active receptors of the G protein-coupled receptor family have been reported by several groups to be involved in certain human diseases. These constitutively active receptors have residues that have been substituted in various regions of their seven membrane-spanning structure (reviewed in (23) ). The in vitro mutation of residues in the C terminus of the third intracellular loop of the adrenergic receptor family resulted in constitutively active receptors(14, 15, 16) . That region has been shown to be important in receptor coupling to its G protein (24) and some of its sites to be essential in keeping the receptor in a constrained, inactive conformation in absence of agonist(14, 15, 16) .
We have mutated
Ala and Leu
of the C terminus of the third
intracellular loop of the human PAF receptor into Glu
and
Arg
, respectively. These non-conservative mutations were
chosen to maximize chances of obtaining a constitutive mutant
receptor(15) . The major findings of the present study are that
the substitutions of two immediately adjacent residues, Ala
and Leu
, surprisingly led to completely opposite
phenotypes; the A230E mutant receptor is totally unresponsive and has a
decreased affinity for PAF; and the L231R mutated receptor shows
constitutive activity and displays a higher affinity for PAF than the
WT receptor.
According to the extended ternary complex model, G
protein-coupled receptors exist in an equilibrium between
``inactive'' and ``active'' conformational states:
R R
, respectively (16) . The agonist is
thought to stabilize the active conformation by virtue of a
preferentially higher affinity for R
, thereby shifting the
equilibrium to the right and resulting in productive receptor-G protein
coupling(15) .
The lack of response of the A230E mutant may
be coming from an incapacity to interact productively with a G protein
suggested by the absence of IP accumulation at high agonist
concentrations and by the competition binding curves that are
monophasic in the CHO expression system and unaffected by guanyl
nucleotides. To ascertain whether the A230E mutant properties were
conferred by the negative charge of the Glu residue brought into the
basic microenvironment of the C terminus of the third intracellular
loop of the PAF receptor, the Ala residue was also
mutated to the Glu isoster, Gln. The A230Q mutant was a 100-fold less
sensitive to PAF in IP production than the WT receptor. In terms of
agonist binding, the A230Q mutant displayed a
10- and
100-fold
decreased affinity for PAF when compared to the WT receptor for the low
and high affinity states, respectively. The A230Q mutant was still
able, however, to interact with G proteins as shown by the IP
production, by its biphasic binding isotherm and by the percentage of G
protein-coupled receptors which was similar to the WT receptors. The
decreased response following PAF stimulation of this receptor mutant
seems to be due to its decreased affinity for the agonist.
It is
interesting to note that both the A230E and the A230Q mutants had lower
intrinsic affinities for PAF than the WT receptor but that only the
A230E mutant seemed unable to interact with G proteins. It appears that
the Ala position is critical in regulating the PAF
receptor conformation, as judged by the agonist binding affinities of
the mutants at this position. Moreover, our results show that the
charge of the residue found at this position is determinant in the PAF
receptor ability to interact with G proteins. The charge of the
Glu
residue of the A230E mutant could prevent the R
R
isomerization in this receptor, as supported by
the very low basal IP accumulation, and/or by interfering in the
interaction between the receptor and the G protein.
Mutations
conferring a constitutively active phenotype make the R to R transition agonist-independent and should bestow a higher
affinity for agonists(15) . The L231R mutated receptor showed a
basal activity approximately 10 times higher than the WT receptor.
Similar results were obtained in a study where a constitutively active
-adrenergic receptor accumulated cAMP about 10 times
more than the WT receptor when the levels of expression were considered (16) . Constitutive mutants of the
-adrenergic
receptor also resulted in basal activity approximately 10 times higher
when the Thr
residue was substituted by a Cys or a Lys
residue (14) . Unlike the L231R mutant, the constitutively
active
- and
-adrenergic receptors
had basal activities that were similar to the stimulated WT
receptors(14, 16) . When stimulated with
10
M PAF, the L231R mutant accumulated
3-fold higher levels of IPs than the WT receptor. If the stimulated IPs
levels are considered as relative increases above the elevated basal
levels, the WT receptor accumulates 52 times more IPs when stimulated
as compared to basal activity whereas the stimulated L231R mutant has a
relative increase of 16. Such differences may be explained by a higher
fraction of the L231R mutant receptors already engaged in coupling to a
G protein (basal activity) that cannot be further stimulated, resulting
in a lower relative increase. Nonetheless, at any given agonist
concentration, the L231R mutant accumulated more total IPs than the WT
receptor and this accumulation did not reach a plateau within the
agonist concentrations used. Hypersensitivity of biological responses
has also been reported for other receptors such as the
-adrenergic (25) and the yeast a-factor (26) receptors.
