(Received for publication, May 22, 1995; and in revised form, July 10, 1995)
From the
Identification of the docking interactions by which peptide
agonists activate their receptors is critical for understanding signal
transduction at the molecular level. The human and Xenopus thrombin
receptors respond selectively to their respective hexapeptide agonists,
SFLLRN and TFRIFD. A systematic analysis of human/Xenopus thrombin
receptor chimeras revealed that just two human-for-Xenopus amino acid
substitutions, Phe for Asn in the Xenopus receptor's
amino-terminal exodomain and Glu for Leu
in the second
extracellular loop, conferred human receptor-like specificity to the
Xenopus receptor. This observation prompted complementation studies to
test the possibility that Arg
in the human agonist peptide
might normally interact with Glu
in the human receptor.
The mutant agonist peptide SFLLEN was a poor agonist at the wild type
human receptor but an effective agonist at a mutant human receptor in
which Glu
was converted to Arg. An ``arginine
scan'' of the receptor's extracellular surface revealed
additional complementary mutations in the vicinity of position 260 and
weak complementation at position 87 but not elsewhere in the receptor.
Strikingly, a double alanine substitution that removed negative charge
from the Glu
region of the human receptor also
effectively complemented the SFLLEN agonist. The functional
complementation achieved with single Arg substitutions was thus due at
least in part to neutralization of a negatively charged surface on the
receptor and not necessarily to introduction of a new salt bridge. By
contrast, charge neutralization did not account for the gain of
responsiveness to SFLLRN seen in the human/Xenopus receptor chimeras.
Thus two independent approaches, chimeric receptors and arginine
scanning for complementary mutations, identified the Glu
region and to a lesser degree Phe
as important
determinants of agonist specificity. These extracellular sites promote
receptor responsiveness to the ``correct'' agonist and
inhibit responsiveness to an ``incorrect'' agonist. They may
participate directly in agonist binding or regulate agonist access to a
nearby docking site.
Seven transmembrane domain G protein-coupled receptors respond to a structurally diverse set of ligands to regulate a host of biological processes. Catecholamines and certain other small ligands elicit responses by binding to their receptors' transmembrane regions(1) . The docking interactions by which peptide agonists activate their receptors are less well-characterized(2, 3, 4, 5, 6, 7) . The thrombin receptor can be viewed as a specialized peptide receptor that contains its own ``tethered ligand.'' Thrombin activates its receptor by cleaving the receptor's amino-terminal exodomain. This limited proteolysis unmasks a new amino terminus which then functions as a tethered peptide agonist, binding to the body of the receptor to cause signaling (8, 9, 10) . Synthetic peptides which mimic the tethered ligand domain behave as peptide agonists, activating the receptor independent of thrombin. Identification of the interactions by which the thrombin receptor's tethered ligand domain triggers transmembrane signaling is central to understanding signaling by this and perhaps other peptide receptors. Such information may also aid the development of novel pharmaceuticals for inhibiting thrombotic, inflammatory, and proliferative actions of thrombin(11, 12) .
Three-dimensional structures for seven transmembrane domain G protein-coupled receptors are currently of low resolution, especially in the extramembranous regions(13) . Thus structures that reveal the details of agonist docking, particularly for agonists that interact with the receptor's extracellular loops, are unlikely to be available in the near future. Mutagenesis studies which examine functional end points remain a valuable approach that provides constraints for model building and analysis of future structural data.
We exploited the specificity of the human and Xenopus thrombin receptor homologues for their respective agonist peptides to identify the receptor domains which distinguish between these agonists(14) . A chimeric receptor in which the Xenopus receptor's extracellular surface (its amino-terminal exodomain and three extracellular loops) was replaced with that of the human receptor showed a remarkable gain of responsiveness to the human agonist and loss of responsiveness to the Xenopus agonist, resulting in human receptor-like agonist specificity(14) . More limited replacement of Xenopus with human receptor sequence suggested that two regions accounted for this change in specificity: residues 244-268 in the second extracellular loop and residues 76-93 located in the amino-terminal exodomain near the start of transmembrane domain 1(14) . An antiserum which recognized the latter region blocked receptor activation by agonist peptide, consistent with a role for this region in agonist function(15, 16) .
