Extracellular domain chimeras of the TSH and LH/CG receptors reveal the mid-region (amino acids 171-260) to play a vital role in high affinity TSH binding

We constructed a series of TSH-LH/CG receptor chimeras by homologous substitution of relatively small regions of the TSH receptor extracellular domain for the corresponding region of the extracellular domain of the LH/CG receptor. Constructs were stably expressed in Chinese hamster ovary cells. Of the five chimeric receptors, only TSH-LHR-14, which contains mid-region domain C (amino acid residues 171-260) of the extracellular component of the TSH receptor, exhibited TSH binding of relatively high affinity. Consistent with this TSH binding, chimera TSH-LHR-14 was the only one that demonstrated a functional response to TSH stimulation in terms of intracellular cAMP generation. These data indicate that domain C plays a vital role in TSH receptor function.     

Cloning, sequencing and expression of human TSH receptor

Complementary cDNA clones encoding the TSH (thyroid stimulatory hormone) receptor were isolated from a human thyroid lambda gt10 library using Iow stringency hybridization with LH/hCG (luteinizing hormone-human choriogonadotropic hormone) receptor probes. Sequencing of the clones showed a 764 amino acid open reading frame. The first 21 amino acids probably correspond to a signal peptide, the mature protein thus contains 743 amino acids (calculated molecular weight: 84,501 daltons). Its putative structure consists of a 394 amino acid extracellular domain, a 266 amino acid membrane spanning domain with 7 putative transmembrane segments and a 83 amino acid intracellular domain. A high degree of homology is observed with LH/hCG receptor suggesting the definition of a new subfamily of G-protein coupled receptors. Computer search showed the presence in the putative third intracellular loop of a motif resembling that described in the non receptor type protein tyrosine kinases (c-src, c-yes, c-fgr, etc...). RNA blots showed that the receptor messenger RNA consists of two major species of 4300 and 3900 nucleotides. The cDNA was inserted into an expression vector and after transfection into COS 7 cells it was shown to produce a functional TSH receptor.     

Amino-terminal leucine-rich repeats in gonadotropin receptors determine hormone selectivity.

Recombinant expression of truncated receptors for luteinizing hormone/chorionic gonadotropin (LH/CG) revealed that the amino-terminal leucine-rich repeats 1-8 of the extracellular receptor domain bind human chorionic gonadotropin (hCG) with an affinity (Kd = 0.72 +/- 0.2 nM) similar to that of the native LH/CG receptor (Kd = 0.48 +/- 0.05 nM). LH/CG receptor leucine-rich repeats 1-8 were used to replace homologous sequences in the closely related receptor for follicle stimulating hormone (FSH). Cells expressing such chimeric LH/CG-FSH receptors bind hCG and show elevated cylic AMP levels when stimulated by hCG but not by recombinant human FSH (rhFSH). Similarly, a chimeric LH/CG receptor in which leucine-rich repeats 1-11 originated from the FSH receptor is activated by rhFSH but not by hCG. For this chimera, no residual [125I] hCG binding was observed in a range of 2 pM to 10 nM. Our results demonstrate that specificity of gonadotropin receptors is determined by a high affinity hormone binding site formed by the amino-terminal leucine-rich receptor repeats.     

Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity

The lutropin-choriogonadotropin (LH/CG) receptor is a cell surface receptor comprised of two domains of roughly equivalent size. The amino-terminal half of the receptor is relatively hydrophilic and is located extracellularly, whereas the carboxyl-terminal half of the receptor shares amino acid homology with other receptors that couple to G proteins and is similarly thought to span the plasma membrane seven times, ending with a relatively short carboxyl-terminal tail. In order to test the role of the extracellular domain in binding hormone, we constructed a mutated rat luteal LH/CG receptor cDNA (termed pCLHR-D2), which encodes for only the extracellular domain, and used it to transiently transfect human kidney 293 cells. Here we report that the expressed extracellular domain of the LH/CG receptor is capable of binding human CG with a high affinity, comparable with that of the full-length receptor. Thus, not only is the extracellular domain of the glycoprotein hormone receptors involved in binding hormone, but it alone is capable of conferring high affinity binding. Unexpectedly, it was also found that this truncated receptor is not secreted into the culture media but remains trapped within the cells.     

Towards understanding the glycoprotein hormone receptors.

