(Received for publication, September 7, 1995; and in revised form, December 22, 1995)
From the
The human platelet thromboxane A receptor is a
member of the G-protein-coupled superfamily of receptors. Previous
pharmacologic studies examining the effects of biochemical reduction,
oxidation, or sulfhydryl alkylation on thromboxane receptors have
suggested a role for cysteines in determining receptor binding
characteristics. To characterize the roles of individual cysteines, we
employed site-directed mutagenesis to substitute serines for cysteines
at seven positions throughout the human K562 thromboxane receptor and
analyzed mutant receptor radioligand
([1S-(1
,2
(5Z),3
(1E,3S),4
]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptane-2-yl]-5-heptenoic
acid) binding and calcium signaling. Replacing cysteines in the amino
terminus (amino acid position 11), and transmembrane domains two and
six (positions 68 and 257) had little effect on thromboxane receptor
binding or signaling. Introduction of serines for cysteines in the
first (position 105) or the second (position 183) extracellular loop
eliminated thromboxane receptor binding, consistent with the existence
of a critical disulfide bond between these positions. Mutation of a
second cysteine in extracellular loop one (position 102) resulted in a
receptor with decreased binding affinity and low binding capacity that
transduced only a low amplitude calcium signal, suggesting the
involvement of a free sulfhydryl group at this location in
receptor-ligand interactions. Finally, mutation of the cysteine at
position 223, located in intracellular loop three, resulted in a
receptor with normal ligand binding characteristics, but which did not
transduce a calcium signal. Some additional amino acid substitutions in
this region of the receptor (Cys-223
Ala, Thr-221
Met)
resulted in receptors that had normal binding but transduced low
amplitude calcium signals, while other mutations in the same region
(His-224
Arg and His-227
Arg) exhibited normal binding
and calcium signaling characteristics. These findings demonstrate that
cysteines in extracellular loops one and two contribute to proper
ligand binding to thromboxane receptors and show the importance of
discrete amino acid sequences in the third intracellular loop,
especially cysteine 223, in thromboxane receptor-effector coupling.
Physiologic consequences of thromboxane agonism such as platelet
shape change/aggregation and vascular smooth muscle contraction are
mediated via a high affinity interaction of thromboxane A or prostaglandin H
with specific target cell membrane
receptors(1, 2) . Thromboxane receptor-mediated
activation of phospholipase C hydrolyzes phosphatidylinositol
bisphosphate and forms inositol 1,4,5-trisphosphate, which stimulates
release of calcium from intracellular stores(3, 4) .
Recently, a mutant human platelet thromboxane receptor with a single
amino acid substitution in the first intracellular loop (Arg-60
Leu) was identified as causing an inherited hemorrhagic disorder
characterized by defective thromboxane receptor coupling to
phospholipase C(5) . The existence of this mutant thromboxane
receptor and its associated disease illustrates the importance of
proper thromboxane receptor function to normal hemostasis and
emphasizes the need to identify structural determinants of thromboxane
receptor ligand binding and receptor-effector coupling.
A comparison
of the deduced amino acid sequences of various members of the
superfamily of G-protein-coupled receptors reveals several highly
conserved cysteine residues, suggesting that cysteines help to
determine receptor function as related to receptor structure. Cysteines
can modulate secondary and tertiary receptor structure due to their
unique ability to form intramolecular disulfide bonds. The human
platelet (K562) receptor contains eight cysteine residues(6) ,
and identifying a role for these amino acids in thromboxane receptor
ligand binding formed the rationale for a prior study that investigated
the effects of disulfide and sulfhydryl reactive agents on thromboxane
receptors(7) . In that study the effects of thromboxane
receptor binding on disulfide bond reduction with dithiothreitol,
oxidation with dithionitrobenzoic acid, and alkylation with N-ethylmaleimide were assessed. Dithiothreitol treatment
decreased ligand binding capacity, an effect that was reversed by
dithionitrobenzoic acid, suggesting that (one or more) disulfide bonds
were crucial for proper binding of thromboxane to its receptor.
Alkylation of free sulfhydryl groups with N-ethylmaleimide
also reduced
[1S-(1,2
(5Z),3
-(1E,3S),4
]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptane-2-yl]-5-heptenoic
acid ([
I]BOP) (
)binding capacity,
further suggesting that a cysteine(s) was involved in facilitating
binding of thromboxane to its receptor independent of disulfide bond
formation.