With
the long term goal of defining the agonist-receptor interactions which
mediate receptor activation, we analyzed progressively finer chimeras
to identify the specific amino acids in the human and Xenopus thrombin
receptors which distinguish their cognate agonists. Ultimately, two
single human for Xenopus amino acid substitutions in the Xenopus
receptor, Phe for Asn in the amino-terminal exodomain and
Glu for Leu
in the second extracellular loop, proved
sufficient to confer robust responses to human agonist. The gain of
function conferred by the Leu
Glu substitution prompted
complementation studies to test the hypothesis that Arg
in
the human agonist peptide might normally interact with Glu
in the human receptor (Fig. 1). These studies again
pointed to the receptor domains surrounding Glu
and to a
lesser degree Phe
but revealed that these domains can
contribute to receptor specificity by excluding activation by unwanted
agonists instead of, or in addition to, providing direct docking
interactions. Overall, our results strongly suggest that these
extracellular receptor regions of the thrombin receptor participate in
receptor activation by binding agonist directly or by regulating
agonist access to a nearby docking site.
Figure 1:
Comparison of human and
Xenopus thrombin receptor agonists and the domains that distinguish
between them. The cleavage site at which thrombin acts to unmask the
tethered ligand or ``agonist peptide'' domain is indicated by
the small arrow. After cleavage, this domain binds
intramolecularly to the body of the receptor to cause signaling.
Synthetic peptides mimicking this domain are full agonists at the
thrombin receptor, bypassing the need for cleavage by thrombin. The
human and Xenopus tethered ligand sequences are compared as are the
receptor regions found to be important in distinguishing between these
two agonists. The important Phe and Glu
regions are indicated in bold. Bold arrows indicate possible complementary
changes.
Responses of wild type and mutant thrombin receptors to
peptide agonists were determined in the Xenopus oocyte expression
system using previously described procedures(8, 14) .
Briefly, cDNAs encoding mutant human and Xenopus thrombin receptors
were generated by standard techniques (17, 18) and
confirmed by dideoxy sequencing(19) . All wild type and mutant
receptors used in this study were epitope tagged with the FLAG sequence
present at the amino terminus of the mature receptor
protein(14, 20) . To describe the various mutants, we
use the amino acid number of the human thrombin receptor sequence with
the start methionine designated as one to refer to both human and their
cognate Xenopus receptor residues(8, 14) ; the
relevant alignment is shown in Fig. 1and Table 1. cRNAs
for the wild type and mutant receptors were transcribed from cDNAs
subcloned into pFROG(8) . 12.5 ng of wild type cRNA and
12.5-25 ng of mutant receptor cRNAs were injected/oocyte (8, 14) . After culture for 24 h, receptor expression
on the oocyte surface was measured as specific binding of antibody to
the FLAG epitope(14) . The quantity of cRNA injected was
adjusted to maintain surface expression of mutant receptors between 50
and 150% of wild type. Responses to wild type and mutant human and
Xenopus agonist peptides were measured as agonist-induced Ca efflux which reflects phosphoinositide hydrolysis in
these cells(8, 14) . For determining EC
s,
four to five different concentrations of each agonist (from sub
EC
to maximally activating) were employed. All
determinations of EC
values for mutant receptor activation
included concentration response curves for the appropriate wild type
receptor(s) in the same experiment. All experiments were replicated at
least twice.
The peptide agonists used in these studies were synthesized as the carboxy amide forms and were purified by high performance liquid chromatography before use(14, 21) .
The tethered ligand domains of the human and Xenopus thrombin receptors are strikingly different: SFLLRN for human and TFRIFD for Xenopus (14) (Fig. 1). Synthetic peptides mimicking these domains show specificity for their respective receptors. Indeed, the human agonist peptide is approximately 1000-fold more potent at the human receptor than at the Xenopus receptor. Our previous studies showed that replacement of the Xenopus thrombin receptor's amino-terminal exodomain and second extracellular loop with the corresponding human sequences resulted in a chimeric receptor with human receptor-like agonist specificity. Individually, these two substitutions resulted in substantial but less dramatic gains of responsiveness to the human agonist(14) .