Lutropin (LH), follitropin (FSH) and thyrotropin (TSH), as well as choriogonadotropin (CG, which binds to the LH receptor) constitute the glycoprotein hormone family. Their 3 receptors have been cloned during the last few months. They belong to the large group of G-protein coupled membrane proteins, with their specific N-terminal domain likely to bind the hormone and the characteristic 7 membrane-spanning segments in their C-terminal moiety. The present review discusses the main results of amino acid sequence analysis performed on the glycoprotein hormone receptors. The putative extracellular head exhibits less than 45% homology over the 3 receptors, while approximately 70% residue conservation is found in the transmembrane moiety. Here only, limited sequence homologies (approximately 20%) can be found with other G-protein coupled receptors. The secondary structure predictions performed on the 3 receptors revealed that the polypeptide sequence predicted as ordered (either alpha-helix or beta-strand) were repeated evenly throughout the extracellular head with a period of approximately 25 amino acids. This analysis helped to define the intervening loops between this ordered stretches as potential candidates for bearing at least part of the binding site of the hormones. Some of the perspectives opened by the cloning of the receptors are described, like the production of the extracellular head of the porcine LH receptor in baculovirus-infected insect cells, and the exploration of the LH receptor's mechanism of functioning as a dimer.     

Isolation of TSH and LH/CG receptor cDNAs from human thyroid: regulation by tissue specific splicing

A TSH receptor (TSH-R) cDNA has been isolated from a human thyroid lambda GT11 library. Unexpectedly, several cDNAs encoding the human LH/CG receptor (LH/CG-R), previously thought to be expressed solely in gonadal cells, were also isolated from the thyroid library. The receptors are structurally related, consisting of a signal sequence, a large extracellular amino terminal domain, seven membrane spanning domains, and a short carboxyl-terminal portion. The TSH-R is encoded by a single 4.2 kilobase mRNA specific to the thyroid. Introns were not present in any hTSH-R cDNAs examined, however, sequencing of several LH/CG-R cDNAs and RNase protection experiments demonstrated that the majority of hLH/CG-R mRNA in the thyroid is incompletely spliced. Consequently, tissue-specific splicing may be an important step in the regulation of the glycoprotein hormone receptor family.     

The nematode leucine-rich repeat-containing, G protein-coupled receptor (LGR) protein homologous to vertebrate gonadotropin and thyrotropin receptors is constitutively active in mammalian cells

The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of an expanding family of homologous leucine-rich repeat-containing, G protein-coupled receptors (LGRs), including the three known glycoprotein hormone receptors; mammalian LGR4 and LGR5; and LGRs in sea anemone, fly, and snail. We isolated nematode LGR cDNA and characterized its gene from the Caenorhabditis elegans genome. This receptor cDNA encodes 929 amino acids consisting of a signal peptide for membrane insertion, an ectodomain with nine leucine-rich repeats, a seven-TM region, and a long C-terminal tail. The nematode LGR has five potential N-linked glycosylation sites in its ectodomain and multiple consensus phosphorylation sites for protein kinase A and C in the cytoplasmic loop and C tail. The nematode receptor gene has 13 exons; its TM region and C tail, unlike mammalian glycoprotein hormone receptors, are encoded by multiple exons. Sequence alignments showed that the TM region of the nematode receptor has 30% identity and 50% similarity to the same region in mammalian glycoprotein hormone receptors. Although human 293T cells expressing the nematode LGR protein do not respond to human glycoprotein hormones, these cells exhibited major increases in basal cAMP production in the absence of ligand stimulation, reaching levels comparable to those in cells expressing a constitutively activated mutant human LH receptor found in patients with familial male-limited precocious puberty. Analysis of cAMP production mediated by chimeric receptors further indicated that the ectodomain and TM region of the nematode LGR and human LH receptor are interchangeable and the TM region of the nematode LGR is responsible for constitutive receptor activation. Thus, the identification and characterization of the nematode receptor provides the basis for understanding the evolutionary relationship of diverse LGRs and for future analysis of mechanisms underlying the activation of glycoprotein hormone receptors and related LGRs.     

