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.

EXPERIMENTAL PROCEDURES

Materials

Construction of Mutant Thromboxane Receptors

HEK 293 Cell Transfection and Selection of Stably Expressing Cell Lines

RNA Analysis

Binding of [I]BOP to Thromboxane A Receptors

Calcium Measurements

Statistical Methods

RESULTS

Construction of Thromboxane Receptor Cysteine Serine Substitution Mutants

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.

Ligand Binding Analysis of Mutant Thromboxane Receptors

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.

Analysis of Mutant Thromboxane Receptor Cell Signaling

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.

Functional Effects of Mutating Other Amino Acids in the Thromboxane Receptor Third Intracellular Loop

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).

DISCUSSION

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 GTPS 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.

FOOTNOTES

*

This work was supported in part by Grants HL49267 and P50 HL52318-01 from the National Institutes of Health, a Merit Review grant from the Veterans Administration, the American Heart Association 1995 Council on Circulation Boots Cardiovascular Research Prize, and a fellowship stipend from the Ohio Affiliate of the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Established Investigator of the American Heart Association, supported with funds contributed in part by its Ohio Affiliate. To whom correspondence should be addressed: University of Cincinnati and the Cincinnati VA Medical Center, 231 Bethesda Ave., ML 542, Cincinnati, OH 45267-0542.

()

The abbreviations and trivial names used are: [I]BOP, [1S-(1,2(5Z),3-(1E,3S),4]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-bute- nyl)-7-oxabicyclo-[2.2.1]heptane-2-yl]-5-heptenoic acid; U46619, 15S-hydroxy-11,9(epoxymethano)-prosta-5Z,13E-dienoic acid; SQ29,548, [[1S]-1,2(5Z)3,4]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid; GTPS, guanosine 5`-3-O-(thio)triphosphate.

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