Characterization of Human A2B Adenosine Receptors: Radioligand Binding, Western Blotting, and Coupling to Gqin Human Embryonic Kidney 293 Cells and HMC-1 Mast Cells

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 ( K D, 36 nM). In competition binding assays, the affinity of agonists is reduced by substitution on either the N 6- or the C-2 position of the adenine ring, whereas 5′-substitutions increase affinity, resulting in the potency order: 5′- N -ethylcarboxamidoadenosine (NECA) ≫ N 6-aminobenzyl-NECA ≈2-chloroadenosine > 2-[4-(2-carboxyethyl)phenethylamino]-NECA (CGS21680) > N 6-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; K I, 32 nM for A2B, 1.4 nM for A2A) and the A1 selective antagonist 8-cyclopentyl-1,3-dipropylxanthine ( K I, 50.5 nM for A2B; 2.5 nM for A1). The K I 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 with K I determinations for inhibition of NECA-stimulated cAMP accumulation in HEK-A2B cells. NECA or N 6-(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.