Asp330 and Tyr331 in the C-terminal cysteine-rich region of the luteinizing hormone receptor are key residues in hormone-induced receptor activation

The luteinizing hormone (LH) receptor plays an essential role in male and female gonadal function. Together with the follicle-stimulating hormone (FSH) and thyroid stimulating hormone (TSH) receptors, the LH receptor forms the family of glycoprotein hormone receptors. All glycoprotein hormone receptors share a common modular topography, with an N-terminal extracellular ligand binding domain and a C-terminal seven-transmembrane transduction domain. The ligand binding domain consists of 9 leucine-rich repeats, flanked by N- and C-terminal cysteine-rich regions. Recently, crystal structures have been published of the extracellular domains of the FSH and TSH receptors. However, the C-terminal cysteine-rich region (CCR), also referred to as the "hinge region," was not included in these structures. Both structure and function of the CCR therefore remain unknown. In this study we set out to characterize important domains within the CCR of the LH receptor. First, we mutated all cysteines and combinations of cysteines in the CCR to identify the most probable disulfide bridges. Second, we exchanged large parts of the LH receptor CCR by its FSH receptor counterparts, and characterized the mutant receptors in transiently transfected HEK 293 cells. We zoomed in on important regions by focused exchange and deletion mutagenesis followed by alanine scanning. Mutations in the CCR specifically decreased the potencies of LH and hCG, because the potency of the low molecular weight agonist Org 41841 was unaffected. Using this unbiased approach, we identified Asp(330) and Tyr(331) as key amino acids in LH/hCG mediated signaling.     

Effects of truncations of the cytoplasmic tail of the luteinizing hormone/chorionic gonadotropin receptor on receptor-mediated hormone internalization.

The LH/CG receptor is a member of the family of G protein-coupled receptors and consists of a large N-terminal extracellular domain (which is responsible for binding hormone) attached to a region that spans the plasma membrane seven times, ending with an intracellularly located C-terminus. Binding of LH or human CG (hCG) to the LH/CG receptor causes a stimulation of adenylyl cyclase, presumably via activation of Gs. The binding of hormone also leads to its subsequent internalization by receptor-mediated endocytosis. In order to investigate the role of the cytoplasmic tail of this receptor in these events, we prepared a series of mutants in which progressively larger portions of the cytoplasmic tail were deleted. Deletion of 58 amino acids from the C-terminus, in which only 11 cytoplasmic residues remain, resulted in a receptor that was not expressed on the plasma membrane. Receptors rat LHR (rLHR)-t653 and rLHR-t631, in which 21 or 43 amino acids were removed, respectively, were properly expressed. These results suggest that a region(s) between residues 616 and 631 of the rLH/CG receptor are required for proper insertion and/or targeting of the receptor into the plasma membrane. Cells expressing rLHR-t653 or rLHR-t631 bound hCG with the same high affinity as cells expressing the full-length receptor, and basal levels of cAMP were the same among the cells. However, cells expressing the truncated receptors responded to hCG with approximately 2-fold greater levels of maximal cAMP accumulation than cells expressing the full-length receptor. Deletion of up to 43 amino acids from the C-terminus of the rLH/CG receptor had no deleterious effect on hCG internalization. In fact, mutants lacking 21 and 43 amino acids exhibited progressively faster rates of hCG internalization as compared to the full-length receptor. Once internalized, hCG was also degraded at a faster rate in cells expressing the truncated LH/CG receptors. Since hCG-stimulated cAMP stimulation and hCG internalization are retained by rLHR-t631, it can be concluded that the residues, not necessarily the same, required for these functions reside within the 26 amino acids of the cytoplasmic tail closest to the seventh transmembrane helix and/or residues within the intracellular loops. Our data show, however, that both hCG-stimulated cAMP production and hCG internalization are enhanced by the removal of the distal portion of the cytoplasmic tail.     

The testicular receptor for follicle stimulating hormone: structure and functional expression of cloned cDNA

Cloned cDNA encoding the rat Sertoli cell receptor for FSH was isolated from a cognate library and functionally expressed in cultured mammalian cells. The FSH receptor (FSH-R), as predicted from the cDNA, is a single 75K polypeptide with a 348 residue extracellular domain which contains three N-linked glycosylation sites. This domain is connected to a structure containing seven putative transmembrane segments which displays sequence similarity to G protein-coupled receptors. Thus, the FSH-R is identical in its structural design to the LH/CG receptor (LH/CG-R). Furthermore, both receptors display 50% sequence similarity in their large extracellular domains and 80% identity across the seven transmembrane segments. Expression of the cloned cDNA in mammalian cells conferred FSH-dependent cAMP accumulation. The selectivity for FSH is attested by the fact that the related human glycoprotein hormones human CG and human TSH do not stimulate adenylyl cyclase in FSH-R expressing cells even when these hormones are present at high concentrations.     