To identify the
specific amino acids in the human and Xenopus thrombin receptors
responsible for distinguishing between their cognate agonists, we
constructed progressively finer chimeras and determined their
responsiveness to human versus Xenopus agonist peptides ( Fig. 1and Table 1). We first focused on the putative
binding region in the receptor's amino-terminal exodomain (14) . Substitution of human receptor amino-terminal exodomain
(HAT) residues 76-93 for the cognate sequence in the Xenopus
receptor yielded some gain of responsiveness to the human agonist but
an exaggerated loss of responsiveness to the Xenopus agonist. More
informative results were obtained by substituting smaller overlapping
segments (76-86, 82-90, and 87-94). The
HAT82-90 chimera exhibited a substantial gain of responsiveness
to the human agonist. HAT76-86 had agonist specificity that
differed little from the Xenopus wild type receptor and HAT87-94
was uninformative, showing loss of responsiveness to both human and
Xenopus agonists (Table 1). The contrasting phenotypes of the
HAT76-86 and HAT82-90 chimeras pointed to residues
87-90 as mediating gain of responsiveness to human agonist.
Indeed, HAT86-90 showed a more than 10-fold lower EC for the human agonist compared to the wild type Xenopus receptor.
Within this region, HAT87 also yielded a more than 10-fold gain of
responsiveness to the human agonist and HAT89,90 a 5-fold gain. HAT86
showed no gain, and Ile
is conserved in the two receptors.
The HAT87 human-for-Xenopus amino acid exchange disrupted a
consensus sequence for N-linked glycosylation by substituting
a Phe for Asn (NIT, Fig. 1). Substitution of Ala
for Asn
in the Xenopus receptor produced no gain of
responsiveness to human agonist peptide (HAT87A, Table 1), thus
the gain of responsiveness to human agonist seen with HAT87 was due to
introduction of the phenyl side chain and not to ablation of an N-linked glycosylation site. Taken together, the data
presented above show that human thrombin receptor amino-terminal
exodomain residues 87-90 play an important role in defining
agonist specificity with Phe
being particularly important.
We used a similar approach to identify the amino acids in the
receptor's second extracellular loop that distinguish between the
human and Xenopus agonists. This loop can be divided into two halves
separated by a cysteine thought to participate in a disulfide bridge
between the first two extracellular loops(8) . HECL2,
254-268, a chimera in which the ``second half'' of the
human receptor's second extracellular loop was substituted for
the cognate Xenopus residues, showed a remarkable gain of
responsiveness toward the human peptide (Fig. 1, Table 1).
By contrast, a chimera containing the ``first half'' of this
loop (HECL2, 244-254) had Xenopus receptor-like agonist
specificity (Fig. 1, Table 1). Because residues
254-258 are conserved in the Xenopus and human receptors, we next
analyzed a series of chimeras in which overlapping segments covering
human residues 259-268 were substituted for the corresponding
Xenopus sequences. HECL2, 259-262 showed a greater than 30-fold
decrease in EC to the human agonist; little change was
seen with the HECL2, 261-265 or 264-268 substitutions. To
determine whether the consensus N-linked glycosylation site
introduced by the HECL2, 259-262 substitution played a role in
the gain of responsiveness to human agonist, HECL2, 259-262 AETL
was constructed. This construct, which substituted Ala-Glu-Thr-Leu
(AETL) rather than Asn-Glu-Thr-Leu (NETL) for the cognate Xenopus
residues DLKD, still yielded a remarkable gain of responsiveness to the
human peptide, but less impressive than that seen with
HECL2,259-262. Whether glycosylation per se or the Asn
side chain itself account for Asn's contribution in this context
is not known. Taken together, these data pointed to residues 259 and
260, Asn and Glu in the human receptor versus Asp and Leu in
the Xenopus, as important for agonist specificity (Table 1).
Notably, changing Leu
in the Xenopus receptor to Glu
(HECL2, 260) yielded a gain of responsiveness to the human peptide
equivalent to that achieved with substitution of the entire second
extracellular loop (Table 1).
