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G L Stiles

Publications and source records attributed to G L Stiles.

At least 37 records · Page 2Linked to original sources
PubMed1995

Agonist-dependent phosphorylation and desensitization of the rat A3 adenosine receptor. Evidence for a G-protein-coupled receptor kinase-mediated mechanism.

A3 adenosine receptor (A3AR) activation contributes to both the cardioprotective and antihypertensive effects of adenosine. To date, no studies have examined the mechanisms by which this receptor undergoes rapid homologous desensitization. Therefore, a functional hemagglutinin epitope-tagged A3AR has been stably expressed in Chinese hamster ovary cells, and its regulation by the AR agonist 5'-N-ethylcarboxamidoadenosine (NECA) has been studied. Cellular exposure to NECA induces rapid (t1/2 = approximately 1 min) A3AR phosphorylation on serine and threonine residues. This is associated with a functional desensitization and a 30-40% reduction in the number of high affinity agonist binding sites as determined by radioligand binding assays. Activation of second messenger-regulated kinases could not mimic the effect of NECA, suggesting a role for G-protein-coupled receptor kinases (GRKs). In vitro phosphorylation assays demonstrate that phosphorylation of agonist-occupied A3ARs is enhanced by GRK2 and that cellular pretreatment with NECA dramatically inhibits subsequent GRK2-mediated phosphorylation in vitro. Therefore, the A3AR is phosphorylated in situ by a kinase similar or identical to GRK2, and this may be involved in rapid functional desensitization of the A3AR.

Adenosine↗
PubMed1995

Human adenosine A1 receptor gene: systematic screening for DNA sequence variation and linkage mapping on chromosome 1q31-32.1 using a silent polymorphism in the coding region.

Adenosine is a major inhibitory neuromodulator in the central nervous system. One of the receptors mediating the central effects of adenosine is the adenosine A1 receptor. We performed a systematic mutation scan of the coding region of the adenosine A1 receptor gene to explore its variability in the general population. Investigating 40 unrelated healthy subjects by single-strand conformation analysis no sequence changes of likely functional relevance were observed. We detected, however, a frequent T to G substitution at nucleotide position 716 which constitutes the first variant described in an adenosine receptor gene. It was used for fine scale linkage mapping of the A1 gene. Employing a polymerase-chain-reaction-based restriction assay, we genotyped 7 CEPH families (Centre d'Etude du Polymorphisme Humaine) and mapped the receptor in a gene cluster around the renin gene on chromosome 1q31-32.1. In addition, we utilized the 716T/G polymorphism to demonstrate biallelic expression of the adenosine A1 receptor gene in adult human brain.

Adult↗
PubMed1995

Differential interaction with and regulation of multiple G-proteins by the rat A3 adenosine receptor.

Interaction of the rat A3 adenosine receptor (A3AR) with G-proteins has been assessed using a stably transfected Chinese hamster ovary cell system. The non-selective AR agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased the labeling of a 41-kDa membrane protein by 4-azidoanilido-[alpha-32P]guanosine 5'-triphosphate (AA-[32P]GTP), a photolabile GTP analogue. Subsequent immunoprecipitation of Gi alpha-subunits indicated that NECA stimulated incorporation of label into both Gi alpha-2 and Gi alpha-3. Additional experiments revealed an A3AR stimulation of label into Gq and/or G11 alpha-subunits, albeit to a lesser degree than that elicited by endogenous P2U purinergic receptors. No interaction with Gs could be detected. Sustained cellular exposure to NECA induced A3AR desensitization and specific down-regulation of Gi alpha-3 and G-protein beta-subunits without changing levels of Gi alpha-2, Gs alpha, or Gq+11 alpha-subunits. Therefore the A3AR can interact with Gi alpha-2, Gi alpha-3, and, to some extent, Gq-like proteins, but sustained agonist exposure down-regulates only one of the G-proteins with which it interacts. This is the first description of the differing specificities of A3AR/G-protein coupling versus down-regulation in situ and provides a potential mechanism by which the A3AR could elicit the heterologous desensitization of signaling events mediated by Gi3.

