Many neurotransmitter receptors that occur in the brain inhibit adenylate cyclase through the mediation of a GTP regulatory complex (Ni). Knowledge of the molecular events associated with the regulation of these receptors by endogenous effectors enhances our understanding of neuronal function. The cerebral cortical adenosine (A1) receptor typifies those neurotransmitter receptors that inhibit adenylate cyclase. As yet information is lacking on its molecular associations in membranes or detergent solution and on its regulation by endogenous modulators. The present proposal seeks to provide a molecular description of the adenosine receptor as it is linked to the inhibition of adenylate cyclase in cortical membranes. The approach taken will be to fractionate, characterize and reconstitute each of the components. The precise components of this study are: i) To identify the adenosine receptor by photolabelling using both agonists and antagonists and verify its identity by analogue specificities, tissue distribution and its hydrodynamic behavior compared with that of the reversibly labelled adenosine A1 receptor. ii) To generate monoclonal antibodies against the receptor, verifying their specificity by a range of tests. iii) To purify the receptor by a combination of class-purification and high-resolution purification techniques in CHAPS. iv) Using purified GTP regulatory proteins from brain, conditions will be devised for the reconstitution of the interaction between solubilized receptor and the N proteins, with particular attention to the role of magnesium. v) The interaction between N proteins and activated forms of C, that is presently observed in detergent solution, will be stabilized in phospholipid micelles. vi) A fully integrated adenosine receptor-N protein and activated catalytic unit from cerebral cortex will be assembled in phospholipid micelles. These strategies aim to provide not only structural information but also insights into the regulatory mechanisms of inhibitory regulation of adenylate cyclase by adenosine A1 receptors.
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