When the deduced amino acid sequence of the human platelet thromboxane receptor is compared to that of related eicosanoid receptors, conserved cysteines are found to be present in the extracellular amino terminus, the first and second extracellular loops, and the third intracellular loop, supporting the notion that cysteines at these positions may be especially important in this receptor family. Therefore, we utilized site-directed mutagenesis and stable expression of thromboxane receptor mutants with cysteine amino acid substitutions to identify the roles of individual human platelet thromboxane receptor cysteines in receptor-ligand interactions and receptor coupling to cell signal effectors. Four of the seven thromboxane receptor cysteine mutations examined were dysfunctional; three demonstrated an impaired ability to bind ligand, and one exhibited normal ligand binding characteristics, but crippled cell signaling.
Figure 1:
Position of mutated cysteines within
overall molecular structure of the human thromboxane receptor. Blocked area represents plasma membrane with seven putative
membrane-spanning -helices.
Figure 2:
Comparison of the deduced amino acid
sequences of human platelet and endothelial thromboxane (hum pla and hum endo TXR), rat thromboxane (rat TXR),
human PGE EP1 (hum EP1), human PGF (hum
FP), and human PGI
(hum IP) receptors. Large rectangles indicate putative hydrophobic
transmembrane-spanning domains. Dots indicate absolute
identity, and vertical lines indicate conservative
substitutions. Cysteines widely conserved among the different receptors
are enclosed in small rectangles.
In order to study the role of cysteines in the function of human thromboxane receptors, 7 cysteines (amino acid numbers 11, 68, 102, 105, 183, 223, and 257) were individually altered to serines using site-directed mutagenesis of single nucleotides. The mutant receptors were stably expressed in HEK 293 cells for evaluation of binding pharmacology and cell signaling. Mutation of cysteine at position 35 (transmembrane 1) was not achieved by these methods despite numerous attempts with two independent mutant oligonucleotides.
Figure 3:
Displacement of
[I]BOP from wild type human thromboxane
receptor cDNA-transfected HEK 293 cells. Compounds used are agonist
[
I]BOP (squares), antagonist SQ29548 (circles) and agonist U46619 (triangles). The data
are presented as means ± standard deviation of six paired
experiments per compound. Calculated values are in Table 1.
Three of the mutant receptors
(Cys-102, Cys-105, and Cys-183 Ser) were found to have greatly
diminished or no capacity to bind [
I]BOP,
although Northern blot analysis revealed that the mutant cell lines
expressed full-length (1.6 kilobase pairs) receptor-specific
transcripts (Fig. 4). Thus, diminished
[
I]BOP binding to these cells was not due to
failure to transcribe the transfected mutant thromboxane receptor
cDNAs. Cells expressing Cys-105
Ser and Cys-183
Ser
exhibited no specific binding (Table 1). Cysteines at these
respective positions are widely conserved throughout the superfamily of
G-protein-coupled receptors and are felt to constitute a structurally
necessary disulfide bond between extracellular loops one and
two(15) , and it appears that elimination of this disulfide
bond, by removing either of the two involved cysteines, destroys
thromboxane binding to its receptor.
Figure 4:
Northern analysis of wild type and mutant
thromboxane receptor cDNA-transfected HEK 293 cells. Approximately 30
µg of total RNA from indicated mouse prostaglandin EP3
receptor (EP) or mutant thromboxane receptor expressing HEK293
cells was analyzed. Expression of 1.6 kilobase transcript was detected
in all cell lines. The positions of 18 and 28 S ribosomal RNAs are
indicated. Pictured below are the results of subsequent hybridization
to
-actin.
The other mutant receptor which
exhibited attenuated ligand binding was Cys-102 Ser. Although
the receptor cDNA was transcribed at similar or higher levels than the
other mutant receptors (Fig. 4), Scatchard analysis of
[
I]BOP binding indicated that few receptors
capable of binding ligand were expressed as the binding capacity was
diminished by approximately 72% compared to wild type (Table 1).
To the extent that Cys-102
Ser did bind
[
I]BOP, the affinities for I-BOP and SQ29548
were decreased by 2- and 5-fold, respectively. Together, these findings
indicate that Cys-102 plays a role in both antagonist and agonist
binding.
