Mode of coupling between the β-adrenergic receptor and adenylate cyclase in turkey erythrocytes

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Abstract
The mode of coupling of the .beta.-adrenergic receptor to the enzyme adenylate cyclase in turkey erythrocyte membranes was analyzed in detail. The following experimental techniques were used: measurement of the kinetics of cyclase activation to its permanently active state in the presence of guanylyl imidodiphosphate, as a function of hormone concentration; and measurement of antagonist and agonist binding to the .beta.-adrenergic receptor prior and subsequent to the enzyme activation by hormone and guanylyl imidodiphosphate. On the basis of these 2 approaches, all the models of receptor to enzyme coupling which involve an equilibrium between the enzyme and the receptor could be rejected. The binding and the kinetic data could be fitted by 2 diametrically opposed models of receptor to enzyme coupling. The precoupled enzyme-receptor model is one in which activation of the enzyme occurs, according to the following scheme: .**GRAPHIC**. where H is the hormone, RE is the precoupled receptor-enzyme complex, k1 and k2 are the rate constants describing hormone binding and k is the rate constant characterizing the formation of HRE'' from the intermediate HRE. According to this model, the activated complex is composed of all of the interacting species. The other model is the collision coupling mechanism: .**GRAPHIC**. where KH is the hormone-receptor dissociation constant, k1 is the bimolecular rate constant governing the formation of HRE and k3 the rate constant governing the activation of the enzyme. In this case the intermediate never accumulates and constitutes only a small fraction of the total receptor and adenylate cyclase concentrations. To establish which of the 2 mechanisms governs the mode of adenylate cyclase activation by its receptor, a diagnostic experiment was performed. Progressive inactivation of the .beta.-receptor by a specific affinity label caused a decrease in the maximal binding capacity of the receptor and a proportional decrease in the rate of activation, but no change in the maximum level of activity was attained. Progressive inactivation of the enzyme by p-hydroxymercuribenzoate did not change the rate of activation nor the capacity of the receptor to bind hormone. Only the maximal level of activation decreased. These results are not compatible with the precoupled model of receptor and cyclase nor with floating receptor models in which an intermediate of hormone, receptor and cyclase is in equilibrium with its reactants. The collision coupling is apparently the mode of coupling between the .beta. receptor and cyclase coupling in turkey erythrocyte membranes.