Reconstitution of turkey erythrocyte beta-adrenergic receptors into human erythrocyte acceptor membranes. Demonstration of guanine nucleotide regulation of agonist affinity.
Digitonin-solubilized turkey erythrocyte beta-adrenergic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack beta receptors. Incorporation of turkey beta receptors into acceptor membranes was directly proportional to the quantity of soluble protein added to the reconstitution system. Reconstituted beta receptors demonstrate saturable [125I]iodohydroxybenzylpindolol binding (Bmax = 11.1 +/- 0.8 fmol/mg, K = 77.8 +/- 8.6 pM) and stereospecificity ((-)-propranolol, K = 11.0 nM; (+)-propranolol, K = 2000 nM; (-)-isoproterenol, K = 250 nM; (+)-isoproterenol, K = 82 micro M). Reconstituted beta receptors appear to be incorporated into acceptor membranes as integral proteins. Reconstituted beta receptors cannot be extracted by high salt or pH (3 to 11); detergent is required for resolubilization of reconstituted beta receptors. Adenylate cyclase stimulation was not obtained in reconstituted membranes since acceptor membranes lack a catalytic subunit. However, guanine nucleotide regulation of agonist affinity was observed indicating a functional reconstitution. GTP (100 micro M) produces a 5-fold decrease in the affinity of isoproterenol for reconstituted beta receptors. Experiments with sulfhydryl reagents indicate that the reconstituted beta receptor couples with the guanine nucleotide regulatory protein of the acceptor membranes. These data describe the successful reconstitution of a beta receptor and indicate that the reconstituted beta receptor can interact with the GTP binding protein of human erythrocyte acceptor membranes.
D000818:Animals; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D006801:Humans; D007536:Isomerism; D007545:Isoproterenol / Q000378:metabolism*; D007700:Kinetics; D010869:Pindolol / Q000031:analogs & derivatives* / Q000378:metabolism; D011433:Propranolol / Q000378:metabolism*; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism*; D013045:Species Specificity; D014422:Turkeys
D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; C011878:iodohydroxybenzylpindolol; D006160:Guanosine Triphosphate; D011433:Propranolol; D010869:Pindolol; D007545:Isoproterenol