Structural differences in the hinge region of the glycoprotein hormone receptors: evidence from the sulfated tyrosine residues

Tyrosine sulfation is a late posttranslational modification of proteins that takes place in the Golgi network. In the past few years, this process has been identified as an important modulator of protein-protein interactions. Sulfated tyrosine residues have recently been identified in the C-terminal, so-called hinge region of the ectodomain of glycoprotein hormone receptors [TSH, LH/chorionic gonadotropin (CG), and FSH receptors] and were shown to play an important role in the interaction with their natural ligands. The position of two sulfated tyrosine residues in a Y-D/E-Y motif appears perfectly conserved in the alignment of TSH and LH receptors from different species, and site-directed mutagenesis experiments demonstrated that sulfation of the first residue of this motif was responsible for the functional effect on hormone binding. In contrast, the corresponding motif is not conserved in the FSH receptor, in which the first tyrosine residue is missing: the Y-D/E-Y motif is replaced by F(333)DY(335). We extend here our previous observation that, in this case, it is sulfation of the second sole tyrosine residue in the motif that is functionally important. An LH/CG receptor harboring an F(331)DY(333) motif (i.e. displaying decreased sensitivity to human CG) was used as a backbone in which short portions of the FSH receptor were substituted. Segments from the FSH receptor capable of restoring sensitivity to human CG were identified by transfection of the chimeras in COS-7 cells. These experiments identified key amino acid residues in the hinge region of the FSH receptor associated with the functional role of the second sulfated tyrosine residue in a Y-D/E-Y motif, allowing for efficient hormone binding. The experiments represent strong evidence that structural differences in the hinge regions of FSH and LH/CG receptors play a significant role in hormone-receptor-specific recognition.     

The role of positively charged amino acids in ATP recognition by human P2X(1) receptors

P2X receptors for ATP are a family of ligand-gated cation channels. There are 11 conserved positive charges in the extracellular loop of P2X receptors. We have generated point mutants of these conserved residues (either Lys --> Arg, Lys --> Ala, Arg --> Lys, or Arg --> Ala) in the human P2X(1) receptor to determine their contribution to the binding of negatively charged ATP. ATP evoked concentration-dependent (EC(50) approximately 0.8 microm) desensitizing responses at wild-type (WT) P2X(1) receptors expressed in Xenopus oocytes. Suramin produced a parallel rightward shift in the concentration response curve with an estimated pK(B) of 6.7. Substitution of amino acids at positions Lys-53, Lys-190, Lys-215, Lys-325, Arg-202, Arg-305, and Arg-314 either had no effect or only a small change in ATP potency, time course, and/or suramin sensitivity. Modest changes in ATP potency were observed for mutants at K70R and R292K/A (20- and 100-fold decrease, respectively). Mutations at residues K68A and K309A reduced the potency of ATP by >1400-fold and prolonged the time course of the P2X(1) receptor current but had no effect on suramin antagonism. Lys-68, Lys-70, Arg-292, and Lys-309 are close to the predicted transmembrane domains of the receptor and suggest that the ATP binding pocket may form close to the channel vestibule.     

Studies on the role of amino acids 38-45 in the expression of a functional thyrotropin receptor.

We previously reported that deletion or substitution of a unique eight-amino acid tract (residues 38-45) in the extracellular domain of the human TSH receptor led to the loss of specific ligand binding to the surface of transfected cells. In the present study we analyzed this region in more detail. Using site-directed mutagenesis of the TSH receptor cDNA, we substituted amino acid residues 38-45, either in three overlapping groups of four amino acids each or individually. The resultant TSH receptor mutant cDNAs were stably transfected into Chinese hamster ovary cells, and the cells were tested for their TSH-binding ability. Our data demonstrate that amino acid residues 38-40 and 42-45 in this region of the human TSH receptor can be substituted without alteration in receptor function and are, therefore, not critical in forming or maintaining the TSH-binding site. However, substitution of Cys41, either alone or together with adjacent amino acids, leads to the loss of TSH binding to its receptor. These data suggest a central role for the amino acid in position 41 in preserving the biological function of the TSH receptor.     

