Human Adenosine Receptor Research Articles

Adenosine receptors and their ligands.

The regulatory actions of adenosine are mediated via four subtypes of G protein-coupled receptors distinguished as A1, A2A, A2B and A3 receptors. Their presence on basically every cell makes them an interesting target for the pharmacological intervention in many pathophysiological situations. A large number of ligands have been synthesized over the last two decades and provide agonists and antagonists that are more or less selective for the known receptor subtypes. In addition, many radioligands are available in tritiated or radioiodinated form. The comparative pharmacological characterization of all four human adenosine receptor subtypes revealed that some of the compounds thought to be selective from data in other species have unexpected potencies at human receptors. As a result, compounds that exhibit high affinity to only one subtype are an exception. Although the selection of ligands is immense, it is less than satisfying for most subtypes of adenosine receptors.

Human Adenosine A1, A2A, A2B, and A3 Receptors Expressed in Chinese Hamster Ovary Cells All Mediate the Phosphorylation of Extracellular-Regulated Kinase 1/2

The known diverse effects of adenosine on mitogenesis may be related to changes in mitogen-activated protein kinases. In this study we therefore compared the phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) via the four known human adenosine receptors A(1), A(2A), A(2B), and A(3), stably transfected into Chinese hamster ovary (CHO) cells. The adenosine analog 5'-N-ethylcarboxamidoadenosine (NECA), known to act on all subtypes, had no effect on untransfected CHO cells, but did cause a substantial time- and dose-dependent phosphorylation in CHO cells transfected with each of the receptors. The maximal phosphorylation was highest in A(1) and A(3) receptor-transfected cells, intermediate in A(2A) and low in A(2B) receptor-expressing CHO cells. For all receptors the half-maximal ERK1/2 phosphorylation was observed at 19-115 nM NECA. NECA acting on adenosine A(2B) receptors was much more potent in stimulating ERK1/2 phosphorylation (EC(50) = 19 nM) than cAMP formation (EC(50) = 1.4 microM). Stimulation with the endogenous ligand adenosine resulted in the same pattern of ERK1/2 phosphorylation as NECA. Concentrations of adenosine that occur physiologically caused an increased phosphorylation after 5 min in CHO cells transfected with any one of the four adenosine receptors. Adenosine at levels reached during ischemia (3 microM) induced a more pronounced, but still transient, activation of ERK1/2. In conclusion, this study shows that all the human adenosine receptors transfected into CHO cells are able to activate ERK1/2 at physiologically relevant concentrations of the endogenous agonist.

ChemInform Abstract: Synthesis of Di‐ and Trisubstituted Adenosine Derivatives (V) and (VIII) and Their Affinities at Human Adenosine Receptor Subtypes.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis of Di- And Tri-Substituted Adenosine Derivatives and Their Affinities at Human Adenosine Receptor Subtypes

The synthesis of 2-(hex-l-ynyl)adenosine derivatives substituted at the N6- and/or 5′-position was carried out on the basis that 2-(hex-l-ynyl)adenosine-5′-N-ethyluronamide (HENECA, 2) showed good affinity and different degree of selectivity for rat adenosine receptors. All new compounds were tested in radioligand binding and adenylyl cyclase assays with recently cloned human A1, A2A, A2B, and A3 adenosine receptors.

Palmitoylation of the recombinant human A1 adenosine receptor: enhanced proteolysis of palmitoylation-deficient mutant receptors

Palmitoylation of the recombinant human A1 adenosine receptor (A1AR) expressed in HEK-293 cells is demonstrated by showing that hexahistidine (His6)/Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG) (H/F) A1ARs, purified to homogeneity from cells metabolically labelled with [3H]palmitate, incorporate tritium into a 38-42 kDa receptor glycoprotein. The amount of palmitoylation is not affected by incubation of cells with the A1AR-selective agonist N6-cyclopentyladenosine (CPA). A1AR palmitoylation is abolished by treatment with neutral hydroxylamine or by mutation of Cys-309 to Ala (C309 → A). Based on Western blotting and pulse-chase experiments with [35S]methionine, at least 90% of wild-type receptors are palmitoylated and turn over with a t½ of 6.4 h. Of the C309 → A mutated receptors, 40% appear to turn over like wild-type receptors, with a t½ of 7.1 h, and 60% appear to be rapidly cleaved to form a 25 kDa receptor fragment that turns over with a t½ of 0.8 h. In HEK-293 cell lines expressing similar numbers of wild-type or C309 → A mutant A1Rs, there is little difference in the kinetics of CPA-induced receptor internalization (1 h), down-regulation (24 h), inhibition of forskolin-stimulated cAMP accumulation, or activation of co-transfected G-protein-activated inward rectifier K+/cardiac inward rectifying K+ (GIRK1/CIR K+) channels. Also unaffected by palmitoylation is guanosine 5′-[γ-thio]-triphosphate ([S]GTP)-sensitive binding to membranes by the agonist 125I-labelled aminobenzyladenosine. The results suggest that palmitoylation has little effect on receptor-effector coupling, agonist-induced internalization or down-regulation. We speculate that palmitoylation may divert newly synthesized A1ARs from a pathway leading to rapid degradation.