The best single amino acid
substitutions identified in each of the two ``specificity
regions'' defined above were combined to generate ``HAT87
+ HECL2, 260.'' This chimera had human receptor-like agonist
specificity despite bearing only two human-for-Xenopus amino acid
substitutions ( Fig. 2and Table 1). The corresponding
double alanine substitution mutant had only a small relative gain of
responsiveness to human agonist compared to the wild type Xenopus
receptor. Taken together with our earlier studies(14) , this
completes a systematic analysis of human/Xenopus thrombin receptor
chimeras to identify the specific receptor residues which distinguish
the human from Xenopus agonists. The data raised the possibility that
the agonist peptide might interact directly with the receptor's
extracellular surface at or near positions 87 and 260 and suggested a
candidate docking interaction. In the agonist peptide, position 5
changes from the aromatic Phe in Xenopus to the basic
Arg
in human. In the receptor, position 260 changes from
the hydrophobic Leu
in Xenopus to the acidic Glu
in human. The remarkable gain of responsiveness to human agonist
conferred by replacing Xenopus receptor's Leu
with
Glu thus suggested that Arg
in the human agonist might
dock with Glu
in the human receptor ( Fig. 1and Table 1).
Figure 2:
Two
single amino acid substitutions confer human receptor-like specificity
to the Xenopus thrombin receptor. Human or Xenopus wild type thrombin
receptors or the Xenopus receptor in which amino acids 87 and 260 were
replaced by the cognate human residues (HAT87 + HECL2,
260) were expressed in Xenopus oocytes and responses to the
indicated concentrations of human (SFLLRN) or Xenopus (TFRIFD) agonist peptides were determined. The specific amino
acid substitutions are indicated in single letter code. Data
(mean ± S.D. (n = 4)) are expressed as percent
maximal agonist-induced Ca release and represent four
replicate experiments. Expression levels were similar for the three
receptors. Average maximal responses were also similar and ranged from
25- to 50- fold increases in agonist-induced
Ca release in
the various experiments. Arrows point out the >100-fold
gain in responsiveness (decrease in EC
) to human agonist
and 4-fold loss of responsiveness (increase in EC
) to
Xenopus agonist caused by the two amino acid
substitutions.
This hypothesis was first tested by introducing
potentially complementary amino acid substitutions at these positions
in the human agonist and receptor. Replacement of Arg in
the human agonist peptide with Glu caused a remarkable loss of function
at the wild type human receptor. Strikingly, this was remedied by
replacing Glu
in the human receptor with Arg but not Ala (Fig. 3).
Figure 3:
Complementary mutations in agonist peptide
and receptor. Wild type human thrombin receptor or mutant human
receptors in which Glu was converted to arginine (E260R) or alanine (E260A) were expressed in Xenopus
oocytes and
Ca release in response to the indicated
concentrations of wild type human agonist (SFLLRNPNDK, SFLLRN)
or a mutant agonist peptide in which arginine in position five was
changed to glutamate (SFLLENPNDK, SFLLEN). The longer agonist
peptides were used because the SFLLEN hexapeptide was not sufficiently
soluble. Data (mean ± S.D. (n = 4)) are
expressed as percent maximal agonist-induced
Ca release
and represent four replicate experiments. Expression levels and maximal
responses were similar for the three receptors. Maximal responses
ranged from 35- to 45-fold increases in agonist-induced
Ca
release. Arrows point out the >50-fold decrease in
EC
for the glutamate bearing agonist and the 4-fold
increase to native agonist caused by the Glu
Arg (E260R)
substitution.
