TY - JOUR T1 - Characterization of Human A<sub>2B</sub> Adenosine Receptors: Radioligand Binding, Western Blotting, and Coupling to G<sub>q</sub>in Human Embryonic Kidney 293 Cells and HMC-1 Mast Cells JF - Molecular Pharmacology JO - Mol Pharmacol SP - 705 LP - 713 VL - 56 IS - 4 AU - Joel Linden AU - Tami Thai AU - Heidi Figler AU - Xiaowei Jin AU - Anna S. Robeva Y1 - 1999/10/01 UR - http://molpharm.aspetjournals.org/content/56/4/705.abstract N2 - 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 &gt;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 &gt; 2-[4-(2-carboxyethyl)phenethylamino]-NECA (CGS21680) &gt;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. The American Society for Pharmacology and Experimental Therapeutics ER -