Purification of beta-adrenergic receptors from Beijing duck erythrocyte plasma membranes and their reconstitution with Gs and adenylate cyclase on asolectin liposomes

摘要

Catecholamines such as adrenaline, norepinephrine and isoproterenol regulate a wide variety of physiological responses via their specific binding to adrenergic receptors located in the plasma membrane. Adrenergic receptors have been divided into two major types, alpha and beta. Binding of ligands to P-adrenergic receptors (P-AR) first triggers the activation of stimulatory GTP-binding protein (Gs). The activated Gs then interacts with adenylate cyclase at the intracellular face of the plasma-membraneto stimulate the formation of cAMP, an important intracellular messenger in animal cells, which regulates a variety of biochemical and physiological functions including carbohydrate, lipid and nucleic acid metabolism, as well as synaptic transmission, ion channel function and transcription. To date, beta-adrenergic receptors have been purified from several sources such as frog and turkey erythrocytes and hamster lung, etc. Here we report the purification of P-adrenergic receptors from Beijing duck erythrocyte membranes by affinity chromatography and their reconstitution with Gs and AC from bovine brain cortices on asolectin liposomes to form proteoliposomes with better functional coupling of these three components. Such a reconstituted cAMP signalling pathway would provide an available experimental model for further studies on the molecular mechanism of signal transduction.