To determine the specificity of this
complementation phenomenon, we first tested agonist peptides in which
Glu or Asp replaced the native agonist residues at positions 2-5
for their ability to activate wild type versus GluArg and Phe
Arg mutant human thrombin
receptors. Only the agonists with substitutions at position five were
effectively complemented by the Glu
Arg mutation (Fig. 4A). Asp and Glu substitutions behaved similarly,
consistent with a role for the negatively charged side chain. It was
possible that the side chains of the aspartates or glutamates
introduced at agonist positions 2, 3, or 4 formed an intramolecular
salt bridge with the Arg
side chain in this series of
peptides, making these side chains unavailable to participate in
interactions with the receptor. To examine this explanation of the lack
of complementation seen for agonists with substitutions at positions
2-4, we tested a second series of agonists (SFLLFNPNDK,
SFLLDNPNDK, SFLDFNPNDK, SFDLFNPNDK, and SDLLFNPNDK) that lacked
Arg
. In this series, SFLLFNPNDK was an effective agonist
at the wild type receptor, all aspartate substitutions caused loss of
agonist function at the wild type receptor, and only SFLLDNPNDK was
effectively complemented by the Glu
Arg receptor mutation
(data not shown). We cannot exclude the possibility that other
intra-agonist interactions specific to positions 2-4 make
negatively charged side chains introduced at these positions
unavailable to the receptor. However, at face value, these data suggest
that receptor position 260 looks primarily at agonist position 5.
Figure 4:
Specificity of complementary mutations. A, mutations in agonist suggest specificity for position 5.
Synthetic peptides representing wild type sequence SFLLRNPNDK or
peptides with the indicated substitutions were assayed for activity at
the wild type human thrombin receptor (HWT, open
bars), the GluArg (E260R, hatched
bars), and the Phe
Arg (F87R, closed
bars) mutant receptors expressed in Xenopus oocytes. Peptides were
added 100 µM and agonist-induced increased in
Ca release determined. Data shown are mean ± S.D. (n = 2). This experiment was replicated once. Note the
loss of function of the SFLLENPNDK and SFLLDNPNDK peptides at the wild
type receptor which was remedied by the Glu
Arg mutation
and to a lesser extent by the Phe
Arg mutation. B,
arginine scan of receptor. Xenopus oocytes expressing wild type human
thrombin receptor (HWT) or receptors bearing the indicated
single arginine substitutions were assayed for responsiveness to
SFLLENPNDK (100 µM) versus SFLLRNPNDK (10
µM). All mutant receptors were expressed at comparable
levels on the oocyte surface. (*) indicates ``loss of
function'' mutations; these mutant receptors yielded responses to
both wild type and mutant agonists that were less than 15% of those
seen with the wild type receptor. All other mutant receptors had
responses to wild type agonist that were indistinguishable from wild
type receptor. Accordingly, responses to the mutant peptide SFLLENPNDK
were expressed as percent of the wild type receptor's response to
wild type agonist determined in parallel in each experiment. The data
shown are the means of duplicate determinations. Similar results were
obtained in two or more experiments with each mutant receptor.
Receptors showing ``positive'' complementation were
arbitrarily defined as those that conferred responses to maximal
concentrations of SFLLENPNDK that were >50% of the maximal response
of wild type receptor to wild type agonist. C, arginine scan
summary: location of mutations and their phenotypes. The
receptor's extracellular loops are shown; Phe
and
Glu
are indicated. Closed spheres indicate
positions at which arginine substitution caused a significant gain of
responsiveness to the SFLLEN agonist. Gray spheres indicate
sites at which arginine substitution caused loss of function to both
agonists (see * in B), and open spheres indicate
sites at which arginine substitution had little effect. Note that the
``complementation sites'' indicated by the closed spheres map to the same regions that were identified in the chimera
studies and that these are surrounded by loss of function
sites.
Our second test of specificity was an ``arginine scan'' of
the receptor's extracellular surface, a search for mutant
receptors that would respond to the SFLLEN mutant agonist (Fig. 4, B and C). Remarkably, complementary
mutations were found only in the regions previously identified as
important for receptor specificity by the chimera studies. Arg
substitutions at Glu and Glu
provided
strong complementation and Arg substitutions at nearby residues 261,
263, 265, 268, and 269 caused lesser but reproducible gains in
responsiveness to SFLLEN relative to SFLLRN (Fig. 4). Arg
substitution at position 87, the other site identified in the chimera
studies, provided weak complementation. By contrast, Arg substitutions
in extracellular loops 1 and 3 and in the first half of loop 2, all
sites found to be unimportant for agonist specificity in the chimera
studies, failed to yield complementation. These findings again suggest
that the Glu
region and possibly Phe
are
positioned to interact with agonist position five.