Animals↗
PubMed1995

Structure-activity relationships of 9-alkyladenine and ribose-modified adenosine derivatives at rat A3 adenosine receptors.

9-Alkyladenine derivatives and ribose-modified N6-benzyladenosine derivatives were synthesized in an effort to identify selective ligands for the rat A3 adenosine receptor and leads for the development of antagonists. The derivatives contained structural features previously determined to be important for A3 selectivity in adenosine derivatives, such as an N6-(3-iodobenzyl) moiety, and were further substituted at the 2-position with halo, amino, or thio groups. Affinity was determined in radioligand binding assays at rat brain A3 receptors stably expressed in Chinese hamster ovary (CHO) cells, using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-(N-methyluronamide)), and at rat brain A1 and A2a receptors using [3H]-N6-PIA ((R)-N6-phenylisopropyladenosine) and [3H]CGS 21680 (2-[[[4-(2-carboxyethyl)-phenyl]ethyl]amino]-5'- (N-ethylcarbamoyl)adenosine), respectively. A series of N6-(3-iodobenzyl) 2-amino derivatives indicated that a small 2-alkylamino group, e.g., methylamino, was favored at A3 receptors. N6-(3-Iodobenzyl)-9-methyl-2-(methylthio)adenine was 61-fold more potent than the corresponding 2-methoxy ether at A3 receptors and of comparable affinity at A1 and A2a receptors, resulting in a 3-6-fold selectivity for A3 receptors. A pair of chiral N6-(3-iodobenzyl) 9-(2,3-dihydroxypropyl) derivatives showed stereoselectivity, with the R-enantiomer favored at A3 receptors by 5.7-fold. 2-Chloro-9-(beta-D-erythrofuranosyl)-N6-(3-iodobenzyl)adenine had a Ki value at A3 receptors of 0.28 microM. 2-Chloro-9-[2-amino-2,3-dideoxy-beta-D-5-(methylcarbamoyl)- arabinofuranosyl]-N6-(3-iodobenzyl)adenine was moderately selective for A1 and A3 vs A2a receptors. A 3'-deoxy analogue of a highly A3-selective adenosine derivative retained selectivity in binding and was a full agonist in the inhibition of adenylyl cyclase mediated via cloned rat A3 receptors expressed in CHO cells. The 3'-OH and 4'-CH2OH groups of adenosine are not required for activation at A3 receptors. A number of 2',3'-dideoxyadenosines and 9-acyclic-substituted adenines appear to inhibit adenylyl cyclase at the allosteric "P" site.

Adenine↗
PubMed1995

Adenosine receptor subtypes: characterization and therapeutic regulation.

Adenosine receptors (ARs) are members of the G protein-coupled receptor family and mediate the multiple physiological effects of adenosine. Currently, four AR subtypes have been cloned: A1AR, A2aAR, A2bAR, and A3AR. All subtypes are distinctly distributed throughout the body and AR agonists and antagonists have potential therapeutic utility. Knowledge of AR amino acid structure has been utilized in mutagenesis studies to identify specific receptor regions that interact with distinct classes of ligands. Cloning of ARs has also permitted receptor regulatory processes such as desensitization to be studied in greater detail, in particular, the molecular mechanisms underlying this event. Cloning of the human A1AR has revealed that alternate splicing generates distinct receptor transcripts. The existence of a particular transcript in a tissue or cell apparently regulates the level of A1AR expression in the tissue. This review focuses on these aspects of AR structure and function and their therapeutic regulation.

Adenosine↗
PubMed1995

125I-4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol, a high affinity antagonist radioligand selective for the A2a adenosine receptor.