Palmitoylation of the recombinant human A1 adenosine receptor: enhanced proteolysis of palmitoylation-deficient mutant receptors

Palmitoylation of the recombinant human A(1) adenosine receptor (A(1)AR) expressed in HEK-293 cells is demonstrated by showing that hexahistidine (His(6))/Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG) (H/F) A(1)ARs, purified to homogeneity from cells metabolically labelled with [(3)H]palmitate, incorporate tritium into a 38-42 kDa receptor glycoprotein. The amount of palmitoylation is not affected by incubation of cells with the A(1)AR-selective agonist N(6)-cyclopentyladenosine (CPA). A(1)AR palmitoylation is abolished by treatment with neutral hydroxylamine or by mutation of Cys-309 to Ala (C(309)-->A). Based on Western blotting and pulse-chase experiments with [(35)S]methionine, at least 90% of wild-type receptors are palmitoylated and turn over with a t1/2 of 6.4 h. Of the C(309)-->A mutated receptors, 40% appear to turn over like wild-type receptors, with a t1/2 of 7.1 h, and 60% appear to be rapidly cleaved to form a 25 kDa receptor fragment that turns over with a t1/2 of 0.8 h. In HEK-293 cell lines expressing similar numbers of wild-type or C(309)-->A mutant A(1)Rs, there is little difference in the kinetics of CPA-induced receptor internalization (1 h), down-regulation (24 h), inhibition of forskolin-stimulated cAMP accumulation, or activation of co-transfected G-protein-activated inward rectifier K(+)/cardiac inward rectifying K(+) (GIRK1/CIR K(+)) channels. Also unaffected by palmitoylation is guanosine 5'-[gamma-thio]-triphosphate ([S]GTP)-sensitive binding to membranes by the agonist (125)I-labelled aminobenzyladenosine. The results suggest that palmitoylation has little effect on receptor-effector coupling, agonist-induced internalization or down-regulation. We speculate that palmitoylation may divert newly synthesized A(1)ARs from a pathway leading to rapid degradation.

2-Substituted N-ethylcarboxamidoadenosine derivatives as high-affinity agonists at human A3 adenosine receptors.

A number of 2-substituted 5'-N-ethylcarboxamidoadenosine (NECA) derivatives was investigated for their affinity and selectivity at human A3 adenosine receptors. The compounds were tested in radioligand competition studies and modulation of adenylyl cyclase activity on membranes from CHO cell lines stably transfected with the four human adenosine receptor subtypes. In binding studies the most potent compound, 2-(3-hydroxy-3-phenyl)propyn-1-yl-NECA (PHPNECA), exhibited a subnanomolar affinity for A3 adenosine receptors with a Ki value of 0.4 nM. As opposed to the limited A3 selectivity of PHPNECA, a 100-fold selectivity compared to both A1 and A2A receptors was found for 2-(2-phenyl)ethynyl-NECA (PENECA; Ki 6 nM). The EC50 values for activation of adenylyl cyclase via A2A adenosine receptors were in good agreement with the respective Ki values from binding experiments. In contrast, IC50 values for A1 and A3 receptor-mediated inhibition of adenylyl cyclase were shifted to higher values compared to the respective affinities determined in radioligand competition studies. Similar discrepancies between binding and functional data have been observed for the inhibitory A1 adenosine receptor in previous studies. Therefore, the same A3 selectivity of PENECA compared to A1 receptors was found in binding and adenylyl cyclase inhibition whereas the selectivity compared to A2A receptors that was detected in ligand binding was obscured in the functional assay. The series of compounds presented in this study identifies 2-substitution of the purine system as a promising target for the development of A3-selective high-affinity ligands.

