Molecular mechanism of Zn2+ agonism in the extracellular domain of GPR39

Ala substitution of potential metal-ion binding residues in the main ligand-binding pocket of the Zn2+-activated G protein-coupled receptor 39 (GPR39) receptor did not decrease Zn2+ potency. In contrast, Zn2+ stimulation was eliminated by combined substitution of His17 and His19, located in the N-terminal segment. Surprisingly, substitution of Asp313 located in extracellular loop 3 greatly increased ligand-independent signaling and apparently eliminated Zn2+-induced activation. It is proposed that Zn2+ acts as an agonist for GPR39, not in the classical manner by directly stabilizing an active conformation of the transmembrane domain, but instead by binding to His17 and His19 in the extracellular domain and potentially by diverting Asp313 from functioning as a tethered inverse agonist through engaging this residue in a tridentate metal-ion binding site.     

Solution structure of the second extracellular loop of human thromboxane A2 receptor

Thromboxane A(2) receptor (TP receptor), a prostanoid receptor, belongs to the G protein-coupled receptor family, composed of three intracellular loops and three extracellular loops connecting seven transmembrane helices. The highly conserved extracellular domains of the prostanoid receptors were found in the second extracellular loop (eLP(2)), which was proposed to be involved in ligand recognition. The 3D structure of the eLP(2) would help to further explain the ligand binding mechanism. Analysis of the human TP receptor model generated from molecular modeling based on bacteriorhodopsin crystallographic structure indicated that about 12-14 A separates the N- and C-termini of the extra- and intracellular loops. Synthetic loop peptides whose termini are constrained to this separation are presumably more likely to mimic the native loop structure than the corresponding loop region peptide with unrestricted ends. To test this new concept, a peptide corresponding to the eLP(2) (residues 173-193) of the TP receptor has been made with the N- and C-termini connected by a homocysteine disulfide bond. Through 2D nuclear magnetic resonance (NMR) experiments, complete (1)H NMR assignments, and structural construction, the overall 3D structure of the peptide was determined. The structure shows two beta-turns at residues 180 and 185. The distance between the N- and C-termini of the peptide shown in the NMR structure is 14.2 A, which matched the distance (14.5 A) between the two transmembrane helices connecting the eLP(2) in the TP receptor model. This suggests that the approach using the constrained loop peptides greatly increases the likelihood of solving the whole 3D structures of the extra- and the intracellular domains of the TP receptor. This approach may also be useful in structural studies of the extramembrane loops of other G protein-coupled receptors.     

Identification of a disulfide bridge connecting the alpha-subunits of the extracellular domain of the insulin receptor.

The alpha 2 beta 2 structure of the insulin receptor has previously been shown to involve one disulfide bridge between the alpha-subunits in the region containing Cys435, Cys468 and Cys524. We have digested the soluble extracellular domain of the insulin receptor with succinylated trypsin, partially separated the resulting peptides, and sequenced a number of fractions. The peptides containing Cys435 and Cys468 appeared in the same fraction, indicating that these two form a disulfide bond, and in another fraction we found the sequence of the peptide containing Cys524. Since it has been shown that the extracellular domain of the insulin receptor has no free thiols and since no other sequences containing cysteine were found in these fractions, we conclude that Cys524 forms a disulfide bond to the Cys524 in the other alpha-subunit.     

Sauvagine cross-links to the second extracellular loop of the corticotropin-releasing factor type 1 receptor

Contact sites between the corticotropin-releasing factor receptor type 1 (CRFR1), the sauvagine (SVG) radioligands [Tyr(0),Gln(1)]SVG ((125)I-YQS) and [Tyr(0),Gln(1), Leu(17)]SVG ((125)I-YQLS) were examined. (125)I-YQLS or (125)I-YQS was cross-linked to CRFR1 using the chemical cross-linker, disuccinimidyl suberate (DSS), which cross-links the epsilon amino groups of lysine residues that have a molecular distance of 11.4 A. DSS specifically and efficiently cross-linked (125)I-YQLS and (125)I-YQS to CRFR1. CRFR1 contains 5 putative extracellular lysine residues (Lys(110), Lys(111), Lys(113), Lys(257), and Lys(262)) that can cross-link to the 4 lysine residues (Lys(16), Lys(22), Lys(25), and Lys(27)) of the radioligands. Identification of the CNBr-cleaved fragments of CRFR1 cross-linked to (125)I-YQLS or (125)I-YQS established that the second extracellular loop of CRFR1 cross-links to Lys(16) of YQS. Additionally, site-directed mutagenesis (changing Lys to Arg in CRFR1 individually and in combination) revealed that Lys(257) in the second extracellular loop of CRFR1 is an important cross-linking site. In conclusion, it was shown that in SVG-bound CRFR1, Lys(257) of CRFR1 lies in close proximity (11.4 A) to Lys(16) of SVG.     