The A2a adenosine receptor (AR) mediates several important physiological effects of adenosine, including vasodilation and inhibition of platelet aggregation. Until recently, no antagonist radioligand of sufficient selectivity or affinity was available. We describe the synthesis and characterization by radioligand binding of 125I-4-(2-[7-amino-2-{2-furyl}-{1,2,4}triazolo{2,3-a}- {1,3,5}triazin-5-yl-amino]ethyl)phenol (125I-ZM241385) in membranes from two cell types that express A2a ARs. Membranes from Chinese hamster ovary (CHO) cells expressing a recombinant canine A2a AR bound 125I-ZM241385 with high affinity, and agonist competition experiments with 2-(p-carboxyethyl)-phenylamino-5'-N-carboxamidoadenosine, 5'-N-ethylcarboxamidoadenosine, and (-)-N6[(R)-phenylisopropyl]adenosine revealed a potency order characteristic of an A2a AR binding site. Membranes from bovine striatum, which contain a native A2a AR, also bound 125I-ZM214385 with similarly high affinity and also displayed a pharmacological profile for displacement of radioligand binding that was consistent with that of an A2a AR. Also, under conditions in which 125I-ZM241385 bound with high affinity to a recombinant rat A2a AR expressed in CHO cells, no specific binding was detectable in membranes from CHO cells expressing functional rat A1, A2b, or A3 ARs, indicating that over the range of concentrations used in radioligand binding assays, 125I-ZM214385 is a highly selective antagonist radioligand for study of A2a ARs within a given species.

Animals↗
PubMed1995

Separate promoters in the human A1 adenosine receptor gene direct the synthesis of distinct messenger RNAs that regulate receptor abundance.

There are two types of transcripts for the human A1, adenosine receptor. They are expressed in a tissue-specific manner in human tissues and contain distinct exons. Previously, it had appeared that the two transcripts may have occurred through alternative splicing. The transcript beta has two upstream AUG codons, which in transiently transfected COS-7 cells leads to a reduced level of receptor expression. When genomic sequence including sequences 5' to transcriptional start site, exon 1A, intron 1A, exon 1B, intron 1B, exon 2, and coding sequence was inserted into an expression vector (pCMV5/huA1), the resulting transcripts had the same overall structure as the transcripts present in human tissues. Primer extension and 5' rapid amplification of cDNA ends of mRNA from transfected cells revealed the transcription start sites for these two transcripts occurred in what previously had been termed introns. These results were confirmed with similar analysis of mRNA derived from human tissues. Two nonconsensus putative TATA boxes (TTAAGA and TTTAAA) are located upstream of the transcription start sites for transcripts alpha and beta. When the TATA boxes and their flanking sequences were fused to a firefly luciferase gene containing promoterless vector, both demonstrated strong promoter activity in Chinese hamster ovary cells. Promoter A directs the synthesis of transcript alpha, and promoter B directs the synthesis of transcript beta. Promoter A contains a series of AGG elements between the putative TATA box and the transcription start, which accounts for a major portion of the promoter activity based on deletion and mutation analysis. In general, promoter A is more active than promoter B in transfected cells. The nonconsensus TATA box in promoter B plays a more important role in promoter activity than the TATA box in promoter A. The human A1 adenosine receptor gene appears to use two separate promoters to direct synthesis of distinct transcripts, which can then regulate the relative abundance of A1 adenosine receptor in tissues. We have redefined the human A1 adenosine receptor gene structure based on these new data.

Animals↗
PubMed1994

Selective ligands for rat A3 adenosine receptors: structure-activity relationships of 1,3-dialkylxanthine 7-riboside derivatives.