Acyl-Hydrazide Derivatives of a Xanthine Carboxylic Congener (XCC) as Selective Antagonists at Human A2B Adenosine Receptors.

The structure–activity relationships (SAR) of 8-phenyl-1,3-dipropylxanthine derivatives in binding to recombinant human A2B adenosine receptors were explored, in order to identify selective antagonists. Based on the finding of receptor selectivity in MRS 1204, containing an N-hydroxysuccinimide ester attached through the p-position of the 8-phenyl substituent [Jacobson et al. (1999): Drug Dev. Res., 47:45–53], a hydrazide and its more stable imide derivatives were synthesized. The hydrazide of XCC (8-[4-[[[carboxy]methyl]oxy]phenyl]-1,3-dipropylxanthine) was acylated with a variety of mono- and dicarboxylic acids. Ki values were determined in the adenosine receptor binding assays. At recombinant human A2B receptors expressed in membranes of HEK-293 cells, antagonist radioligands used were the xanthine 125I-ABOPX (125I-3-(4-amino-3-iodobenzyl)-8-oxyacetate-1-propyl-xanthine) and the nonxanthine antagonist [3H]ZM 241385 ([3H]4-(2-[7-amino-2-{furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-ylamino-ethyl)phenol). The initial screening utilized rat A1/A2A receptors and human A3 receptors, and selected compounds were examined at the human A1/A2A subtypes. A 1,2-dimethylmaleimide derivative, 14 (MRS 1595), bound to human A2B receptors with a Ki of 19 nM and proved to be selective vs. human A1/A2A/A3 receptors by 160-, 100-, and 35-fold, respectively. Enprofylline (3-propylxanthine) is slightly selective for A2B receptors, suggesting removal of the 1-propyl group; however, combination of the 1-H-3-Pr and 8-phenyl substituents eliminated the selectivity. Other potent and moderately selective A2B antagonists were a tetrahydrophthaloyl derivative 18b (MRS 1614, Ki value 10 nM) and amino acid conjugates of the XCC-hydrazide, i.e., the glutarimide 24b (MRS 1626, Ki value 13 nM), and protected dipeptide 27 (MRS 1615, Ki value 11 nM). Drug Dev. Res. 47:178–188, 1999.

Acyl‐hydrazide derivatives of a xanthine carboxylic congener (XCC) as selective antagonists at human A2B adenosine receptors

Acyl‐hydrazide derivatives of a xanthine carboxylic congener (XCC) as selective antagonists at human A2B adenosine receptors

1,3-Dialkylxanthine Derivatives Having High Potency as Antagonists at Human A2B Adenosine Receptors.

The structure-activity relationships (SAR) of alkylxanthine derivatives as antagonists at the recombinant human adenosine receptors were explored in order to identify selective antagonists of A2B receptors. The effects of lengthening alkyl substituents from methyl to butyl at 1- and 3-positions and additional substitution at the 7- and 8-positions were probed. Ki values, determined in competition binding in membranes of HEK-293 cells expressing A2B receptors using 125I-ABOPX (125I-3-(4-amino-3-iodobenzyl)-8-(phenyl-4-oxyacetate)-1-propylxanthine), were approximately 10 to 100 nM for 8-phenylxanthine functionalized congeners. Xanthines containing 8-aryl, 8-alkyl, and 8-cycloalkyl substituents, derivatives of XCC (8-[4-[[[carboxy]methyl]oxy]phenyl]-1,3-dipropylxanthine) and XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]-oxy]phenyl]-1,3-dipropylxanthine), containing various ester and amide groups, including L- and D-amino acid conjugates, were included. Enprofylline was 2-fold more potent than theophylline in A2B receptor binding, and the 2-thio modification was not tolerated. Among the most potent derivatives examined were XCC, its hydrazide and aminoethyl and fluoroethyl amide derivatives, XAC, N-hydroxyethyl-XAC, and the L-citrulline and D-p-aminophenylalanine conjugates of XAC. An N-hydroxysuccinimide ester of XCC (XCC-NHS, MRS 1204) bound to A2B receptors with a Ki of 9.75 nM and was the most selective (at least 20-fold) in this series. In a functional assay of recombinant human A2B receptors, four of these potent xanthines were shown to fully antagonize the effects of NECA-induced stimulation of cyclic AMP accumulation.

