J. Biol. Chem., Vol. 255, Issue 11, 5015-5018, Jun, 1980
Reconstitution of turkey erythrocyte beta-adrenergic receptors into human erythrocyte acceptor membranes. Demonstration of guanine nucleotide regulation of agonist affinity
DR Jeffery, RR Charlton and JC Venter
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.
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