1,3-Dibutylxanthine 7-riboside has been found to be a partial agonist at A3 adenosine receptors (van Galen et al. Mol. Pharmacol. 1994, 45, 1101-1111). 1,3-Dialkylxanthine 7-riboside analogues modified at the 1-, 3-, and 8-purine positions and at the ribose 5'-position were synthesized. The nucleoside analogues were examined for affinity in radioligand binding assays at rat brain A3 adenosine receptors stably expressed in CHO cells, using the radioligand [[125I]-4-amino-3-iodobenzyl]adenosine-5'-N-methyluronamide (AB-MECA). Affinity was assayed at rat brain A1 and A2a receptors using [3H]PIA and [3H]CGS 21680, respectively. The affinity of xanthine 7-ribosides at A3 receptors depended on the 1,3-dialkyl substituents in the order: Pent > or = Bu >> Hx > Pr approximately Me. 1,3-Dipentylxanthine 7-riboside was slightly selective for A3 receptors (2-fold vs A1 and 10-fold vs A2a). 8-Methoxy substitution was tolerated at A3 receptors. 2-Thio vs 2-oxo substitution increased potency at all three subtypes and slightly increased A3 vs A1 selectivity. The 5'-uronamide modification, which was previously found to enhance A3 selectivity in N6-benzyladenosine derivatives, was also incorporated into the xanthine 7-ribosides, with similar results. The affinity of 1,3-dialkylxanthine 7-riboside 5'-uronamides at A3 receptors depended on the N-alkyluronamide substituent in the order: MeNH > EtNH >> NH2 >> Me2N. Affinity of the 5'-uronamides at A3 receptors was dependent on the 1,3-dialkyl substitution in the order: Bu > Pent > Hex. 1,3-Dibutylxanthine 7-riboside 5'-N-methylcarboxamide, with a Ki value of 229 nM at A3 receptors, was 160-fold selective for rat A3 vs A1 receptors and > 400-fold selective vs A2a receptors. This derivative acted as a full agonist in the A3 receptor-mediated inhibition of adenylate cyclase.

Animals↗
PubMed1994

2-Substitution of N6-benzyladenosine-5'-uronamides enhances selectivity for A3 adenosine receptors.

Adenosine derivatives bearing an N6-(3-iodobenzyl) group, reported to enhance the affinity of adenosine-5'-uronamide analogues as agonists at A3 adenosine receptors (J. Med. Chem. 1994, 37, 636-646), were synthesized starting from methyl beta-D-ribofuranoside in 10 steps. Binding affinities at A1 and A2a receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells were compared. N6-(3-Iodobenzyl)adenosine was 2-fold selective for A3 vs A1 or A2a receptors; thus it is the first monosubstituted adenosine analogue having any A3 selectivity. The effects of 2-substitution in combination with modifications at the N6- and 5'-positions were explored. 2-Chloro-N6-(3-iodobenzyl)adenosine had a Ki value of 1.4 nM and moderate selectivity for A3 receptors. 2-Chloro-N6-(3-iodobenzyl)adenosine- 5'-N-methyluronamide, which displayed a Ki value of 0.33 nM, was selective for A3 vs A1 and A2a receptors by 2500- and 1400-fold, respectively. It was 46,000-fold selective for A3 receptors vs the Na(+)-independent adenosine transporter, as indicated in displacement of [3H]N6-(4- nitrobenzyl)-thioinosine binding in rat brain membranes. In a functional assay in CHO cells, it inhibited adenylate cyclase via rat A3 receptors with an IC50 of 67 nM. 2-(Methylthio)-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide and 2-(methylamino)-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide were less potent, but nearly as selective for A3 receptors. Thus, 2-substitution (both small and sterically bulky) is well-tolerated at A3 receptors, and its A3 affinity-enhancing effects are additive with effects of uronamides at the 5'-position and a 3-iodobenzyl group at the N6-position.

Adenosine↗
PubMed1994

Role of the second extracellular loop of adenosine receptors in agonist and antagonist binding. Analysis of chimeric A1/A3 adenosine receptors.

Adenosine receptor (AR) agonists and antagonists are approximately 100-fold and 100,000-fold, respectively, more potent at the bovine A1AR as compared to the rat A3AR. To determine regions of ARs involved in ligand recognition, chimeric receptors composed of bovine A1AR and rat A3AR sequence were constructed and their ligand binding properties examined following expression in COS-7 cells. Substitutions of the second extracellular loop or a region encompassing transmembrane domains 6 and 7 of the A1AR into the A3AR resulted in enhanced affinities of both agonists and antagonists compared to wild-type A3AR. The region of the second extracellular loop of the A1AR responsible for this effect was identified as the distal eleven amino acids of the loop. Replacement of this segment of the A3AR with that of the A1AR in combination with the regions encompassing transmembrane domains 6 and 7 resulted in a 50,000-fold increase in the Kd for antagonist radioligand, [3H]1,3-dipropyl-8- cyclopentylxanthine. Agonist affinity at this chimeric was over 100-fold greater than that displayed by wild-type A3AR. Thus, multiple regions of ARs including a segment of the second extracellular loop are involved in ligand recognition, and considerable overlap exists in structural features required for agonist and antagonist binding.