Identification of the Adenine Binding Site of the Human A1 Adenosine Receptor

To provide new insights into ligand-A1 adenosine receptor (A1AR) interactions, site-directed mutagenesis was used to test the role of several residues in the first four transmembrane domains of the human A1AR. First, we replaced eight unique A1AR residues with amino acids present at corresponding transmembrane (TM) positions of A2AARs. We also tested the role of carboxamide amino acids in TMs 1-4, and the roles of Val-87, Leu-88, and Thr-91 in TM3. Following conversion of Gly-14 in TM1 to Thr-14, the affinity for adenosine agonists increased 100-fold, and after Pro-25 in TM1 was converted to Leu-25, the affinity for agonists fell. After conversion of TM3 sites Thr-91 to Ala-91, and Gln-92 to Ala-92, the affinity for N6-substituted agonists was reduced, and binding of ligands without N6 substituents was eliminated. When Leu-88 was converted to Ala-88, the binding of ligands with N6 substituents was reduced to a greater extent than ligands without N6 substituents. Following conversion of Pro-86 to Phe-86, the affinity for N6-substituted agonists was lost, and the affinity for ligands without N6 substitution was reduced. These observations strongly suggest that Thr-91 and Gln-92 in TM3 interact with the adenosine adenine moiety, and Leu-88 and Pro-86 play roles in conferring specificity for A1AR selective compounds. Using computer modeling based on the structure of rhodopsin, a revised model of adenosine-A1AR interactions is proposed with the N6-adenine position oriented toward the top of TM3 and the ribose group interacting with the bottom half of TMs 3 and 7.

Dexamethasone stimulates human A1 adenosine receptor (A1AR) gene expression through multiple regulatory sites in promoter B.

The expression of the human A1 adenosine receptor gene is controlled by two promoters, promoters A and B, and they are located 600 base pairs apart. The characteristics of the two promoters differ by the activity of expression, tissue specificity, and the potential regulatory elements around them. Promoter A is more active but its expression is observed only in selected tissues, whereas promoter B is constitutively expressed but at much reduced levels. In Chinese hamster ovary (CHO) cells transiently transfected with plasmids containing either promoter linked to a reporter gene, dexamethasone (dex) can stimulate (or enhance) the expression of promoter B much more effectively than that of promoter A. Mutation and deletion studies on plasmids containing promoter B have shown that the stimulation is mediated through multiple regulatory sites, including a serum response element, AP1, and TATA box. However, a single-glucocorticoid response element monomer-binding site between promoters A and B does not have significant contribution to dex-regulated expression. The interactions between glucocorticoid receptor (GR) and some regulatory sites are probably occurring via this protein (GR) interacting with other DNA-binding proteins because there is no GR DNA-binding sequence in the sites studied. The stimulation can be eliminated by mifepristone, an antagonist of GR, indicating the involvement of GR in gene regulation. In addition, dex treatment also stimulated the expression of A1 adenosine receptors in CHO cells transfected with the plasmids containing contiguous genomic sequences of promoter B or promoters A and B linked to the receptor-coding sequence. When promoter A is active and both promoter A and B are present in a construct, dex treatment induced a much smaller percentage of stimulation.

Comparison of CGS 15943, ZM 241385 and SCH 58261 as antagonists at human adenosine receptors