The constitutive L231R mutant has a higher
affinity for PAF than the WT receptor both in the coupled and the
uncoupled states. The lower affinity which is considerably higher than
the corresponding state of the WT receptor is an intrinsic property of
the mutant molecule as shown by binding experiments in COS-7 cells
expressing receptors at high levels (16) and by using GTPS
in CHO cells. Such intrinsic higher affinities have been described for
constitutive mutants of the adrenergic receptor
family(14, 15, 16) . The intrinsic high
affinity property of a constitutively activated
-adrenergic receptor was shown not to reflect an
altered interaction with a G protein, since when receptors were
solubilized and purified, the mutant receptor retained its higher
affinity than the WT(16) .
The increase in affinity of the
``high affinity state'' of the L231R mutant compared to the
WT receptor is remarkable and appears to be G protein
coupling-dependent. In fact, we see with the competition binding
studies in CHO cells that the binding curve for the L231R mutant is
biphasic, indicating that there is an equilibrium between the two
states of the L231R receptor, RR
; that is, the
constitutive receptor is not uniquely in a high affinity state and it
needs to couple to a G protein to be so. The ``low affinity
state'' of the L231R mutant is intermediate between the
``high'' and the low affinity states of the WT receptor. This
low affinity state, therefore, does not correspond to the coupled WT
receptor. Moreover, we have shown that this high intrinsic affinity of
the low affinity state appears to be the uncoupled form of the L231R
receptor. Thereby, the elevated basal activity of the L231R mutant
would not result from its high intrinsic affinity, but could result
from a bigger tendency of the L231R mutant to isomerize to
R
, favoring productive interaction with G proteins. This
tendency seems to be reflected in our data by what could be a higher
percentage of receptors coupled to a G protein for the L231R mutant
relative to the WT receptor. The L231R mutation does not confer the
``relaxed'' conformation to the receptors but rather allows
them to achieve more easily the R
state in a dynamic
process; otherwise monophasic high affinity competition binding curves
unaffected by guanyl nucleotides would be obtained. Supporting our
data, computer simulations implied that constitutive mutant receptors
isomerize to an active state more readily than do WT
receptors(16) . At present, we cannot exclude the possibility
that the primary effect of the alterations is to increase coupling to
the G protein that would result in higher activity of the L231R mutant.
But the L231R mutation must influence the PAF receptor molecule in
other ways because of the differences between the high affinity states
of this mutant and of the WT receptor that cannot be explained only by
the intrinsic higher affinity or by increased G protein coupling.
Perhaps the combination of those factors can lead to a conformational
change resulting in this remarkable higher affinity.
There is
considerable evidence that receptor interaction with G proteins is
driven by two factors, conformation and electrostatic
interactions(27) . The introduction of the negatively charged
Glu at position 230 led to total loss of G protein coupling, while
substitution of Gln for Ala brought back the coupling of
the receptor. We might speculate that the L231R mutation is bringing
one more positively charged residue in an already basic
microenvironment (KRRARL) that could favor the attachment of the
G protein. Further experiments will be necessary to delineate the
mechanisms involved in the higher affinity of the L231R mutant, as well
as in its basal and stimulated activities. It must be noted that the
present studies are necessarily performed in a transfected system, in
which the PAF receptor preferentially couples to the
class of G proteins. The implication of the residues described
herein in leukocytes in which
subunits are also
involved, remains to be evaluated.
In summary, both residues at
position 230 and 231 are important in the regulation of the PAF
receptor conformation. Mutation of Leu to Arg
led to higher agonist affinity and higher agonist-independent and
-dependent activity of the PAF receptor. Mutation of Leu
into other residues will reveal whether only the specificity of
the WT sequence at this position is uniquely constraining the activity
of the receptor in the unliganded state. The residue 230 has profound
effects on agonist affinity and its charge is determinant in G protein
coupling. Substitutions by other residues will enlighten the
relationship between amino acid side chains at this position and the
structure-function of the PAF receptor. We have defined two adjacent
residues that when mutated lead to completely opposite phenotypes. Work
is presently underway in our laboratory to determine if a
``hinge'' region has been identified, by substituting
neighboring residues.
This system extends the notion that the C terminus of the third intracellular loop of the G protein-coupled receptors plays a determinant role in the overall conformation of the receptor molecule and the L231R mutant receptor should lend itself to screening for inverse agonists as potential therapeutic tools in PAF pathophysiology.