Backbone-methylated analogues of the principle receptor binding region of human parathyroid hormone. Evidence for binding to both the N-terminal extracellular domain and extracellular loop region

We have used backbone N-methylations of parathyroid hormone (PTH) to study the role of these NH groups in the C-terminal amphiphilic alpha-helix of PTH (1-31) in binding to and activating the PTH receptor (P1R). The circular dichroism (CD) spectra indicated the structure of the C-terminal alpha-helix was locally disrupted around the methylation site. The CD spectra differences were explained by assuming a helix disruption for four residues on each side of the site of methylation and taking into account the known dependence of CD on the length of an alpha-helix. Binding and adenylyl cyclase-stimulating data showed that outside of the alpha-helix, methylation of residues Asp30 and Val31 had little effect on structure or activities. Within the alpha-helix, disruption of the structure was associated with increased loss of activity, but for specific residues Val21, Leu24, Arg25, and Leu28 there was a dramatic loss of activities, thus suggesting a more direct role of these NH groups in correct P1R binding and activation. Activity analyses with P1R-delNT, a mutant with its long N-terminal region deleted, gave a different pattern of effects and implicated Ser17, Trp23, and Lys26 as important for its PTH activation. These two groups of residues are located on opposite sides of the helix. These results are compatible with the C-terminal helix binding to both the N-terminal segment and also to the looped-out extracellular region. These data thus provide direct evidence for important roles of the C-terminal domain of PTH in determining high affinity binding and activation of the P1R receptor.     

Essential role for the second extracellular loop in C5a receptor activation

More than 90% of G protein-coupled receptors (GPCRs) contain a disulfide bridge that tethers the second extracellular loop (EC2) to the third transmembrane helix. To determine the importance of EC2 and its disulfide bridge in receptor activation, we subjected this region of the complement factor 5a receptor (C5aR) to random saturation mutagenesis and screened for functional receptors in yeast. The cysteine forming the disulfide bridge was the only conserved residue in the EC2-mutated receptors. Notably, approximately 80% of the functional receptors exhibited potent constitutive activity. These results demonstrate an unexpected role for EC2 as a negative regulator of C5a receptor activation. We propose that in other GPCRs, EC2 might serve a similar role by stabilizing the inactive state of the receptor.     

Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.     

Proline residue 280 in the second extracellular loop (EC2) of the VPAC2 receptor is essential for the receptor structure

Inspection of the amino acid sequence of the human VPAC1 and the VPAC2 receptors after alignment of the conserved residues indicates that the second extracellular loop (EC2) is one amino acid shorter in the VPAC1 receptor due to the lack of a proline residue in position 294. We hypothesized that this could be of importance for receptor structure and/or for ligand recognition. Insertion by directed mutagenesis of a proline in that position (294 VPAC1) had little consequence on the binding of several agonists but reduced the affinity for the VPAC1 antagonist. Coupling of the 294 VPAC1 receptor to adenylate cyclase was improved, as demonstrated by an increased affinity for VIP and other agonists, and by a shift of the VPAC1 antagonist to partial agonist behavior. Deletion of the proline 280 (DeltaPro280 VPAC2) in the VPAC2 receptor markedly reduced the apparent affinity for all the agonists tested. Replacement of the proline by a glycine residue had a smaller effect on the ligands affinities. The proline residue in the VPAC2 receptor EC2 is thus essential for the receptor structure, and the EC2 domain is involved in ligand recognition and receptor functionality.     

Conformational and molecular interaction studies of glucagon-like peptide-2 with its N-terminal extracellular receptor domain

Glucagon-like peptide-2 (GLP-2) is a therapeutic target used in the treatment of short bowel syndrome. In this paper, we present the three dimensional solution structure of GLP-2 peptide determined using nuclear magnetic resonance (NMR) and molecular modelling. The GLP-2 adopts an α-helical conformation similar to that of secretin family of hormones. In order to understand the molecular details governing the ligand binding and receptor activation, macromolecular docking studies were performed between the N-terminal extracellular domain of GLP-2 receptor and the GLP-2 hormone using a data driven docking program.     

