This proposal is for the continuation of experiments designed to elucidate the mechanism through which ADP brings about its varied effects on platelets. We will attempt to characterize biochemically the receptor which mediates the inhibition of adenylate cyclase, which we have studied by means of kinetics and ligand binding experiments, using ADP analogues that bind to the receptor with higher affinity than the natural nucleotide. Several complimentary approaches to the isolation of the receptor protein will be used, including photoaffinity labeling, affinity chromatography and reversible ligand binding. We will measure the binding of 2-methylthioADP to platelets and to other cells, and to platelet membranes, and determine whether this binding is influenced by those conditions that affect the binding of other agonists that regulate adenylate cyclase through the guanine binding transducer proteins implicated in the hormonal control of this enzyme. Binding of 2-methylthioADP to solubilized membrane proteins isolated by electrophoresis under non-denaturing conditions will be used to identify ADP binding sites. A novel affinity chromatography medium will be used to isolate those ADP binding proteins that have the characteristics of the receptor. For this purpose ADP will be coupled to an insoluble support matrix through substituents at the 2- position of the purine ring. Photoaffinity analogues of ADP in which a photoactivatable azido function is attached through a spacer group to the ADP molecule, also through substitution at the 2- position, will be used to characterize the ADP receptor with respect to its behaviour in a number of analytical separation systems. Proteins isolated by these techniques will be tested for receptor function by their ability to reconstitute an ADP-regulated adenylate cyclase system. The relation between the receptor that regulates adenylate cyclase and the receptor involved in platelet activation by ADP will be investigated.
| Cristalli, G; Mills, D C (1993) Identification of a receptor for ADP on blood platelets by photoaffinity labelling. Biochem J 291 ( Pt 3):875-81 |
