Identification and chemical characterisation of beta-adrenergic receptors in intact turkey erythrocytes.

Abstract

The radiolabelled β-adrenergic antagonist (−)-[3H]dihydroalprenolol bound to two independent classes of sites on intact turkey erythrocytes. About 1300 high affinity and 18,000 low affinity sites per cell were observed. The low affinity sites (Kd = 210 nM) were different from the functional β-adrenergic receptors since the binding of (−)-[3H]dihydroalprenolol could be displaced by β-adrenergic antagonists but not by agonists and since, in marked contrast with the β-adrenergic receptors in membranes, the sites were sensitive to the alkylating agent N-ethylmaleimide and resistant to the reducing agent dithiothreitol. Agonist-displaceable binding of the tracer only occurred to the high affinity sites. The radiolabelled binding properties of these sites (Kd = 2.7 nM) were in close agreement with those earlier reported for the functional receptors in purified membranes. Bound tracer could be stereospecifically displaced from these sites by both β-adrenergic agonists and antagonists. The specific order of potencies for agonists to displace bound tracer (isoproterenol > norepinephrine > epinephrine), as well as the low affinity for α-adrenergic ligands and non-bioactive catechol compounds confirmed the βl-adrenergic nature of these high affinity sites. DTT§ caused a time- and concentration-dependent decline in the number of receptors of intact erythrocytes as well as of purified and solubilised membranes. Binding of β-adrenergic agonists and antagonists protected the receptors against the action of DTT. These findings indicate that essential disulphide bond(s) of the receptor are located at the extracellular face of the membrane, probably at, or near, the hormone binding site. In contrast with earlier findings on membranes, β-adrenergic agonists did not appear to sensitize the intact cell receptors to inactivation by N-ethylmaleimide.