Isolation of a Tripeptide from a Random Phage Peptide Library That Inhibits P1,P4-Diadenosine 5‘-Tetraphosphate Binding to Its Receptor

Abstract

Extracellular P¹,P⁴-diadenosine 5‘-tetraphosphate (Ap₄A) has been implicated as a modulator of cell stress. We have previously demonstrated specific receptors for Ap₄A at the surface of cardiac myocytes (Walker et al., 1993a). In addition, we have isolated a monoclonal antibody (mAb TL4) that recognized the Ap₄A receptor and inhibited binding of Ap₄A to its receptor (Walker & Hilderman, 1993). As part of our effort to characterize the Ap₄A receptor building domain, we screened a random phage peptide library with mAb TL4. After affinity purification of specifically bound phage, we isolated 38 individual phage clones. Twenty-eight of these clones bound mAb TL4 in ELISA and dot blot analyses. Twenty-two of the twenty-eight individual clones contained inserts with an RGS tripeptide sequence. Synthetic RGS peptide specifically inhibits the binding of mAb TL4 to its membrane receptor. Furthermore, the RGS peptide also inhibits [³H]Ap₄A binding to its receptor. These data are consistent with the RGS peptide mimicking part of the mAb TL4 recognition site on the Ap₄A receptor. The RGS peptide may be used to help characterize the Ap₄A receptor binding domain and to help determine the physiological significance of the interaction between Ap₄A and its receptor.