Characterization of human A2B adenosine receptors:: Radioligand binding, Western blotting, and coupling to Gq in human embryonic kidney 293 cells and HMC-1 mast cells

Recombinant human A(2B) adenosine receptors (A(2B)ARs) and receptors extended on the amino terminus with hexahistidine and the FLAG epitope, DYKDDDDK (H/F-A(2B)) were stably overexpressed (to >20,000 fmol/mg protein) in human embryonic kidney 293 cells (HEK-A(2B)). By Western blotting, the H/F-A(2B) receptor runs as a 34.8-kDa glycoprotein. Pharmacological properties of A(2B)ARs were characterized with I-125-3-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 approximate to 2-chloroadenosine > 2-[4-(2-carboxyethyl) phenethylamino]-NECA (CGS21680) > N-6-aminobenzylad-enosine. The A(2B)AR is potently blocked by the A(2A)-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 A(2B), 1.4 nM for A(2A)) and the A(1) selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (K-I, 50.5 nM for A(2B); 2.5 nM for A(1)). The K-I values for the antiasthmatic xanthines, theophylline (7.8 mu M) and enprofylline (6.4 mu M), are below their therapeutic plasma concentrations (20 to 50 mu M), and agree with KI determinations for inhibition of NECA-stimulated cAMP accumulation in HEK-A(2B) cells. NECA or N-6-(2-iodo)benzyl-5'-N-methylcarboxamidodoadenosine (IBMECA) stimulate inositol trisphosphates and calcium accumulation in HEK-A(2B) or HEK-A(3) cells, respectively, but only the A(3) response is prevented by pertussis toxin. In human HMC-1 mast cells, A(2B)AR activation stimulates calcium mobilization and cAMP accumulation. We conclude that HEK-A(2B) cells and HMC-1 mast cells possess A(2B)AR glycoproteins that are coupled to both G(q/11) and G(s).