Activation of the mouse heart adenosine 5',5"'-P1-P4-tetraphosphate receptor.
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.
Walker
J
J
Department of Biological Sciences, Clemson University, South Carolina 29634-1903.
Lewis
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Pivorun
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Hilderman
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D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000138:chemical synthesis / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D051379:Mice; D009206:Myocardium / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015226:Dinucleoside Phosphates; D010761:Phosphorus Radioisotopes; D011956:Receptors, Cell Surface; D015636:Magnesium Chloride; C020733:diadenosine tetraphosphate; D004492:Edetic Acid