Characterization of Human A2B Adenosine Receptors: Radioligand Binding, Western Blotting, and Coupling to Gqin Human Embryonic Kidney 293 Cells and HMC-1 Mast Cells
- Departments of 1Internal Medicine (J.L., T.T., H.F., A.S.R.),2Molecular Physiology and Biological Physics (J.L.), and 3Biochemistry (X.J.), University of Virginia, Charlottesville, Virginia
Abstract
Recombinant human A2B adenosine receptors (A2BARs) and receptors extended on the amino terminus with hexahistidine and the FLAG epitope, DYKDDDDK (H/F-A2B) were stably overexpressed (to >20,000 fmol/mg protein) in human embryonic kidney 293 cells (HEK-A2B). By Western blotting, the H/F-A2Breceptor runs as a 34.8-kDa glycoprotein. Pharmacological properties of A2BARs were characterized with125I-3-aminobenzyl-8-phenyl-(4-oxyacetic acid)-1-propylxanthine (KD, 36 nM). In competition binding assays, the affinity of agonists is reduced by substitution on either the N6- or the C-2 position of the adenine ring, whereas 5′-substitutions increase affinity, resulting in the potency order: 5′-N-ethylcarboxamidoadenosine (NECA) ≫N6-aminobenzyl-NECA ≈2-chloroadenosine > 2-[4-(2-carboxyethyl)phenethylamino]-NECA (CGS21680) >N6-aminobenzyladenosine. The A2BAR is potently blocked by the A2A-selective antagonist 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo-[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol (ZM241385; KI, 32 nM for A2B, 1.4 nM for A2A) and the A1 selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (KI, 50.5 nM for A2B; 2.5 nM for A1). TheKI values for the antiasthmatic xanthines, theophylline (7.8 μM) and enprofylline (6.4 μM), are below their therapeutic plasma concentrations (20 to 50 μM), and agree withKI determinations for inhibition of NECA-stimulated cAMP accumulation in HEK-A2B cells. NECA orN6-(2-iodo)benzyl-5′-N-methylcarboxamidodoadenosine (IB-MECA) stimulate inositol trisphosphates and calcium accumulation in HEK-A2B or HEK-A3 cells, respectively, but only the A3 response is prevented by pertussis toxin. In human HMC-1 mast cells, A2BAR activation stimulates calcium mobilization and cAMP accumulation. We conclude that HEK-A2B cells and HMC-1 mast cells possess A2BAR glycoproteins that are coupled to both Gq/11 and Gs.
Footnotes
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Send reprint requests to: Dr. Joel Linden, Departments of Internal Medicine and Molecular Physiology, Box MR4 6012, Room MR4 6071, Health Sciences Center, University of Virginia, Charlottesville, Virginia. E-mail:jlinden{at}virginia.edu
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This work was supported by National Institutes of Health Grant RO1-HK37942.
- Abbreviations:
- AR
- adenosine receptor
- DPX
- 1,3-diethyl-8-phenylxanthine
- CPX
- 8-cyclopentyl-1,3-dipropylxanthine
- XAL
- 8-(4-((2-aminoethyl)aminocarbonyl-methyloxy)phenyl)-1,3-dipropylxanthine
- ABOPX
- 3-(3,4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propyl-xanthine
- H/F-A2B
- recombinant human A2B adenosine receptors extended on the amino terminus with hexahistidine and the FLAG epitope (DYKDDDDK)
- NECA
- 5′-N-ethylcarboxamidoadenosine
- CPA
- N6-cyclopentyladenosine
- CGS21680
- 2-[4-(2-carboxyethyl)phenethylamino]-5N-N-ethylcarboxamidoadenosine
- XAC
- 8-(4-((2-aminoethyl)aminocarbonylmethyloxy)phenyl)-1,3-dipropylxanthine
- ABA
- N6-aminobenzyladenosine
- BW-A1433
- 8-(4-carboxyethenylphenyl)-1,3-dipropylxanthine
- AB-NECA
- aminobenzyl-5′-N-ethylcarboxamidoadenosine
- DMEM
- Dulbecco’s modified Eagle’s medium
- APE
- 2-[2-(4-amino-phenyl)ethylamino]adenosine
- TBS/T
- Tris-buffered saline/Tween 20
- CHO
- Chinese hamster ovary
- IP3
- inositol trisphosphate
- IB-MECA
- N6-(2-iodo)benzyl-5′-N-methylcarboxamidodoadenosine
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- Received March 5, 1999.
- Accepted June 21, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
