Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Extracellular adenine and uridine nucleotides exert their physiological and pathophysiological responses through a large group of ligand-gated ion channel P2X receptors and G protein-coupled P2Y receptors (Fredholm et al., 1994; Ralevic and Burnstock, 1998). Molecular cloning and characterization studies have so far identified seven functional mammalian P2Y receptor subtypes (P2Y_{1,2,4,6,11,12,13}) (Harden, 1998; King et al., 1998) P2Y receptor subtypes 1, 2, 4, 6, and 11 couple to the stimulation of phospholipase C (PLC), which ultimately results in activation of protein kinase C and mobilization of calcium from intracellular stores. In addition to coupling to PLC, the hP2Y₁₁ receptor also couples to adenylyl cyclase to promote cyclic AMP accumulation (Communi et al., 1997, 1999; Qi et al., 2001). The P2Y₁₂ receptor (also known as P2Y_AC or P2T) was recently cloned and shown to be the elusive platelet ADP receptor coupled to the inhibition of adenylyl cyclase (Foster et al., 2001; Hollopeter et al., 2001). The P2Y₁₃ receptor (6PR86), which is 48% identical to the P2Y₁₂ receptor, is also activated by ADP and coupled to inhibition of adenylyl cyclase (Communi et al., 2001).

Traditionally, G protein-coupled receptor subtypes have been classified by their agonist and antagonist profiles. However, pharmacological classification of G protein-coupled receptors is complicated by the observation that minor structural differences in species homologs can have profound consequences on receptor activity. For example, the rat 5-HT_1B receptor has 93% sequence identity with its human homolog but displays a markedly different rank order of agonist potencies (Voigt et al., 1991; Gerhardt and van Heerikhuizen, 1997; Hoyer and Martin, 1997). In the P2Y receptor family, the rat homolog of the P2Y₄ receptor has a markedly different nucleotide selectivity than its human homolog, even though the two receptors have 83% sequence identity (Bogdanov et al., 1998; Webb et al., 1998; Kennedy et al., 2000). Thus, species homologs, even those with high sequence identity overall, do not necessarily exhibit identical pharmacological properties. This potential for variance in the pharmacological properties of species homologs makes it important to characterize these receptors to determine whether species-specific differences exist, because erroneous conclusions can arise based on the false assumption that species homologs exhibit very similar or identical properties.

We recently cloned a P2Y receptor from Madin-Darby canine kidney epithelial cells that has approximately 70% amino acid identity to the hP2Y₁₁ receptor (Zambon et al., 2001). Initial characterization of this receptor in a canine thymocyte cell line revealed that it promoted both inositol lipid hydrolysis and cyclic AMP accumulation, strongly suggesting that this receptor is the canine homolog of the P2Y₁₁ receptor. In the current study, we stably expressed the canine and human P2Y₁₁ receptors in CHO-K1 and 1321N1 cells and characterized their nucleotide selectivities and second messenger coupling properties. These data demonstrate that the two receptors differ markedly in their nucleotide selectivity, their sensitivity to 2-thioether substitution of the adenine ring, and their coupling efficiency to adenylyl cyclase. We further show that at least part of the difference in nucleotide selectivity of the two receptors can be attributed to the amino acid at position 265 (numbering from the hP2Y₁₁ receptor) located at the juxtaposition of TM 6 and the third extracellular loop. These results thus identify a key residue involved in determining nucleotide binding and activation of this P2Y receptor.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. All cell culture reagents were supplied by the Lineberger Comprehensive Cancer Center tissue culture facility (University of North Carolina, Chapel Hill, NC). Tunicamycin and hexokinase were from Roche Biomedical (Indianapolis, IN). ITP, XTP, diadenosine tetraphosphate, 3-isobutyl-1-methylxanthine, suramin, RB-2, PPADS, and apyrase were purchased from Sigma (St. Louis, MO). ATP, UTP, and GTP were from Pharmacia/Upjohn (Piscataway, NJ). ADP, AMP, UDP, 2MeSATP, 2MeSADP, ATPS, and ADPS were obtained from Calbiochem (La Jolla, CA). Stock solutions of ADP and 2MeSADP in Dulbecco's modified Eagle's high-glucose medium were treated for 2 h with 50 U/ml hexokinase (Kennedy et al., 2000). ATPS and ADPS stock solutions were treated for 1 h with 3 U/ml apyrase, and 2MeSATP was purified by high-performance liquid chromatography.