What is the
physical basis for the functional complementation seen in these
studies? The most dramatic complementation was observed when Arg
replaced Glu at positions 260 or 264. By replacing or neutralizing
negative charges, Arg substitutions at these and nearby positions might
be acting to eliminate repulsive electrostatic interactions with
position five of the SFLLEN peptide rather than (or in addition to)
providing a salt bridge for binding Glu in the mutant
agonist (Fig. 5A). Arginine scanning of the Glu
region did reveal an apparent helical periodicity, with positive
complementation occurring every three to four residues (Fig. 4B), and it was possible that the local structure
of this loop placed the Glu
and Glu
side
chains in proximity. We therefore tested the double alanine
substitution Glu
Ala + Glu
Ala for gain
of responsiveness to the SFLLEN peptide. In contrast to single alanine
substitutions which had little effect ( Fig. 3and data not
shown), the double alanine substitution caused a gain of responsiveness
to the SFLLEN peptide comparable to that seen with the
Glu
Arg substitutions (Fig. 5). This suggests that
the Arg substitutions at positions 260 or 264 had complemented the
SFLLEN peptide's agonist function at least in part by
neutralizing nearby negative charge at positions 264 or 260,
respectively (Fig. 5).
Figure 5:
Charge neutralization in the arginine
substitution mutants and chimeras. Does removal of repulsive
electrostatic interactions between agonist and receptor explain the
gain of function seen in these studies? A, the sequence of the
Glu region in the human wild type (HWT) and relevant
mutant thrombin receptors. Residues that were altered in one or more of
the mutants are shown in bold. The predicted net charge of
this region at physiological pH is indicated at right. Note that the
Glu
region in the wild type human thrombin receptor (HWT) has a net charge of -2 and might repel the SFLLEN
mutant agonist. Arginine substitutions in this region reduced this net
charge, perhaps accounting for the observed gain of responsiveness to
SFLLEN. A double alanine substitution mutant that would also neutralize
the Glu
region (E260A, E264A) was
therefore tested for possible gain of responsiveness to the SFLLEN
agonist (C). B, charged residues in the region of the
Xenopus thrombin receptor corresponding to the human receptor's
Glu
region. Two lysines that might
interfere electrostatically with the action of the SFLLRN agonist are
present at positions 261 and 264. The HECL2,260 substitution, which
caused a gain of responsiveness to SFLLRN, did add a negatively charged
side chain to this region. A double alanine substitution mutant that
removed the potentially inhibitory lysines was tested for possible gain
of responsiveness to SFLLRN (see Table 1). C,
neutralization of negative charges in the Glu
region
causes gain of responsiveness to the SFLLEN mutant agonist. The wild
type human receptor (HWT) or the human receptor in which the
glutamates at positions 260 and 264 were converted to alanines (E260A + E264A) were expressed in Xenopus oocytes and
responses to the indicated concentrations of SFLLRNPNDK (SFLLRN) or
SFLLENPNDK (SFLLEN) were determined. Data shown are the means of
duplicate determinations. Similar results were obtained in five
separate experiments.
-
, E260A + E264A SFLLRN;
- - -
, HWT SFLLRN;
-
E260A + E264A
SFLLEN;
- - -
, HWT SFLLEN.
Does neutralization of repulsive
electrostatic interactions also account for the gain of responsiveness
to the human agonist peptide achieved in the receptor chimera studies?
The LeuGlu replaced a neutral side chain with an acidic
one. Might this acidic side chain be acting by neutralizing nearby
basic lysine side chains to decrease repulsive interactions with
Arg
in the human agonist (Fig. 5B)?
Substitution of alanines for the two lysines near position 260 in the
Xenopus receptor removed the positively charged side chains from this
region but caused neither gain of responsiveness to the human agonist
nor loss of responsiveness to the Xenopus agonist (Table 1). Thus
the gain of responsiveness seen in the chimera studies does not appear
to be due to removal of electrostatic interactions that interfere with
the action of the human agonist on the Xenopus receptor.