Recombinant expression and purification of the N-terminal extracellular domain of the parathyroid hormone receptor

Our goal is to elucidate the nature of the bimolecular interaction of parathyroid hormone (PTH) with its receptor, the parathyroid hormone receptor type-1 (PTHR1). In order to study this interaction, we are aiming to obtain a three-dimensional structure of the PTH-PTHR1 bimolecular complex. Due to the very low expression levels of endogenous PTHR1, a recombinant form is required for structural analysis. However, the extreme hydrophobicity of the transmembrane regions of PTHR1 makes heterologous expression of PTHR1 difficult. Therefore, we sought to express the N-terminal extracellular domain (N-ECD) of PTHR1, a region that plays a pivotal role in ligand interaction. We expressed the N-ECD in both bacterial (Escherichia coli) and insect (Sf9) cells. The form produced in E. coli, a fusion-protein with thioredoxin, is soluble. However, removal of the fusion partner from a partially purified preparation results in dramatic loss of yield of the N-ECD. Expression in Sf9 cells, however, facilitates purification of a soluble form of the N-ECD. Isothermal calorimetry demonstrates that this N-ECD binds PTH-(1-34), albeit with lower affinity than the full-length receptor. This report describes the expression and purification of milligram quantities of the isolated N-ECD of PTHR1. The receptor fragment retains the ability to bind its cognate peptide ligand, an important pre-requisite for subsequent structural studies.     

A structural disulfide of yeast protein-disulfide isomerase destabilizes the active site disulfide of the N-terminal thioredoxin domain

Protein-disulfide isomerase (PDI) is an essential catalyst of disulfide formation and isomerization in the eukaryotic endoplasmic reticulum. PDI has two active sites at either end of the molecule, each containing two cysteines that facilitate thiol-disulfide exchange. In addition to its four catalytic cysteines, PDI possesses two non-active site cysteines whose location and separation distance varies by organism. In higher eukaryotes, the non-active site cysteines are located in the C-terminal half of the protein sequence and are separated by 30 amino acids. In contrast, the internal cysteines of PDI from lower eukaryotes are located near the N-terminal active site and are much closer together in sequence. The function of these cysteines and the significance of their unique location in yeast PDI have been unclear. Previous data (Xiao, R., Wilkinson, B., Solovyov, A., Winther, J. R., Holmgren, A., Lundstrom-Ljung, J., and Gilbert, H. F. (2004) J. Biol. Chem. 279, 49780-49786) suggest that the internal cysteines exist as a disulfide in the endoplasmic reticulum of Saccharomyces cerevisiae. By coupling mass spectrometry with a gel-shift technique that allows us to measure the redox potentials of the PDI active sites in the presence and absence of the non-active site cysteines, we find that the non-active site cysteines form a disulfide that is stable even in a very reducing environment and demonstrate that this disulfide exists to destabilize the N-terminal active site disulfide, making it a better oxidant by 18-fold. Consistent with this finding, we show that mutating the non-active site cysteines to alanines disrupts both the oxidase and isomerase activities of PDI in vitro.     

Role of Arg182 in the second extracellular loop of angiotensin II receptor AT2 in ligand binding.

The phenolic side chain of Tyr(4) present in Ang II is proposed to interact with the side chain of Arg 167 of the AT1 receptor. To determine the contribution of the analogous Arg182 in the ligand-binding properties of the AT2, we replaced the Arg182 with Glu and Ala, and analyzed the ligand-binding properties. Our results suggest that replacing Arg182 with either Glu or Ala abolished the ability of the AT2 receptor to bind the nonspecific peptidic ligands, (125)I-Ang II and [(125)I-Sar(1)-Ile(8)]Ang II, as well as the AT2 receptor-specific peptidic ligand (125)I-CGP42112A. We have shown previously that replacing the positively charged side chain of Lys215 with the negatively charged side chain of Glu in the fifth TMD did not alter the high affinity binding of (125)I-CGP42112A to the AT2 receptor. However, ligand-binding properties of the Arg182Glu mutant suggest that positively charged side chain of Arg182 located in the junction of second ECL and the fourth TMD is critical for high affinity binding of all three peptidic ligands to the AT2 receptor.     

The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily.     