Cells expressing wild
type platelet thromboxane receptors responded to (thromboxane mimetic)
U46619 stimulation with a dose-dependent increase in intracellular free
calcium having an EC value of 56 ± 7 nM (Fig. 5A, Table 1). As expected, the two
mutant receptors that failed to specifically bind
[
I]BOP (Cys-105
Ser and Cys-183
Ser) also showed no calcium response to thromboxane stimulation (Table 1). Cys-102
Ser, which had greatly reduced binding
capacity, transduced only a small calcium signal at maximal
concentrations of U46619 (10 ± 2% of wild type signal n = 3), which was not of sufficient amplitude for accurate
determination of the concentration-response characteristics.
Figure 5:
A, representative analog tracing of
U46619-induced increases in intracellular free calcium in wild type
human thromboxane receptor cDNA-transfected HEK 293 cells. Each tracing
represents a single experiment with addition of U46619 at arrow in concentrations indicated. Inset,
concentration-response relationship to U46619-stimulated wild type
thromboxane receptor calcium signaling. Each point is mean ±
standard error for five separate experiments. EC was 56
± 7 nM with maximum U46619-stimulated increase in
calcium of 108 ± 12 nM. B, comparison of
U46619-induced increases in intracellular free calcium in wild type and
Cys-223
Ser mutant human thromboxane receptor expressing HEK 293
cells. Each tracing represents a single experiment with addition of 1
µM U46619 at arrow. No U46619-induced rise in
intracellular free calcium is seen in HEK293 cells expressing Cys-223
Ser mutant human thromboxane
receptors.
Introduction of serine at position 223 in the third intracellular
loop resulted in a receptor that did not transduce a U46619-induced
calcium signal at concentrations of U46619 up to 5 µM (Fig. 5B and Table 2), even though it had
normal ligand binding characteristics (see above). This finding
suggested that Cys-223 was critically important for proper thromboxane
receptor coupling to G-protein effectors. To determine if defective
signaling by the Cys-223 Ser thromboxane receptor was
specifically due to elimination of the cysteine at that position, or
instead resulted from introduction of serine, we constructed a second
mutation changing Cys-223 to alanine. When transiently expressed in
HEK293 cells, Cys-223
Ala had similar
[
I]BOP binding characteristics to wild type and
Cys-223
Ser (Table 2). In fura-2 studies, Cys-223
Ala exhibited an attenuated U46619-induced calcium transient compared
to cells transiently expressing wild type receptor (Table 2).
Together, the results with Cys-223
Ser and Cys-223
Ala
indicate that a cysteine at amino acid position 223, while not
absolutely critical for receptor-effector coupling, is necessary for
normal efficiency of thromboxane receptor coupling to calcium
signaling.
Figure 6: Amino acid sequence of transmembrane domain five, the third intracellular loop, and transmembrane domain six of the human platelet thromboxane receptor. Filled blocked area represents plasma membrane. Position of cysteine 223 is noted. Arrows indicate positions of other mutated amino acids (Table 2).
In this study we have used substitution mutagenesis to define
the roles for cysteine residues in human platelet thromboxane receptor
function. Introduction of serine for cysteine in the amino terminus
(Cys-11) or transmembrane domains two or six (Cys-68 and Cys-257) of
the thromboxane receptor had no effect on agonist binding or cell
signaling. In contrast, substitution of serine for cysteines in the
first (Cys-105) or the second (Cys-183) extracellular loops eliminated
thromboxane receptor binding, and mutation of a second cysteine in
extracellular loop one (Cys-102) resulted in a receptor with decreased
affinity for both an agonist and an antagonist and low levels of
agonist binding despite high level expression of mRNA transcripts. The
most interesting result derived from analysis of a cysteine residue in
the third intracellular loop (Cys-223), which, when changed to serine
or alanine, resulted in a receptor with ligand binding properties
similar to wild type, but with impaired calcium signaling. The
importance of this region of the thromboxane receptor's third
intracellular loop, and of Cys-223 in particular, was supported by
mutational analysis of additional amino acids in the region of Cys-223,
one of which (Thr-221 Met) also resulted in a receptor with
diminished cell signaling. We believe that these studies are the most
comprehensive to date investigating the structure-function
relationships for ligand binding and cell signaling of the human
thromboxane receptor.
