Activation of the mouse heart adenosine 5',5'''-P1-P4-tetraphosphate receptor

Abstract

We have previously demonstrated that mouse brain membrane fractions have a specific, saturable receptor for diadenylated nucleotides. Binding is specific for two adenosines, and the length of the phosphate bridge is critical, with four phosphates being optimal [Hilderman et al. (1991) J. Biol. Chem. 266, 6915-6918]. In this report, we demonstrate that adenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding to its receptor is dependent upon an activation step that requires divalent cations and a serine protease. Monoclonal antibodies (Mabs) are identified that inhibit Ap4A binding to its membrane receptor. These antibodies recognize a 212-kDa membrane protein. However, SDS-PAGE analysis of Ap4A cross-linked to membrane fractions reveals that Ap4A is not attached to the 212-kDa peptide but to a 30-kDa polypeptide. Appearance of the 30-kDa polypeptide is dependent on the activation step, and one of the inhibitory antibodies blocks its appearance. We suggest that the protease-dependent processing step involves cleavage of the 212-kDa component with the appearance of an active 30-kDa receptor.

The activation of P1‐ and P2‐purinoceptors in the guinea‐pig left atrium by diadenosine polyphosphates

“…A receptor that specifically binds adenine dinucleotides rather than ATP has been isolated from Swiss CDl mice cardiac muscle membranes (Walker et al, 1993b;Hilderman et al, 1994). Unfortunately, no functional study has been carried out to determine what action this receptor mediates.…”

Regulation of rat mesangial cell growth by diadenosine phosphates.

“…As to the type of receptors, there are divergent findings in literature. Hilderman et al identified a unique receptor for diadenosine phosphates ( 13,14), but on the other hand a subtype of purinergic receptors, the P2A receptor, may be activated by diadenosine phosphates (15,16).…”

A peptide isolated from a random phage peptide library is a structural mimic to the P1, P4‐diadenosine 5′‐tetraphosphate binding site on its receptor

“…Our rationale is that the screening of random peptide libraries with mAb TL4, which has been shown to inhibit Ap 4 A binding to its membrane receptor [20,22], should select fusionphage clones whose inserts contain amino acid sequences mimicking the Ap 4 A-binding domain of the receptor. We screened a 15-residue library with mAb TL4 and isolated 24 individual clones whose DNA were sequenced.…”

“…However, the nature of the receptor involved in Ap 4 A-mediated cellular signaling is unclear. We have demonstrated in cardiac tissue that Ap 4 A appears to act on a unique dipurinoceptor which is highly specific for Ap 4 A, Ap 5 A, and Ap 6 A (P 2D ) [20]. This receptor undergoes an activation step which enhances only Ap 4 A binding [21].…”

“…Receptor activation is required for binding of Ap 4A to its receptor. Specific Ap 4 A binding is enhanced threefold to fivefold when the membrane homogenates are incubated in binding buffer (67 mM Tris/ HCl pH 7.7 and 0.1 mM MgCl 2) for 60 min at 29°C prior to the assay [20]. We refer to this incubation as receptor activation.…”