Solution structure of the third extracellular loop of human thromboxane A2 receptor

The extracellular domains of the thromboxane A2 receptor (TP receptor) were found to be involved in the specific ligand recognition. Determination of the three-dimensional (3D) structure of the extracellular loops would help to explain the mechanism of the ligand binding to its receptor with regard to the tertiary structure. Based on our previous studies on the extracellular loop of the human TP receptor, the synthetic loop peptides, whose termini are constrained to 10 to 14-A separations, are more likely to mimic the native structure of the extracellular loops. In this study, a peptide with the sequence of the third extracellular loop (eLP3, residues 271-289) of the TP receptor was synthesized, and its termini were constrained by the formation of a disulfide bond between the additional homocysteines located at both ends. Fluorescence spectroscopic studies showed that the fluorescence intensity of this constrained loop peptide could be increased by the addition of SQ29,548, a TP receptor antagonist, which indicated the interaction between the peptide and the ligand. The structure of this peptide was then studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. 1H NMR assignments of the peptide were obtained and structure constraints were derived from nuclear Overhauser effects and J-coupling constants. The solution structure of the peptide was then calculated based on these constraints. The overall structure shows a beta turn from residues 278 to 281. It also shows a distance of 9.45A between the ends of the N and C termini of the peptide, which agrees with the distance between the two residues at the ends of the transmembrane helices connecting the eLP3 on the TP receptor working model generated using molecular modeling, based on the crystal structure of bovine rhodopsin. These results provide valuable information for the characterization of the complete 3D structure of the extracellular domains of the human TP receptor.     

Role of the second extracellular loop of human C3a receptor in agonist binding and receptor function

The C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor with an unusually large second extracellular loop (e2 loop, approximately 172 amino acids). To determine the function of this unique structure, chimeric and deletion mutants were prepared and analyzed in transfected RBL-2H3 cells. Whereas replacement of the C3aR N-terminal segment with that from the human C5a receptor had minimal effect on C3a binding, substitution of the e2 loop with a smaller e2 loop from the C5a receptor (C5aR) abolished binding of 125I-C3a and C3a-stimulated calcium mobilization. However, as much as 65% of the e2 loop sequence (amino acids 198-308) may be removed without affecting C3a binding or calcium responses. The e2 loop sequences adjacent to the transmembrane domains contain multiple aspartate residues and are found to play an important role in C3a binding based on deletion mutagenesis. Replacement of five aspartate residues in the e2 loop with lysyl residues significantly compromised both the binding and functional capabilities of the C3a receptor mediated by intact C3a or by two C3a analog peptides. These data suggest a two-site C3a-C3aR interaction model similar to that established for C5a/C5aR. The anionic residues near the N and C termini of the C3aR e2 loop constitute a non-effector secondary interaction site with cationic residues in the C-terminal helical region of C3a, whereas the C3a C-terminal sequence LGLAR engages the primary effector site in C3aR.     

Identification of residues important for ligand binding of thromboxane A2 receptor in the second extracellular loop using the NMR experiment-guided mutagenesis approach

The second extracellular loop (eLP2) of the thromboxane A(2) receptor (TP) had been proposed to be involved in ligand binding. Through two-dimensional (1)H NMR experiments, the overall three-dimensional structure of a constrained synthetic peptide mimicking the eLP2 had been determined by our group (Ruan, K.-H., So, S.-P., Wu, J., Li, D., Huang, A., and Kung, J. (2001) Biochemistry 40, 275-280). To further identify the residues involved in ligand binding, a TP receptor antagonist, SQ29,548 was used to interact with the synthetic peptide. High resolution two-dimensional (1)H NMR experiments, NOESY, and TOCSY were performed for the peptide, SQ29,548, and peptide with SQ29,548, respectively. Through completed (1)H NMR assignment and by comparing the different spectra, extra peaks were observed on the NOESY spectrum of the peptide with SQ29,548, which implied the contacts between residues of eLP2 at Val(176), Leu(185), Thr(186), and Leu(187) with SQ29,548 at position H2, H7, and H8. Site-directed mutagenesis was used to confirm the possible ligand-binding sites on native human TP receptor. Each of the four residues was mutated to the residues either in the same group, with different structure or different charged. The mutated receptors were then tested for their ligand binding activity. The receptor with V176L mutant retained binding activity to SQ29,548. All other mutations resulted in decreased or lost binding activity to SQ29,548. These mutagenesis results supported the prediction from NMR experiments in which Val(176), Leu(185), Thr(186), and Leu(187) are the possible residues involved in ligand binding. This information facilitates the understanding of the molecular mechanism of thromboxane A(2) binding to the important receptor and its signal transduction.     