A special role for cysteines in determining
the functional characteristics of G-protein-coupled receptors was first
suggested by chemical studies of -adrenergic receptors, where
disulfide reducing agents resulted in loss of ligand binding sites and
reduction in binding affinity(19, 20) . Similar
results were later described for human platelet thromboxane receptors,
where reduction with dithiothreitol or sulfhydryl alkylation with N-ethylmaleimide resulted in reduced thromboxane receptor
ligand binding, implying that cysteines could determine receptor
binding characteristics through disulfide bridging as well as due to
the contribution of one or more free sulfhydryl groups(7) .
Therefore, we undertook the present studies to more clearly determine
the effect on thromboxane receptor function of altering individual
cysteines.
Replacement of cysteine by serine at positions 11, 68,
and 257 did not affect the ability of thromboxane receptors to bind
thromboxane agonist or to increase intracellular free calcium levels,
suggesting that these amino acids do not play an important role in
receptor-agonist or receptor-G-protein interactions (although Cys-11
Ser and Cys-257
Ser had minimally decreased affinity for
the antagonist SQ29548 and may play minor roles in antagonist binding).
Furthermore, if any of these cysteines had been involved in formation
of a disulfide bond, then substitution by serine should have disrupted
that bond and impaired receptor function in some regard. It is not
surprising that Cys-11, in the extracellular amino terminus of the
receptor, does not contribute in a critical manner to thromboxane
receptor binding or cell signaling. However, our results with Cys-68
and Cys-256 demonstrate the different functions that analogous cysteine
residues can perform in thromboxane receptors compared to other
G-protein-coupled receptors (21, 22) .
Cysteine 68
is located in the second transmembrane-spanning domain of the
thromboxane receptor. While Cys-68 Ser behaved normally in
binding and calcium signaling studies, mutation of the analogous
cysteine (amino acid position 69) in the M
muscarinic
acetylcholine receptor impaired agonist binding, but not antagonist
binding(21) . In contrast, mutation of the analogous cysteine
(amino acid position 77) of the
adrenergic receptor,
like the thromboxane receptor, did not alter receptor function in any
measurable way(22) . Thus, a sulfhydryl group in the second
transmembrane domain appears to contribute to normal ligand binding of
muscarinic, but not adrenergic or thromboxane receptors.
The only
other cysteines located within transmembrane domains of the thromboxane
receptor are at positions 34 and 257. Despite multiple attempts at
mutagenesis of Cys-34 using two different oligonucleotides, this mutant
could not be made. In any case, a cysteine in the first
transmembrane-spanning domain is not common among G-protein-coupled
receptors, or even among closely related members of the prostanoid
receptor family (Fig. 2). A cysteine located within the sixth
transmembrane domain is present in -adrenergic receptors,
thromboxane receptors, and human prostaglandin E
,
F
, and I
receptors (Fig. 2). Our
finding that replacement of Cys-257 did not alter thromboxane receptor
agonist binding or calcium signaling contrasts with replacement of the
analogous cysteine (amino acid position 285) of the
adrenergic receptor, which resulted in a receptor with normal
ligand binding, but impaired cell signaling as measured by cAMP
accumulation (22) . The variable effects of mutating sixth
transmembrane domain cysteine residues in thromboxane and
adrenergic receptors points to differences in the structural
requirements for receptor-effector coupling in the eicosanoid and
adrenergic receptor families.
While mutational substitution of most
of the cysteines in the thromboxane receptor did not affect ligand
binding, substitution of serine for any of the three cysteines in
extracellular loops one and two had rather dramatic effects on the
[I]BOP binding properties of the receptor.