Three structurally related non-xanthine compounds, CGS 15943, ZM 241385 and SCH 58261, are potent A2A adenosine receptor antagonists and have been used as tools in many pharmacological studies. We have now characterized their affinity and selectivity profile on human adenosine receptors stably transfected into either CHO cells (A1 and A2B receptors) or HEK-293 cells (A2A and A3 receptors). In binding studies using [3H]SCH 58261 as a radioligand, the three compounds were equally potent at A2A receptors, their K(i) value being less than 1 nM. Affinity for A1 and A3 receptors was measured using [3H]DPCPX and [125I]AB-MECA as radioligands. Given the lack of selective ligands, interaction with A2B receptors was assessed using the cAMP accumulation assay following stimulation by the adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA). CGS 15943 was almost as potent at A1 receptors (K(i)3.5 nM) as at A2A receptors, showed moderate affinity for A3 receptors (K(i) 95 nM) and also interacted with A2B receptors (K(i) 44 nM; pA2 7.5). ZM 241385 showed little affinity for A1 receptors (K(i) 255 nM), and did not interact with A3 receptors (K(i)>10 microM); however, it displayed moderate affinity for A2B receptors (K(i) 50 nM; pA2 7.3). SCH 58261 had weak affinity for A1 receptors (K(i) 287 nM), no interaction with A3 receptors (K(i)>10 microM), and showed negligible interaction with A2B receptors (K(i) 5 microM; pA2 6.0). These data indicate that SCH 58261 is the most selective A2A antagonist currently available. Moreover, the different receptor selectivity of these three chemically related compounds provides useful information to progress with structure-activity relationship studies.

Characterization of potent ligands at human recombinant adenosine receptors

The four adenosine receptor subtypes have been stably transfected into Chinese hamster ovary (CHO) cells allowing for comparative studies in a similar cellular background, using radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). We are currently using the transfected CHO cells for extensive screening of nucleosides and purine derivatives of our library. Screening of a number of 2-alkynyl analogs of 5′-N-ethylcarboxamidoadenosine (NECA) indicated that introduction of particular substituents, such as the racemic 2-phenylhydroxypropynyl group, led to a highly potent, nonselective agonist at A1, A2A, and A3 subtypes (PHPNECA, Ki in the low nanomolar range at the three subtypes). In the A2B functional assay, it has been found that PHPNECA (EC50 A2B = 0.88 μM) is threefold more potent than NECA. This article is the first report in which the introduction of a bulky group in the 2-position of NECA led to a compound that is active as an agonist at the human A2B subtype. On the other hand, the presence of a phenyl ring conjugated to the triple bond as in phenylethynylNECA (PENECA) enhanced selectivity for the A3 subtype. In the purine series (potential antagonists), 8-bromo-9-ethyladenine (8-BEA) showed good affinity toward all adenosine receptor subtypes (Ki A1 = 0.28 μM, Ki A2A = 0.052 μM, Ki A2B = 0.84 μM, Ki A3 = 27.8 μM). On the other hand, the introduction of alkynyl chains in the 8-position resulted in an increased affinity at the A3 receptor (8-hexynyl-9-ethyladenine, 8-HEEA, Ki A3 = 0.62 μM and 8-phenylethynyl-9-ethyladenine, 8-PEEA, Ki A3 = 0.086 μM). Drug Dev. Res. 45:176–181, 1998. © 1998 Wiley-Liss, Inc.

Antagonists of the Receptor-G Protein Interface Block Gi-coupled Signal Transduction

The carboxyl terminus of heterotrimeric G protein alpha subunits plays an important role in receptor interaction. We demonstrate that peptides corresponding to the last 11 residues of Galphai1/2 or Galphao1 impair agonist binding to A1 adenosine receptors, whereas Galphas or Galphat peptides have no effect. Previously, by using a combinatorial library we identified a series of Galphat peptide analogs that bind rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366). Native Galphai1/2 peptide as well as several analogs were tested for their ability to modulate agonist binding or antagonist-agonist competition using cells overexpressing human A1 adenosine receptors. Three peptide analogs decreased the Ki, suggesting that they disrupt the high affinity receptor-G protein interaction and stabilize an intermediate affinity state. To study the ability of the peptides to compete with endogenous Galphai proteins and block signal transduction in a native setting, we measured activation of G protein-coupled K+ channels through A1 adenosine or gamma-aminobutyric acid, type B, receptors in hippocampal CA1 pyramidal neurons. Native Galphai1/2, peptide, and certain analog peptides inhibited receptor-mediated K+ channel gating, dependent on which receptor was activated. This differential perturbation of receptor-G protein interaction suggests that receptors that act on the same G protein can be selectively disrupted.

