Evidence for a G protein-coupled diadenosine-5′,5"′-P1,P4-tetraphosphate (Ap4A) receptor binding site in lung membranes from rat

Nucleotide receptors are of considerable importance in the treatment of lung diseases, such as cystic fibrosis. Because diadenosine polyphosphates may also be of significance as signalling molecules in lung, as they are in a variety of tissues, in the present work we investigated the binding sites for [H-3]diadenosine-5',5"'-P-1, P-4-tetraphosphate (Ap(4)A) in plasma membranes from rat lung and studied their possible coupling to G proteins. We present evidence for a single high-affinity binding site for [H-3]Ap(4)A with similar affinity for other diadenosine polyphosphates Ap(n)A (n = 2 to 6). Displacement studies with different nucleotides revealed that the [H-3]Ap(4)A binding site was different from P2X and P2Y(2) receptor binding sites. Pretreatment of lung membranes with GTP gamma S or GTP in the presence of Mg2+ increased the K-i for Ap(4)A from 91 nM to 5.1 mu M, which is indicative of G protein coupling. The putative coupling to G proteins was further confirmed by the enhancement of [S-35]GTP gamma S binding (to G(alpha) proteins) to lung membranes by Ap(4)A (63% increase over basal) in a concentration-dependent manner. Therefore, our data for the first time provide evidence of a G protein-coupled Ap(4)A binding site in lung membranes. (C) 1999 Elsevier Science B.V. All rights reserved.