The human growth hormone receptor. Secretion from Escherichia coli and disulfide bonding pattern of the extracellular binding domain

A gene fragment encoding the extracellular domain of the human growth hormone (hGH) receptor from liver was cloned into a plasmid under control of the Escherichia coli alkaline phosphatase promoter and the heat-stable enterotoxin (StII) signal peptide sequence. Strains of E. coli expressing properly folded hGH binding protein were identified by blotting colonies with 125I-hGH. The E. coli strain capable of highest expression (KS330) secreted 10 to 20 mg/liter of culture of properly processed and folded hGH receptor fragment into the periplasmic space. The protein was purified to near homogeneity in 70 to 80% yield (in tens of milligram amounts) using ammonium sulfate precipitation, hGH affinity chromatography, and gel filtration. The unglycosylated extracellular domain of the hGH receptor has virtually identical binding properties compared to its natural glycosylated counterpart isolated from human serum, suggesting glycosylation is not important for binding of hGH. The extracellular binding domain codes for 7 cysteines, and we show that six of them form three disulfide bonds. Peptide mapping studies show these disulfides are paired sequentially to produce short loops (10-15 residues long) as follows: Cys38-Cys48, Cys83-Cys94, and Cys108-Cys122. Cys241 is unpaired, and mutagenic analysis shows that the extreme carboxyl end of the receptor fragment (including Cys241) is not essential for folding or binding of the protein to hGH. High level expression of this receptor binding domain and its homologs in E. coli will greatly facilitate their detailed biophysical and structural analysis.     

Multiple residues in the second extracellular loop are critical for M3 muscarinic acetylcholine receptor activation

Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cis-retinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys(220) residue, Gln(207), Gly(211), Arg(213), Gly(218), Ile(222), Phe(224), Leu(225), and Pro(228) were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance.     

A profile of the residues in the second extracellular loop that are critical for ligand recognition of human prostacyclin receptor

The residues in the second extracellular loop (eLP2) of the prostanoid receptors, which are important for specific ligand recognition, were previously predicted in our earlier studies of the thromboxane A2 receptor (TP) using a combination of NMR spectroscopy and recombinant protein approaches. To further test this hypothesis, another prostanoid receptor, the prostacyclin receptor (IP), which has opposite biological characteristics to that of TP, was used as a model for these studies. A set of recombinant human IPs with site-directed mutations at the nonconserved eLP2 residues were constructed using an Ala-scanning approach, and then expressed in HEK293 and COS-7 cells. The expression levels of the recombinant receptors were six-fold higher in HEK293 cells than in COS-7 cells. The residues important for ligand recognition and binding within the N-terminal segment (G159, Q162, and C165) and the C-terminal segment (L172, R173, M174, and P179) of IP eLP2 were identified by mutagenesis analyses. The molecular mechanisms for the specific ligand recognition of IP were further demonstrated by specific site-directed mutagenesis using different amino acid residues with unique chemical properties for the key residues Q162, L172, R173, and M174. A comparison with the corresponding functional residues identified in TP eLP2 revealed that three (Q162, R173, and M174) of the four residues are nonconserved, and these are proposed to be involved in specific ligand recognition. We discuss the importance of G159 and P179 in ligand recognition through configuration of the loop conformation is discussed. These studies have further indicated that characterization of the residues in the eLP2 regions for all eight prostanoid receptors could be an effective approach for uncovering the molecular mechanisms of the ligand selectivities of the G-protein-coupled receptors.     

Agonist versus antagonist action of ATP at the P2Y4 receptor is determined by the second extracellular loop

UTP is a potent full agonist at both the human P2Y(4) (hP2Y(4)) and rat P2Y(4) (rP2Y(4)) receptor. In contrast, ATP is a potent full agonist at the rP2Y(4) receptor but is a similarly potent competitive antagonist at the hP2Y(4) receptor. To delineate the structural determinants of agonism versus antagonism in these species homologues, we expressed a series of human/rat P2Y(4) receptor chimeras in 1321N1 human astrocytoma cells and assessed the capacity of ATP and UTP to mobilize intracellular Ca(2+). Replacement of the NH(2) terminus of the hP2Y(4) receptor with the corresponding region of the rP2Y(4) receptor resulted in a receptor that was activated weakly by ATP, whereas replacement of the second extracellular loop (EL2) of the hP2Y(4) receptor with that of the rP2Y(4) receptor yielded a chimeric receptor that was activated fully by UTP and near fully by ATP, albeit with lower potencies than those observed at the rP2Y(4) receptor. These potencies were increased, and ATP was converted to a full agonist by replacing both the NH(2) terminus and EL2 in the hP2Y(4) receptor with the corresponding regions from the rP2Y(4) receptor. Mutational analysis of the five divergent amino acids in EL2 between the two receptors revealed that three amino acids, Asn-177, Ile-183, and Leu-190, contribute to the capacity of EL2 to impart ATP agonism. Taken together, these results suggest that the second extracellular loop and the NH(2) terminus form a functional motif that plays a key role in determining whether ATP functions as an agonist or antagonist at mammalian P2Y(4) receptors.