Expression of hP2Y₁₁ and cP2Y₁₁ Receptors in CHO-K1 and 1321N1 Cells. CHO-K1 cells have been used by us and others to characterize the hP2Y₁₁ receptor (Communi et al., 1997, 1999; Qi et al., 2001). Although CHO-K1 cells express a P2Y₂ receptor that responds to ATP and UTP (Iredale and Hill, 1993), exogenous expression of either the cP2Y₁₁ or hP2Y₁₁ receptor in these cells gave rise to ATP-promoted increases in inositol phosphate accumulation 20-fold higher than those in vector-infected cells (Qi et al., 2001). This allowed us to carry out pharmacological studies of the P2Y₁₁ receptor in CHO-K1 cells. In some experiments, P2Y₁₁ receptors were expressed in 1321N1 cells. Expression in CHO-K1 cells resulted in higher receptor levels than in 1321N1 human astrocytoma cells, as indicated by the lower EC₅₀ values of all nucleotides in CHO-K1 cells for promotion of inositol phosphate and cyclic AMP accumulation. No differences in the rank order of potency of nucleotides in the two cell lines were observed (Qi et al., 2001), but the higher levels of expression in CHO-K1 cells allowed full concentration-effect curves to be generated for many of the lower potency agonists (especially in cyclic AMP accumulation experiments with the hP2Y₁₁ receptor). In some experiments (Figs. 6, 7, and 8), receptors were HA-tagged to estimate cell surface expression by RIA. Inclusion of an HA-tag at the N terminus of the P2Y₁₁ receptor had no effect on the nucleotide selectivity or signaling properties (data not shown).

Assays of Inositol Phosphate and Cyclic AMP Accumulation. Cells stably expressing the hP2Y₁₁ or cP2Y₁₁ receptor were seeded in 24-well plates at 5 × 10⁴ cells/well (1 × 10⁵ cells/well for 1321N1 cells) and assayed 3 days later when confluent. Inositol lipids were radiolabeled by overnight incubation of the cells with 200 µl of inositol-free Dulbecco's modified Eagle's medium containing 4.5 g/l glucose and 0.4 µCi of myo-[³H]inositol. No changes of medium were made subsequent to the addition of [³H]inositol. Agonists or antagonists were added at 5× concentration in 50 µl of 50 mM LiCl and 250 mM HEPES, pH 7.25. After a 5-min incubation at 37°C, the medium was aspirated, and the assay was terminated by adding 0.75 ml of boiling EDTA, pH 8.0. [³H]Inositol phosphates were resolved by Dowex AG1-X8 columns as described previously (Lazarowski et al., 1995).

Intracellular [Ca²⁺] Measurements. Agonist-promoted increases in intracellular [Ca²⁺] were quantified under constant superfusion as described previously (Kennedy et al., 2000). These studies were carried out in 1321N1 cells expressing the human or canine P2Y₁₁ receptor, because the endogenous P2Y₂ receptor in CHO-K1 cells, although not a problem when measuring inositol phosphate or cyclic AMP accumulation of exogenously expressed P2Y₁₁ receptors, interferes with Ca²⁺ mobilization studies. 1321N1 cells do not express endogenous P2Y receptors and thus are well suited for nucleotide-promoted [Ca²⁺] measurements. Agonists were applied for 30 s in the superfusate (1.4 ml/min) and the change in intracellular [Ca²⁺] was measured in 7 to 16 individual cells per coverslip and averaged. To generate concentration-effect curves, each concentration of nucleotide was applied only once to each individual coverslip (to avoid receptor desensitization) and the average response from 7 to 16 cells per coverslip was measured from four to six coverslips. Data were recorded and processed using an InCyt IM2 digital imaging system (Intracellular Imaging Inc., Cincinnati, OH).

Mutagenesis. Mutations were incorporated into the human and canine P2Y₁₁ receptor by four-primer polymerase chain reaction (Ho et al., 1989). To account for differences in receptor expression, wild-type and mutant receptor cDNA containing an HA epitope tag immediately after the start codon served as template in the polymerase chain reactions. After amplification, the mutant receptors were cloned into the EcoRI and XhoI sites of pLXSN, and the presence of the mutations was verified by sequencing.