Tyrosine sulfation is required for agonist recognition by glycoprotein hormone receptors

The glycoprotein hormone receptors (thyrotrophin receptor, TSHr; luteinizing hormone/chorionic gonadotrophin receptor, LH/CGr; follicle-stimulating hormone receptor, FSHr) constitute a subfamily of rhodopsin-like G protein-coupled receptors (GPCRs) with a long N-terminal extracellular extension responsible for high-affinity hormone binding. These ectodomains contain two cysteine clusters flanking nine leucine-rich repeats (LRR), a motif found in several protein families involved in protein-protein interactions. Similar to the situation described recently in CCR5, we demonstrate here that the TSHr, as it is present at the cell surface, is sulfated on tyrosines in a motif located downstream of the C-terminal cysteine cluster. Sulfation of one of the two tyrosines in the motif is mandatory for high-affinity binding of TSH and activation of the receptor. Site-directed mutagenesis experiments indicate that the motif, which is conserved in all members of the glycoprotein hormone receptor family, seems to play a similar role in the LH/CG and FSH receptors.     

Human choriogonadotropin binds to a lutropin receptor with essentially no N-terminal extension and stimulates cAMP synthesis.

The lutropin (LH) receptor, which belongs to the family of G-protein coupled receptors, consists of an extracellular hydrophilic N-terminal extension of 341 amino acids and a membrane-embedded C-terminal region of 333 amino acids. This C-terminal region comprises a short N terminus, seven transmembrane domains, three cytoplasmic loops, three exoplasmic loops, and a C terminus. Recently, it was reported that the N-terminal extension of the LH receptor alone or a naturally occurring variant LH receptor similar to the N-terminal extension is capable of binding the hormone with an affinity slightly higher than that of the native receptor. This finding raises a question as to whether the N-terminal extension represents the entire hormone binding site and, if so, how is hormone binding transduced to the activation of a G-protein? In an attempt to answer this important question, we have prepared truncated receptors containing an N-terminal extension as short as 10 amino acids. Surprisingly, the truncated receptors were not only capable of binding the hormone, albeit with low affinities, but also capable of stimulating cAMP synthesis. These results suggest a possibility that the hormone, at least in part, interacts with the membrane-embedded C-terminal region and modulates it to activate adenylate cyclase. The low hormone binding affinities of the truncated receptors taken together with high affinity hormone binding to the N-terminal extension of the LH receptor indicate the existence of two or more contact points between the receptor and the hormone.     

A low molecular weight agonist signals by binding to the transmembrane domain of thyroid-stimulating hormone receptor (TSHR) and luteinizing hormone/chorionic gonadotropin receptor (LHCGR)

Many cognate low molecular weight (LMW) agonists bind to seven transmembrane-spanning receptors within their transmembrane helices (TMHs). The thienopyrimidine org41841 was identified previously as an agonist for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and suggested to bind within its TMHs because it did not compete for LH binding to the LHCGR ectodomain. Because of its high homology with LHCGR, we predicted that thyroid-stimulating hormone receptor (TSHR) might be activated by org41841 also. We show that org41841 is a partial agonist for TSHR but with lower potency than for LHCGR. Analysis of three-dimensional molecular models of TSHR and LHCGR predicted a binding pocket for org41841 in common clefts between TMHs 3, 4, 5, 6, and 7 and extracellular loop 2 in both receptors. Evidence for this binding pocket was obtained in signaling studies with chimeric receptors that exhibited improved responses to org41841. Furthermore, a key receptor-ligand interaction between the highly conserved negatively charged E3.37 and the amino group of org41841 predicted by docking of the ligand into the three-dimensional TSHR model was experimentally confirmed. These findings provide the first evidence that, in contrast to the ectodomain binding of cognate ligands, a LMW agonist can bind to and activate glycoprotein hormone receptors via interaction with their transmembrane domain.     

Analysis of parathyroid hormone (PTH)/secretin receptor chimeras differentiates the role of functional domains in the pth/ pth-related peptide (PTHrP) receptor on hormone binding and receptor activation.