New substituted 9-alkylpurines as adenosine receptor ligands

In the present study an investigation of the structure–activity relationships in 9-ethylpurine derivatives, aimed at preparing A 1, A 2A, A 2B, and A 3 selective adenosine receptor antagonists, was undertaken. Our synthetic approach was to introduce various substituents (amino, alkoxy and alkynyl groups) into the 2-, 6-, or 8-positions of the purine ring. The starting compounds for each series of derivatives were respectively: 2-iodo-9-ethyladenine ( 9), obtained from 2-amino-6-chloropurine ( 5); 9-ethyl-6-iodo-9 H-purine ( 11), 8-bromo-9-ethyl-adenine ( 3) and 8-bromo-9-ethyl-6-iodo-9 H-purine ( 13), obtained from 9-ethyl-adenine ( 2). The synthesized compounds were tested in in vitro radioligand binding assays at A 1, A 2A, and A 3 human adenosine receptor subtypes. Due to the lack of a suitable radioligand the affinity of the 9-ethyladenine derivatives at A 2B adenosine receptors was determined in adenylyl cyclase experiments. In general, the series of 9-ethylpurine derivatives exhibited a similar pharmacological profile at A 1 and A 2A receptors whereas some differences were found for the A 3 and the A 2B subtypes. 8-Bromo-9-ethyladenine ( 3) showed higher affinity for all receptors in comparison to the parent compound 2, and the highest affinity in the series for the A 2A and A 2B subtypes ( K i=0.052 and 0.84 μM, respectively). Analyzing the different substituents, a phenethoxy group in 2-position ( 10a) gave the highest A 2A versus A 2B selectivity (near 400-fold), whereas a phenethylamino group in 2- and 6-position ( 10b and 12b, respectively) improved the affinity at A 2B receptors, compared to the parent compound 2. The presence of a hexynyl substituent in 8-position led to a compound with good affinity at the A 3 receptor ( 4d, K i=0.62 μM), whereas (ar)alkynyl groups are detrimental for the potency at the A 2B subtype. These differences give raise to the hope that further modifications will result in the development of currently unavailable leads with good affinity and selectivity for A 2B adenosine receptors.

A single-stranded DNA binding site in the human A1 adenosine receptor gene promoter.

Human A1 adenosine receptor gene expression is controlled by two independent promoters. The upstream promoter, promoter A, is subject to tissue specific regulation because not all cells express the mRNA associated with this promoter. One potential regulatory sequence located downstream of the TATA box is an AGG element appearing in a tandem repeat. In a previous study, transient transfection assays showed that mutations made in those AGG elements substantially reduced promoter activity. In the current study, DNase I footprinting indicated nuclear protein binding to this sequence between the TATA box and transcriptional start site. Electrophoretic mobility shift assay confirmed further the presence of an AGG element binding protein (AGBP) in human brain nuclear protein extracts. This binding protein has much higher affinity for single-stranded than for double-stranded DNA, and the binding is sequence specific. A series of assays also showed that AGBP is not related to the nuclear factor SP1 and the binding does not require metal cofactors. Therefore, AGBP is likely to be a specific single-stranded DNA binding protein that is required for the full expression of A1 adenosine receptor gene and particularly abundant in brain tissue.

An endogenous A2B adenosine receptor coupled to cyclic AMP generation in human embryonic kidney (HEK 293) cells.

1. Cyclic AMP generation by adenosine analogues was examined in human embryonic kidney (HEK 293) cells by use of a [3H]-adenine pre-labelling methodology. 2. Adenosine analogues showed the following rank order of potency (pD2 value): 5'-N-ethylcarboxamidoadenosine (NECA, 5.24)>2-chloroadenosine (4.41) > or = adenosine (4.19)= N6-(2-(4-aminophenyl)-ethylamino)adenosine (APNEA, 4.11). The A2A-selective agonist CGS21680 failed to elicit a significant stimulation of cyclic AMP generation at concentrations below 30 microM. 3. Of these agents, NECA was observed to exhibit the greatest intrinsic activity, while in comparison maximal responses to adenosine (76+/-8% NECA response), 2-chloroadenosine (70+/-6%) and APNEA (40+/-3%) were significantly reduced. 4. Antagonists of the NECA-evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA2 value): CGS 15943 (7.79)=XAC (7.74)>DPCPX (7.01)=PD115199 (6.93) 8FB-PTP (6.80)>KF 17837 (5.98)>3-propylxanthine (5.13). 5. Agarose gel electrophoresis of the products of the polymerase chain reaction, with cDNA generated from HEK 293 cell total RNA showed virtually identical patterns and nucleotide sizes in comparison with the vector for the full length human brain A2B adenosine receptor. 6. We concluded that HEK 293 cells express an endogenous adenosine receptor coupled to cyclic AMP generation which is of the A2B subtype.

