Functional implantation of a solubilized beta-adrenergic receptor in the membrane of a cell.
When the beta-adrenergic receptor of turkey erythrocytes was solubilized by deoxycholate, it retained its potential to activate an adenylate cyclase system. Electron microscopy showed that true solubilization had apparently been achieved; no residual membrane or vesicle structure was found. After removal of deoxycholate and addition of phospholipid, the reprecipitated beta-adrenergic receptor was implanted in the cell membrane of Friend erythroleukemia cells by using a chemical fusion method recently developed. Membranes prepared from the cells demonstrated 30-fold stimulation of the Friend cell adenylate cyclase by the implanted beta-adrenergic receptor. The function of the indigenous prostaglandin E(1) receptor of the Friend cells was not much affected by the implantation of large amounts of the foreign receptor. Activity mediated by the beta-adrenergic receptor reached 60% of the activity obtained with fluoride. The implanted receptor is therefore considered to be efficiently coupled to the adenylate cyclase system. The major difficulties hitherto preventing solubilization of hormone receptors and subsequent reconstitution of their function have been overcome by the approach developed in the present work. Conditions of solubilization need preserve only the receptor because all other components, even those unidentified as yet, can be supplied in excess by the adenylate cyclase system of the cell in which the receptor will be implanted. Subsequent recoupling of the receptor to the adenylate cyclase is performed in the native insoluble state of these molecules. Thus, the components need not be subjected to the hazards of solubilization in a common detergent as is usually required in reconstitution procedures. The importance of using implantation as an assay for a functional receptor in the course of purification and the likelihood that the procedure can be adapted to other receptors for hormones and neurotransmitters are discussed.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000648:ultrastructure; D005622:Friend murine leukemia virus; D007545:Isoproterenol / Q000494:pharmacology; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D051379:Mice; D010743:Phospholipids; D011941:Receptors, Adrenergic / Q000502:physiology*; D011943:Receptors, Adrenergic, beta / Q000502:physiology*; D012995:Solubility; D014422:Turkeys
D010743:Phospholipids; D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; D000262:Adenylyl Cyclases; D007545:Isoproterenol