Replacement of either Cys-105 or Cys-184 resulted in no displaceable
binding to the receptor, although mRNA transcript levels were as high
or higher than in the other mutant receptor expressing cell lines. The
most likely explanation for this observation is that these two
cysteines form an intramolecular disulfide bond that is necessary for
proper receptor folding, expression in the membrane, ligand binding, or
all three. Although it was not possible to measure and localize
receptor protein in our studies, and therefore definitively state that
the mutant receptor proteins were expressed in levels comparable to the
observed levels of mRNA, there is ample evidence supporting the notion
that cysteines in these positions are necessary for normal receptor
function. A cysteine in the first and second extracellular loops
appears to be a common structural feature of nearly all members of the
G-protein-coupled receptor family. In the
-adrenergic receptor,
mutagenic substitution of the analogous cysteines (amino acid positions
106 and 184) has previously been shown to affect agonist, but not
antagonist binding(23) , while mutation of the analogous
cysteines (amino acid positions 98 and 178) in the M
muscarinic receptor, like the thromboxane receptor, resulted in a
receptor with no binding or signaling(21) . In the case of the
thromboxane receptor, it has been demonstrated that the
oxidation/reduction state of the receptor can affect radioligand
binding in a manner similar to that observed in the present study when
we mutated these two cysteines(7) . Of particular interest are
the recent observations that naturally occurring mutations which
disrupt disulfide bond formation of rhodopsin and V
vasopressin receptors cause human diseases characterized
biochemically by improperly folded or non-functional
receptors(24, 25, 26) . Taken together, these
studies clearly implicate disulfide bonds between cysteines as being
critical for normal G-protein-coupled receptor expression and function.
It is interesting to note that Cys-102, only three amino acids away
from Cys-105, does not appear from our results to be capable of
substituting for Cys-105 and forming a disulfide bond with Cys-184: at
least not a disulfide bond that results in a functional thromboxane
receptor. This indicates that only a single form of disulfide bond
between the first and second extracellular loops is adequate for normal
thromboxane receptor expression and function.
The other thromboxane
receptor mutant that exhibited altered ligand binding was Cys-102
Ser. In this case, receptor affinity for thromboxane agonist and
antagonist was decreased. Additionally, the receptor agonist binding
capacity was much lower than one would expect of thromboxane receptors
expressed at a level corresponding to the quantity of mRNA transcript
we observed by Northern analysis. This result is similar to the
previous observation that alkylation of platelet thromboxane receptors
with N-ethylmaleimide diminished receptor ligand binding
capacity(7) . Together, these observations strongly suggest
that Cys-102 exists as a free sulfhydryl that contributes to
thromboxane receptor ligand binding. A cysteine at this relative
position is not conserved in the G-protein-coupled receptor
superfamily, nor is it present in the closely related receptors for
prostaglandins E
, F
or I
,
indicating that the requirement for a free sulfhydryl is specific to
the thromboxane receptor. The first extracellular loop where Cys-102 is
located has previously been implicated as being important in ligand
binding to this receptor(27, 28) . Halushka's
group used N-bromosuccinimide or diethylpyrocarbonate to
biochemically modify histidine residues in the thromboxane receptor and
found altered receptor binding affinity. The platelet thromboxane
receptor contains four histidine residues, two of which are in the
first extracellular loop (amino acid positions 89 and 96), while the
other two are in the third intracellular loop (amino acid positions 224
and 227). In the present study, the third loop histidines were mutated
to arginine with no alteration of I-BOP binding, suggesting that the
effects of N-bromosuccinimide and diethylpyrocarbonate are due
to modification of histidines in the first extracellular loop. These
results, together with our own, indicate that Cys-102 and surrounding
amino acids in the first extracellular loop of the platelet thromboxane
receptor play critical, but as yet unspecified roles in ligand binding.
Mutation of Cys-223 in the third intracellular loop between
transmembrane domains five and six completely abolished thromboxane
mimetic-stimulated calcium signaling without appreciably altering the
ligand binding properties of the receptor. The third intracellular loop
has previously been implicated in coupling of adrenergic receptors to
G-proteins(29) , and studies using chimeric
-adrenergic/
-adrenergic or
-adrenergic/M
muscarinic receptors have
indicated that specificity of G-protein coupling to these receptors is
determined by amino acids at the amino-terminal portion of
intracellular loop three(16, 30) . Platelet
thromboxane receptors are coupled to calcium signaling and
phospholipase C via interactions with G-proteins of the G
and G
families (31, 32, 33, 34) . Studies of other
phospholipase C-coupled receptors, although relatively few in number,
indicate that the third intracellular loop is also important in
G-protein coupling to these receptors. Site-directed mutagenesis of
amino acids in intracellular loop three of the angiotensin II type 1A
receptor has shown that this region is required for proper coupling to
phospholipase C(18) . This notion is further supported by
demonstration that synthetic peptides corresponding to the amino half
of angiotensin II 1A receptor intracellular loop three can mimic the
effects of agonist-occupied receptor in GTP
S binding
assays(35) .