RIA for Detection of HA-Tagged Receptors. 1321N1 cells expressing HA-tagged wild-type and mutated P2Y₁₁ receptors were seeded at 1 × 10⁵ cells/well in a 24-well plate. Assays to quantitate the expression of HA-tagged receptors were performed on confluent cells 3 days after plating essentially as described previously (Brinson and Harden, 2001). Briefly, cells were fixed in 4% paraformaldehyde for 15 min at room temperature. After the washing and blocking steps, cells were incubated for 1 h at 37°C with a 1:1000 dilution of mouse anti-HA monoclonal antibody (clone HA.11; Covance Research Products, Denver, PA). Cells were washed twice with Hanks' balanced salt solution (20 mM HEPES, pH 7.4, and 150 mM NaCl) containing Ca²⁺ and Mg²⁺, followed by addition (typically 1 × 10⁵ cpm/well) of ¹²⁵I-labeled rabbit anti-mouse antibody. After a 2-h incubation at room temperature, the cells were washed twice with Hanks' balanced salt solution containing Ca²⁺ and Mg²⁺. Cells were then solubilized with 1 M NaOH and transferred to glass tubes for quantitation of radioactivity by -counting.

Statistics. Data are expressed as the mean ± S.D. or geometric mean. Concentration-response curves were fitted to the data by logistic (Hill equation), nonlinear regression analysis with DeltaGraph software (SPSS, Chicago, IL). Data were compared using one-way analysis of variance and Tukey's comparison or by Student's t test with P < 0.05 considered to be statistically significant.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Adenine Nucleotide Selectivities of hP2Y₁₁ and cP2Y₁₁ Receptors. Both the cP2Y₁₁ and hP2Y₁₁ receptor were stably expressed in CHO-K1 cells, and their nucleotide selectivities and second messenger signaling properties were determined. As was observed previously with the hP2Y₁₁ receptor, ATP promoted both inositol phosphate and cyclic AMP accumulation in CHO-K1 cells expressing the cP2Y₁₁ receptor.¹ Adenosine triphosphate nucleotides were considerably more potent and efficacious as agonists at the hP2Y₁₁ receptor than their corresponding diphosphate nucleotides for promotion of both inositol phosphate and cyclic AMP accumulation (Fig. 1; Table 1). The potency order of these nucleotides was ATPS  2MeSATP  ATP  ADPS > 2MeSADP > ADP. In addition, ADP, ADPS, and 2MeSADP were partial agonists at the hP2Y₁₁ receptor, with apparent efficacies that were 60 to 80% of maximal response to ATP (Table 1). As reported previously in CHO-hP2Y₁₁ cells (Qi et al., 2001), cyclic AMP responses to ADP and 2MeSADP (both up to 300 µM) did not reach a clear maximum and an EC₅₀ value could not be determined (Fig. 1B).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   The capacity of adenine nucleotides to promote inositol phosphate and cyclic AMP accumulation in CHO-hP2Y11 cells. Nucleotide-promoted increases in inositol phosphate (A) or cyclic AMP (B) accumulation were measured in CHO-hP2Y11 cells. Responses were normalized to the maximal response obtained with ATP (300 µM). Data shown are the mean of triplicate assays from three to six separate experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   The capacity of adenine nucleotides to promote inositol phosphate and cyclic AMP accumulation in CHO-cP2Y11 cells. Nucleotide-promoted increases in inositol phosphate (A) or cyclic AMP (B) accumulation were measured in CHO-cP2Y11 cells. Responses were normalized to the maximal response obtained with ADP (100 µM). Data shown are the mean of triplicate assays from three to six separate experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   The capacity of nucleotides to increase intracellular [Ca2+] in 1321N1 cells expressing either the hP2Y11 or cP2Y11 receptor. Intracellular [Ca2+] in response to the indicated nucleotides was measured under constant superfusion in 1321N1-hP2Y11 cells (A) and 1321N1-cP2Y11 cells (B). Each point was determined from a minimum of four coverslips, with 9 to 16 cells monitored per coverslip, as described under Experimental Procedures.