βγ Subunits of Pertussis Toxin-sensitive G Proteins Mediate A1 Adenosine Receptor Agonist-induced Activation of Phospholipase C in Collaboration with Thyrotropin: A NOVEL STIMULATORY MECHANISM THROUGH THE CROSS-TALK OF TWO TYPES OF RECEPTORS

COS-7 cells were transiently transfected with human thyrotropin receptor and dog A1 adenosine receptor cDNAs. An A1 agonist, N6-(L-2-phenylisopropyl) adenosine (PIA), which is ineffective alone, enhanced the thyrotropin (TSH)-induced inositol phosphate production, reflecting phospholipase C (PLC) activation, but inhibited the TSH-induced cAMP accumulation, reflecting adenylyl cyclase inhibition. These PIA-induced actions were completely inhibited by pertussis toxin (PTX) treatment. Moreover, in the cells expressing a PTX-insensitive mutant of Gi2alpha or Gi3alpha, in which a glycine residue was substituted for a cysteine residue to be ADP-ribosylated by PTX, at the fourth position of the C terminus, PIA effectively exerted both stimulatory and inhibitory effects on the TSH-induced actions although the cells were treated with the toxin. Overexpression of the betagamma subunits of the G proteins enhanced the TSH-induced inositol phosphate production without any significant effect on the cAMP response; under these conditions, PIA did not further increase the elevated inositol phosphate response to TSH. On the contrary, overexpression of a constitutively active mutant of Gi2alpha, in which the guanosine triphosphatase activity is lost, inhibited the TSH-induced cAMP accumulation but hardly affected the inositol phosphate response; under these conditions, PIA never exerted further inhibitory effects on the cAMP response to TSH. In contrast to the case of the TSH-induced inositol phosphate response, the response to a constitutively active G11alpha mutant was not appreciably affected, and that to NaF was rather inhibited by PIA and overexpression of the betagamma subunits. Taken together, these results suggest that a single type of PTX-sensitive G protein mediates the A1 adenosine receptor-linked modulation of two signaling pathways in collaboration with an activated thyrotropin receptor; alpha subunits of the PTX-sensitive G proteins mediate the inhibitory action on adenylyl cyclase, and the betagamma subunits mediate the stimulatory action on PLC. In the case of the latter stimulatory action on PLC, the betagamma subunits may not directly activate PLC. The possible mechanism by which betagamma subunits enhance the TSH-induced PLC activation is discussed.

Mutagenesis reveals structure-activity parallels between human A2A adenosine receptors and biogenic amine G protein-coupled receptors.

Structure-affinity relationships for ligand binding at the human A2A adenosine receptor have been probed using site-directed mutagenesis in the transmembrane helical domains (TMs). The mutant receptors were expressed in COS-7 cells and characterized by binding of the radioligands [3H]CGS21680, [3H]NECA, and [3H]XAC. Three residues, at positions essential for ligand binding in other G protein-coupled receptors, were individually mutated. The residue V(3.32) in the A2A receptor that is homologous to the essential aspartate residue of TM3 in the biogenic amine receptors, i.e., V84(3.32), may be substituted with L (present in the A3 receptor) but not with D (in biogenic amine receptors) or A. H250(6.52), homologous to the critical N507 of rat m3 muscarinic acetylcholine receptors, may be substituted with other aromatic residues or with N but not with A (Kim et al. J. Biol. Chem. 1995, 270, 13987-13997). H278(7.43), homologous to the covalent ligand anchor site in rhodopsin, may not be substituted with either A, K, or N. Both V84L(3.32) and H250N(6.52) mutant receptors were highly variable in their effect on ligand competition depending on the structural class of the ligand. Adenosine-5'-uronamide derivatives were more potent at the H250N(6.52) mutant receptor than at wild type receptors. Xanthines tended to be close in potency (H250N(6.52)) or less potent (V84L(3.32)) than at wild type receptors. The affinity of CGS21680 increased as the pH was lowered to 5.5 in both the wild type and H250N(6.52) mutant receptors. Thus, protonation of H250(6.52) is not involved in this pH dependence. These data are consistent with a molecular model predicting the proximity of bound agonist ligands to TM3, TM5, TM6, and TM7.