Alanine
scanning of the Phe and Glu
regions in the
human receptor was undertaken to define the importance of individual
side chains for responsiveness to agonist. Phe
Ala caused
a small loss of responsiveness to human agonist, a 13-fold increase in
EC
. Ala substitutions for the adjacent Ile
and Ser
caused 40- and 23-fold increases while
substitutions at Glu
and Asp
had little
effect (Table 2). In the second extracellular loop, little or no
loss of function occurred with alanine substitutions at residues
259-265. Thus in general, individual alanine substitutions at the
residues that the chimera and complementation studies pointed to as
important for maintaining receptor specificity caused little loss of
responsiveness to agonist (Table 2). However, larger deletions
and substitutions in either region did cause substantial loss of
receptor function ( Table 2and data not shown). The lack of
dramatic loss of function from single alanine substitutions in the
receptor's specificity regions is perhaps not surprising. In the
agonist itself, positions 3-6 presumably mediate receptor
specificity ( Fig. 1and (14) ) Single alanine
substitutions at these positions caused only partial loss of activity (14, 21) , and each amino acid in the agonist probably
interacts with several in the receptor.
In contrast to the
individual alanine or arginine substitutions within the specificity
regions of the receptor, single substitutions at aromatic or
hydrophobic residues bounding these domains caused substantial loss of
function ( Table 2and Fig. 4). Similarly, in the agonist
peptide, in contrast to ``specificity'' positions 3-6,
alanine substitution for the conserved Phe at agonist position 2
ablates agonist function(14, 21, 22) . The
finding of critical hydrophobic residues next to specificity residues
in both receptor and agonist is tantalizing. Wells and colleagues (23) have recently found that the majority of the binding
energy for growth hormone-receptor docking is contributed by
hydrophobic interactions, with nearby charged and hydrophilic residues
apparently helping to maintain specificity. Moreover, the docking
interactions of thrombin's anion-binding exosite with the
thrombin receptor itself relies on the interactions of aromatic and
hydrophobic residues that are surrounded by charged
surfaces(9, 24) . These analogies prompt the
speculation that the critical Phe in the thrombin
receptor's agonist peptide may dock in a hydrophobic pocket
bounded by the Glu
and perhaps the Phe
specificity regions. Candidates for contributing to this putative
hydrophobic pocket include the conserved aromatic
cluster(266-271) at the carboxyl end of the thrombin
receptor's second extracellular loop and/or Ile
in
its amino-terminal exodomain; point mutations at these sites cause
dramatic of loss of function and both sequences are continuous with the
specificity regions.
In summary, a systematic analysis of
human-Xenopus chimeric thrombin receptors identified two small regions,
residues 82-90 in the amino-terminal exodomain and 259-262
in the second extracellular loop, as important for distinguishing the
human versus Xenopus agonist peptides. Within these domains,
Phe and Glu
made major contributions. These
gain of function studies suggested the possibility of direct
interactions between these regions and the human agonist peptide and
raised the hypothesis that Arg
in the agonist might
interact with Glu
. This prompted a systematic arginine
scan of the thrombin receptor's extracellular surface in search
of mutations which would complement an Arg
Glu mutant
agonist peptide. Strikingly, this scan again identified the Glu
region and to a lesser degree Phe
, but not other
sites in the receptor. The successful complementation of the
Arg
Glu mutant agonist peptide by arginine substitutions in
the receptor's Glu
region appeared to be due to
removal of repulsive electrostatic interactions. By contrast, the gain
of function to human agonist seen in the chimera studies could not be
accounted for by such a mechanism.
Taken together, our data suggest
two alternative models. The Phe and Glu
regions of the receptor may simply play a gatekeeper role,
regulating agonist access to a nearby binding site that is responsible
for receptor activation. Alternatively, Arg
and perhaps
nearby residues in the human agonist peptide may dock directly with the
Glu
region and/or Phe
in the receptor and
contribute to the conformational change that causes receptor
activation. Our data show that these regions can both inhibit receptor
activation by an ``incorrect'' agonist and promote receptor
activation by the ``correct'' agonist, both features expected
for a docking site. Moreover, our recent finding that mutations in the
Glu
region can cause constitutive activation of the human
thrombin receptor support a direct role for this region in receptor
activation. (
)