Agonist Activities of Other Nucleotides. To investigate whether the cP2Y₁₁ receptor is activated by a broader range of natural nucleotides than the hP2Y₁₁ receptor, we tested the capacities of UTP, UDP, GTP, ITP, XTP, and AMP to promote inositol phosphate accumulation in CHO-hP2Y₁₁ and CHO-cP2Y₁₁ cells. None of these nucleotides exhibited significant agonist activity at either the hP2Y₁₁ or cP2Y₁₁ receptor (data not shown). At a concentration of 300 µM, apyrase-treated Ap₄A promoted inositol phosphate accumulation at 34 ± 4.6% of the ATP response at the hP2Y₁₁ receptor, and 24 ± 3.0% of the ADP response at the cP2Y₁₁ receptor (basal subtracted, n = 3, data not shown). These data indicate that both the cP2Y₁₁ and hP2Y₁₁ receptors are highly adenine nucleotide-specific.

Coupling Efficiencies of Human and Canine P2Y₁₁ Receptors to Inositol Lipid Hydrolysis and Cyclic AMP Accumulation. We have previously demonstrated that although the hP2Y₁₁ receptor promotes both inositol phosphate and cyclic AMP accumulation, it promotes inositol lipid hydrolysis with much higher efficiency than cyclic AMP accumulation (Qi et al., 2001). In contrast to the hP2Y₁₁ receptor, the cP2Y₁₁ receptor activates both second messenger pathways with very similar potencies (Fig. 2). Thus, agonist potencies for promotion of inositol phosphate accumulation at the hP2Y₁₁ receptor were 7- to 19-fold greater than for promotion of cyclic AMP accumulation (Table 1), whereas agonist potencies for promotion of the two second messenger responses differed by only 2- to 4-fold at the cP2Y₁₁ receptor (Table 2). These data indicate that the cP2Y₁₁ receptor couples to cyclic AMP accumulation with higher efficiency than does the hP2Y₁₁ receptor.

Sensitivity to P2Y Receptor Antagonists. We also determined the sensitivities of the two species homologs to nonspecific P2Y antagonists. Three antagonists, suramin, RB-2, and PPADS, were examined for their ability to block ATP- or ADP-promoted inositol phosphate and cyclic AMP accumulation in CHO-hP2Y₁₁ and CHO-cP2Y₁₁ cells, respectively. Whereas PPADS had little to no effect at either receptor, both suramin and RB-2 behaved as antagonists with similar potencies (Fig. 4). These data suggest that there is very little difference between canine and human P2Y₁₁ receptors in their sensitivity to nonselective P2Y antagonists.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Sensitivity of human and canine P2Y11 receptors to nonselective P2Y receptor antagonists. Inhibition by suramin, reactive blue 2 (RB-2), and PPADS of inositol phosphate accumulation (A and B) or cyclic AMP accumulation (C and D) promoted by either 300 µM ATP in CHO-hP2Y11 cells (A and C) or 100 µM ADP in CHO-cP2Y11 cells (B and D). The diamonds in each graph represent the basal [3H]inositol phosphate or cyclic AMP accumulation obtained in the absence of nucleotides. Data shown are the mean ± standard deviation of triplicate assays from three separate experiments.

The Role of Arg-265 in Triphosphate versus Diphosphate Selectivity in the P2Y₁₁ Receptor. Comparison of the deduced human and canine P2Y₁₁ sequences reveals two changes in the cP2Y₁₁ receptor (His-262 to Tyr and Arg-265 to Gln) located at the juxtaposition of TM 6 and the third extracellular loop that potentially underlie the distinct nucleotide selectivities of the two receptors (Fig. 5). In all other P2Y receptors, there are basic residues at these two positions. These positively charged amino acids have been predicted to interact with the phosphate moiety of nucleotide agonists (Erb et al., 1995; Jiang et al., 1997). To further examine their roles in nucleotide selectivity of the P2Y₁₁ receptor, we mutated His-262 and Arg-265 in the hP2Y₁₁ receptor, both individually and together, to their amino acid counterparts in the cP2Y₁₁ receptor. In addition, both wild-type and mutant receptors were HA-tagged to give an indication of the relative levels of expression.