Adenosine↗
PubMed1994

Structure-activity relationships of 1,3-dialkylxanthine derivatives at rat A3 adenosine receptors.

1,3-Dialkylxanthine analogues containing carboxylic acid and other charged groups on 8-position substituents were synthesized. These derivatives were examined for affinity in radioligand binding assays at rat brain A3 adenosine receptors stably expressed in CHO cells using the new radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide), and at rat brain A1 and A2a receptors using [3H]PIA and [3H]CGS 21680, respectively. A synthetic strategy for introducing multiple carboxylate groups at the 8-position using iminodiacetic acid derivatives was explored. The presence of a sulfonate, a carboxylate, or multiple carboxylate groups did not result in a significant enhancement of affinity at rat A3 receptors, although as previously observed an anionic group tended to diminish potency at A1 and A2a receptors. The rat A3 receptor affinity was not highly dependent on the distance of a carboxylate group from the xanthine pharmacophore. 2-Thio vs 2-oxo substitution favored A3 potency, and 8-alkyl vs 8-aryl substitution favored A3 selectivity, although few derivatives were truly selective for rat A3 receptors. 1,3-Dimethyl-8-(3-carboxypropyl)-2-thioxanthine was 7-fold selective for A3 vs A2a receptors. 1,3,7-Trimethyl-8-(trans-2-carboxyvinyl)xanthine was somewhat selective for A3 vs A1 receptors. For 8-arylxanthines affinity at A3 receptors was enhanced by 1,3-dialkyl substituents, in the order dibutyl > dipropyl > diallyl.

Adenosine↗
PubMed1994

Identification of an adenosine receptor domain specifically involved in binding of 5'-substituted adenosine agonists.

The bovine A1 adenosine receptor (A1AR) and rat A3 adenosine receptor (A3AR) display distinct agonist and antagonist binding properties. To identify regions involved in ligand recognition, A1AR/A3AR chimeric receptors were created, expressed in COS-7 cells, and analyzed by radioligand binding. A chimeric receptor in which the third intracellular loop of the A1AR was replaced with that of the A3AR bound agonists and the antagonist, [3H]xanthine amine congener, with affinities identical to wild-type A1AR. A chimeric receptor with the fifth transmembrane domain (TM5) and third intracellular loop of the A1AR replaced with that of the A3AR displayed antagonist affinity similar to wild-type A1AR. However, relative to the A1AR, this chimeric demonstrated much greater affinity for 5'-substituted adenosine analogs, whereas affinity for N6-substituted compounds was unaffected. Substitution of a 6-amino acid cassette of the exofacial half of TM5 of the A3AR into the A1AR produced enhanced binding of exclusively a 5'-substituted analog, indicating involvement of this specific region in ligand recognition. These findings suggest that the 5'- and N6-substituents of adenosine agonists bind to distinct regions of ARs and that TM5 of the A3AR interacts more favorably with 5'-substituted compounds than does that of the A1AR.

Adenosine↗
PubMed1994

Posttranscriptional mRNA processing as a mechanism for regulation of human A1 adenosine receptor expression.