It was unexpected that mutation of Cys-223 to
Ser would result in a receptor essentially devoid of calcium signaling
activity. Although serine was chosen to substitute for cysteine in our
mutagenesis protocols because the hydroxyl of serine and the sulfhydryl
of cysteine resemble each other in mass and charge, we wished to test
the possibility that insertion of the hydroxyl, rather than removal of
the sulfhydryl, accounted for the dramatic loss of receptor-effector
coupling. Therefore, we constructed a Cys-223 Ala mutant
receptor. This mutant, like Cys-223
Ser, had normal ligand
binding affinity. Interestingly, however, its ability to transduce a
calcium signal was neither as efficient as wild type nor as impaired as
Cys-223
Ser, but was intermediate between the two. This
indicates that Cys-223 is important for normal thromboxane receptor
coupling to G-protein effectors, but that substitution with a serine at
this position has additional deleterious consequences on
receptor-effector coupling.
To further explore the roles of
individual amino acids in the amino terminus of the thromboxane
receptor third intracellular loop, we constructed an additional three
substitution mutant receptors, each of which altered a different amino
acid near Cys-223. Mutation of an amino acid closer to the fifth
transmembrane-spanning segment (Thr-221 Met), like the Cys-223
Ala mutation, resulted in a receptor with normal ligand binding
properties, but cell signaling properties intermediate between wild
type and Cys-223
Ser. Interestingly, substitution of arginine
for His-224 or His-227 generated a receptor that was not significantly
different from wild type binding or signal transduction. Thus, specific
amino acids in the portion of the third intracellular loop closest to
transmembrane-spanning domain five appear to play roles in maintaining
normal thromboxane receptor coupling to G-protein effectors.
The
exact molecular structure responsible for the third intracellular loop
involvement in receptor G-protein interaction is not well understood.
Site-directed mutagenesis studies of the hamster -adrenergic
receptor have identified hydrophobic, but not hydrophilic, residues at
the amino end of intracellular loop three, which are critical for
G-protein coupling(36) . Structural analysis indicates this
region may form an
-helix with hydrophobic residues along one
side, similar to the G-protein activating wasp venom peptide
mastoparan(37) . Interestingly, we have mutated amphipathic
(Cys and Thr) residues resulting in impaired signal transduction.
However, a conformational preference analysis of the amino acids in the
thromboxane receptor third intracellular loop reveals a higher
preference for the carboxyl segment of the loop to form an
-helix
compared to the region near the fifth transmembrane
domain(38) .
The first intracellular loop of the thromboxane
receptor has also been implicated in receptor-G-protein coupling. A
natural mutation has been identified, which changes Arg-60 in the first
intracellular loop to leucine resulting in a hemorrhagic
disorder(5) . Similar to our Cys-223 Ser mutant, the
Arg-60
Leu mutation resulted in a receptor unable to activate
phospholipase C. These studies together show that multiple receptor
determinants are necessary for proper thromboxane receptor coupling to
effectors.
A second human thromboxane receptor cDNA has recently
been cloned from endothelial cells(39) . This form of the
thromboxane receptor arises from alternative splicing of the
thromboxane receptor mRNA and results in a receptor protein identical
to the platelet receptor except for a alternate carboxyl tail. The
carboxyl tail for the endothelial receptor is 47 amino acids longer
than the platelet receptor and contains three cysteines. The role of
these cysteines has not been tested, but one may act as a site for
palmitoylation forming a forth intracellular loop. In
-adrenergic receptors the presence of a palmitoylated
cysteine in the carboxyl tail is critical for normal agonist-induced
down-regulation(40) .
In summary, we have determined the roles of individual cysteine residues in the human platelet thromboxane receptor that help to determine normal receptor-ligand and receptor-effector interactions. We have identified cysteines in the first and second extracellular loops, which, through formation of disulfide bonds between Cys-105 and Cys-184 and via a free sulfhydryl on Cys-102, play roles in agonist and antagonist binding. We have also shown that amino acids, including Cys-223, at the amino end of the third intracellular loop are important for coupling to calcium signaling. These studies represent an initial step toward a full understanding of how thromboxane receptor structure determines its ligand binding and cell signaling characteristics.