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Alignment of the amino acid sequences of P2Y receptors at the juxtaposition of TM 6 and the third extracellular loop. The arrows denote the two residues of the hP2Y11 receptor (His-262 and Arg-265) that are different in the cP2Y11 receptor. See text for more details.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Quantitation of the expression of mutant receptor constructs. Radioimmunoassay showing the relative expression of HA-tagged wild-type and mutant P2Y11 receptors in 1321N1 cells.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   The capacity of ATP, ADP, and 2MeSADP to promote inositol phosphate accumulation in 1321N1 cells expressing wild-type and mutant P2Y11 receptors. The data shown are the mean of triplicate assays from one experiment repeated at least three times with similar results.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Increase in intracellular [Ca2+] in 1321N1 cells expressing wild-type and mutant P2Y11 receptors in response to 300 µM ADP and ATP. Each bar represents the mean increase in intracellular [Ca2+] from five coverslips, with 7 to 14 cells monitored per coverslip, as described under Experimental Procedures. *, P < 0.05, **, P < 0.01, relative to the response to ATP.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

We demonstrate in this study that the cP2Y₁₁ receptor is an adenosine diphosphate-preferring receptor, in contrast to its adenosine triphosphate-preferring human homolog. The triphosphate versus diphosphate preferences of the two receptors were confirmed in studies monitoring nucleotide-promoted increases in intracellular [Ca²⁺] under conditions that minimized nucleotide breakdown and interconversion. Although differences in nucleotide selectivity between species homologs of P2Y receptors have been noted (Li et al., 1998; Kennedy et al., 2000), this is the first example of species homologs that change their preference for diphosphate versus triphosphate nucleotides.

We also show that the cP2Y₁₁ receptor is markedly more sensitive to 2-methylthioether substitution of the adenine ring than the hP2Y₁₁ receptor and that the cP2Y₁₁ receptor couples to cyclic AMP accumulation with greater efficiency than the hP2Y₁₁ receptor. The increased coupling of the cP2Y₁₁ receptor to cyclic AMP accumulation relative to inositol lipid hydrolysis may be due to either changes in the primary structure of the cP2Y₁₁ receptor that facilitate a better coupling efficiency to Gs [there are multiple amino acid differences in the putative intracellular regions of the two receptors (Zambon et al., 2001)] or perhaps reflects the ability of the cP2Y₁₁ receptor to adopt a conformation upon binding of nucleotides that is more efficient at activating Gs than the one adopted by the agonist-occupied hP2Y₁₁ receptor. It should be noted that interaction of the P2Y₁₁ receptor with Gs is inferred, because there is no unequivocal evidence that the receptor directly activates Gs.

Because the canine receptor has only 70% sequence identity to the hP2Y₁₁ receptor and displays a markedly different nucleotide selectivity, it might be argued that the canine receptor is a distinct P2Y receptor subtype and not the species homolog of the hP2Y₁₁ receptor. However, several properties of this receptor make this possibility unlikely. First, both the human and canine receptors couple simultaneously to inositol lipid hydrolysis and cyclic AMP synthesis. The P2Y₁₁ receptor is the only P2Y receptor and one of only a few G protein-coupled receptors that are dually coupled to both of these signaling pathways (Qi et al., 2001). Secondly, although the receptors have distinct selectivity profiles, both receptors are highly adenine nucleotide-selective and have similar sensitivities to nonselective P2Y receptor antagonists. Taken together, these data strongly suggest that the cP2Y₁₁ receptor is the canine homolog of the hP2Y₁₁ receptor. Similar arguments have been made that the avian p2y3 receptor is the species homolog of the mammalian P2Y₆ receptor, even though the two receptors share only 65% amino acid identity and show differences in their nucleotide selectivities (Li et al., 1998).

The marked differences in nucleotide potencies of the human and canine homologs of the P2Y₁₁ receptor have important ramifications on physiological and pharmacological studies compared across species. Studies of native P2Y₁₁ receptors in cells and tissues derived from other mammalian species must now be conducted with caution. Until the P2Y₁₁ receptors from these species are cloned and characterized, their nucleotide selectivities and signaling properties cannot be inferred simply from data on the two molecularly identified P2Y₁₁ receptors. Thus, it will be important to clone the P2Y₁₁ receptors from rat, mouse and other mammalian species and to determine their pharmacological properties. In addition, these studies may also help to understand more clearly the structural and functional aspects of receptor-nucleotide interaction.