The human A1 adenosine receptor gene contains six exons with exons 1, 2, 3, 4, and part of 5 representing 5' untranslated regions. Reverse transcription-PCR with exon-specific primers showed two distinct transcripts containing either exons 3, 5, and 6 or exons 4, 5, and 6, with exons 3 and 4 being mutually exclusive. No mature mRNAs containing exons 1 and 2 have been detected. All human tissues that express any A1 receptors contain mRNA with exons 4, 5, and 6. Tissues which express high levels of A1 receptors contain mRNA with exons 3, 5, and 6. Exon 4 contains two upstream ATG codons whereas exon 3 contains none. COS cells transfected with expression vectors containing exon 4 (exons 1-6, 3-6, or Ex4-6) express much lower levels of A1 receptors than vectors without exon 4 (exons 3, 5, and 6). Mutation of upstream ATG codons in exon 4 leads to 3- to 7-fold increased A1 receptor expression, up to the level seen with the construct containing exons 3, 5, and 6. Thus, in human tissues "basal" levels of A1 receptors can be expressed by use of mRNA containing exons 4, 5, and 6, but when high levels are needed, alternative transcripts with exons 3, 5, and 6 are produced.

Alternative Splicing↗
PubMed1994

Characterization of the human A1 adenosine receptor gene. Evidence for alternative splicing.

To approach transcriptional and translational regulation of adenosine receptors, we have isolated cDNA and genomic clones of the human A1 adenosine receptor (A1AR). The cDNA, when inserted into the pCMV5 expression vector and transfected into COS-7 or CHO cells, leads to the expression of a functional A1AR that displayed all the appropriate pharmacologic properties. The human A1AR gene consists of at least six exons and five introns. A single intron interrupts the coding sequence, while the remaining introns are within the 5'-untranslated region. Comparison of our cDNA with one by Libert et al. (Libert, F., Van Sande, J., Lefort, A., Czernilofsky, A., Dumont, J. E., Vasart, G., Ensinger, H. A., and Mendla, K. D. (1992) Biochem. Biophys. Res. Commun. 187, 919-926) reveals that exon 4 in the 5'-untranslated region was completely missing from their sequence. Study of mRNAs from a range of human tissues by reverse transcription-polymerase chain reaction using a variety of primers revealed clear evidence for alternative splicing. Transcripts containing exons 4, 5, and 6 were found in all tissues expressing A1AR, while a separate transcript with exons 3, 5, and 6 was seen only in selected tissues. No transcript contains both exons 3 and 4. No evidence for expression of exons 1 and 2 could be discerned. Exon 4 contains two AUG initiation codons with reasonable Kozak consensus, suggesting the possibility of translational regulation. Exon 3 contains no AUG initiation codons.

Alternative Splicing↗
PubMed1994

Desensitization of the canine A2a adenosine receptor: delineation of multiple processes.

Stable cell lines that express the canine-derived A2a adenosine receptor (A2aAR) have been generated. Using a previously characterized anti-A2aAR antibody probe, we have identified the recombinant receptor protein and examined the desensitization process of this G protein-coupled receptor. Agonist exposure induced a rapid desensitization of A2aAR-stimulated adenylyl cyclase activity. This was associated with reduced affinity of the receptor for the A2aAR-selective agonist [3H]CGS21680 and agonist-stimulated phosphorylation of the receptor protein. Agonist-stimulated A2aAR sequestration into a light membrane fraction was also detected over the same time frame but, whereas inhibition of this process did not affect the extent of desensitization, the rapid recovery normally observed after short term agonist exposure was dramatically reduced. Long term agonist treatment resulted in the down-regulation of A2aARs and up-regulation of Gi alpha 2 and Gi alpha 3, as determined by immunoblotting. Recovery of A2aAR function after agonist removal required several hours and was associated with the return of receptor levels to control values. In contrast, inactivation of Gi proteins by pertussis toxin treatment did not alter the extent of agonist-induced desensitization observed. Neither short nor long term desensitization could be mimicked by elevation of intracellular cAMP levels alone. Therefore, these data suggest that A2aAR desensitization is mediated by multiple, temporally distinct, agonist-dependent processes. Agonist-stimulated phosphorylation of the receptor may induce short term desensitization by impairing receptor-Gs coupling, whereas long term down-regulation of receptor number and up-regulation of inhibitory G proteins mediate long term adaptation.

Adenosine↗