The type 1 parathyroid hormore receptor (PTH1r) belongs to the class II family of G protein-coupled receptors. To delineate the sites in the PTH1r's N-terminal region, and the carboxy-core domain (transmembrane segments + extracellular loops) involved in PTH binding, we have evaluated the functional properties of 27 PTH1-secretin chimeras receptors stably expressed in HEK-293 cells. The wild type and chimeric receptors were analyzed for cell surface expression, binding for PTH and secretin, and functional responsiveness (cAMP induction) toward secretin and PTH. The expression levels of the chimeric receptors were comparable to that of the PTH1r (60-100%). The N-terminal region of PTH1r was divided into three segments that were replaced either singly or in various combinations with the homologous region of the secretin receptor (SECr). Substitution of the carboxy-terminal half (residues 105-186) of the N-terminal region of PTH1r for a SECr homologous segment did not reduced affinity for PTH but abolished signaling in response to PTH. This data indicate that receptor activation is dissociable from high affinity hormone binding in the PTH1r, and that the N-terminal region might play a critical role in the activation process. Further segment replacements in the N-termini focus on residues 105-186 and particularly residues 146-186 of PTH1r as providing critical segments for receptor activation. The data obtained suggest the existence of two distinct PTH binding sites in the PTH1r's N-terminal region: one site in the amino-terminal half (residues 1-62) (site 1) that participates in high-affinity PTH binding; and a second site of lower affinity constituted by amino acid residues scattered throughout the carboxy-terminal half (residues 105-186) (site 2). In the absence of PTH binding to site 1, higher concentrations of hormone are required to promote receptor activation. In addition, elimination of the interaction of PTH with site 2 results in a loss of signal transduction without loss of high-affinity PTH binding. Divers substitutions of the extracellular loops of the PTH1r highlight the differential role of the first- and third extracellular loop in the process of PTH1r activation after hormone binding. A chimera containing the entire extracellular domains of the PTH1r and the transmembrane + cytoplasmic domains of SECr had very low PTH binding affinity and did not signal in response to PTH. Further substitution of helix 5 of PTH1r in this chimera increased affinity for PTH that is close to the PTH affinity for the wild-type PTH1r but surprisingly, did not mediate signaling response. Additional substitutions of PTH1r's helices in various combinations emphasize the fundamental role of helix 3 and helix 6 on the activation process of the PTH1r. Overall, our studies demonstrated that several PTH1r domains contribute differentially to PTH binding affinity and signal transduction mechanism and highlight the role of the N-terminal domain and helix 3 and helix 6 on receptor activation.     

ATP binding at human P2X1 receptors. Contribution of aromatic and basic amino acids revealed using mutagenesis and partial agonists

P2X receptors comprise a family of ATP-gated ion channels with the basic amino acids Lys-68, Arg-292, and Lys-309 (P2X(1) receptor numbering) contributing to agonist potency. In many ATP-binding proteins aromatic amino acids coordinate the binding of the adenine group. There are 20 conserved aromatic amino acids in the extracellular ligand binding loop of at least 6 of the 7 P2X receptors. We used alanine replacement mutagenesis to determine the effects of individual conserved aromatic residues on the properties of human P2X(1) receptors expressed in Xenopus oocytes. ATP evoked concentration-dependent (EC(50) approximately 1 microm) desensitizing currents at wild-type receptors and for the majority of mutants there was no change (10 residues) or a <6-fold decrease in ATP potency (6 mutants). Mutants F195A and W259A failed to form detectable channels at the cell surface. F185A and F291A produced 10- and 160-fold decreases in ATP potency. The partial agonists 2',3'-O-(4-benzoyl)-ATP (BzATP) and P(1),P(5)-di(adenosine 5')-pentaphosphate (Ap(5)A) were tested on a range of mutants that decreased ATP potency to determine whether this resulted predominantly from changes in agonist binding or gating of the channel. At K68A and K309A receptors BzATP and Ap(5)A had essentially no agonist activity but antagonized, or for R292A potentiated, ATP responses. At F185A receptors BzATP was an antagonist but Ap(5)A no longer showed affinity for the receptor. These results suggest that residues Lys-68, Phe-185, Phe-291, Arg-292, and Lys-309 contribute to ligand binding at P2X(1) receptors, with Phe-185 and Phe-291 coordinating the binding of the adenine ring of ATP.