The residues involved in nucleotide recognition in the P2Y₁₁ receptor have not been delineated. In other P2Y receptors, studies have demonstrated a marked decrease in nucleotide potencies after mutation of both charged and uncharged residues located between TMs 3 and 7 of P2Y₁ (Jiang et al., 1997) and P2Y₂ (Erb et al., 1995) receptors. A subsequent study also showed that several residues in the putative second and third extracellular loops of the P2Y₁ receptor are involved in nucleotide binding and/or receptor activation (Hoffmann et al., 1999). However, outside of a few highly conserved charged amino acids within the TM regions, data from these studies are hard to apply to the P2Y₁₁ receptor due to its low sequence identity to other P2Y receptors. Because of the marked differences in nucleotide selectivity of hP2Y₁₁ and cP2Y₁₁ receptors, we compared the two sequences to identify amino acid changes that might account for these differences.

Inspection of the sequences identified two basic amino acids, His-262 and Arg-265, located at the juxtaposition of TM6 and the third extracellular loop in the hP2Y₁₁ receptor, that are changed to tyrosine and glutamine, respectively, in the cP2Y₁₁ receptor. These residues have been proposed to form part of the nucleotide binding pocket in other P2Y receptors and to form an electrostatic interaction with the phosphate moiety of the nucleotide (Erb et al., 1995; Jiang et al., 1997). Our data indicate that, whereas mutation of His-262 in the hP2Y₁₁ receptor to tyrosine had no effect on nucleotide selectivity, mutation of Arg-265/Gln-268 had marked effects on the relative potencies and efficacies of ATP and ADP in the two receptors. Thus, mutation of Arg-265 to glutamine in the hP2Y₁₁ receptor increased both the relative efficacy and potency of ADP, which is a lower potency partial agonist at the wild-type receptor, to the same as ATP. Likewise, mutation of Gln-268 to arginine in the cP2Y₁₁ receptor increased the relative potency and efficacy of ATP to the same levels as ADP. These data are consistent with the idea that Arg-265 in the hP2Y₁₁ residue is involved in nucleotide binding and functions in part to discriminate between triphosphate and diphosphate nucleotides. Unfortunately, in the absence of a reliable radioligand binding assay for the P2Y₁₁ receptor, it is not possible to demonstrate directly whether Arg-265 is involved in nucleotide binding. Even with this limitation, however, it is clear that the Arg-265/Gln-268 residue plays a significant role in defining whether the receptor prefers triphosphate or diphosphate nucleotides.

Previous studies by Jiang et al. (1997) showed that mutation of His-277 and Lys-280 in the P2Y₁ receptor (the "equivalent" residues to His-262 and Arg-265 in the hP2Y₁₁ receptor; Fig. 5) to alanine resulted in a marked decrease in potency of both 2MeSATP and 2MeSADP, although the effects of mutating Lys-280 were more dramatic than those for mutation of His-277. No changes in the triphosphate versus diphosphate selectivities of the mutant receptors were observed. Similarly, mutation of the residues in the P2Y₂ receptor equivalent to His-262 and Arg-265 to leucine (Erb et al., 1995) markedly decreased the potency of both UTP and ATP, also with no change in their selectivities for triphosphate and diphosphate nucleotides. Our results are very different from these earlier studies and suggest that in the P2Y₁₁ receptor, the identity of the residue at position 265 plays a major role in determining whether the receptor is more readily activated by ATP or ADP nucleotides. These data further suggest that the P2Y₁₁ receptor binds nucleotides in a subtly different manner than either the P2Y₁ or P2Y₂ receptor.

Importantly, neither of these mutations was sufficient to convert completely the selectivity of one homolog to the other, which suggests that other regions of the receptor must also contribute to defining diphosphate versus triphosphate selectivity. Likewise, these mutations had no effect on the sensitivity to 2-thioether substitution of the adenine base (Fig. 7), again suggesting that the amino acid(s) conferring this property must reside in other regions. Interestingly, the two receptors differ most noticeably in their putative extracellular regions, especially in the N terminus and the second and third extracellular loops. These regions may be involved in binding the adenine base and thus may mediate the differential sensitivity to 2-thioether substitution. The reasonably high sequence identity between the two receptors should facilitate a chimeric receptor approach to pinpoint the important regions and residues involved in these differences. These studies are in progress.