Characterization of ATP and P 2 agonists binding to the cardiac plasma membrane P1, P4-diadenosine 5′-tetraphosphate receptor

Abstract

P 1, P 4-Diadenosine 5′-tetraphosphate (Ap 4A) acts as an extracellular modulator through its interaction with purinoceptors. Our laboratory has demonstrated the presence of an Ap 4A receptor in cardiac tissue [ 1,2]. Due to the rapid hydrolysis of ATP by cardiac membranes the relationship of ATP and Ap 4A binding to purinoceptors on cardiac membranes has not been characterized. In this communication we used two approaches to determine the relationship of ATP to the Ap 4A receptor. Radioligand binding carried out with [α- 32P]Ap 4A and adenosine 5′- O-{3-thiotriphosphate} ([γ- 35S]ATPγS) demonstrates the presence of a single high affinity binding site for Ap 4A and the presence of two binding sites for ATPγS. The second approach utilized immunoaffinity purified Ap 4A receptor that was shown to be free of ATPase and Ap 4Aase activities. Non-radiolabeled Ap 4A and ATPγS effectively inhibited photocrosslinking of [α- 32P]8-N 3Ap 4A to the receptor polypeptide while ATP was a much less effective inhibitor. Furthermore, on plasma membranes [α- 32P]8-N 3Ap 4A photocrosslinked to only a 50 kDa polypeptide. These data are consistent with Ap 4A interacting with a homogeneous population of receptors on cardiac plasma membranes but with ATP having a low affinity for the receptor.