The thyrotrophin receptor in the guinea-pig thyroid

Abstract

The interaction of thyrotrophin (TSH) with receptors in guinea-pig thyroid tissue was investigated using 125l-labelled bovine TSH preparations.With thyroid tissue slices in vitro, saturable time-dependent accumulation of labelled TSH was demonstrated. Specificity of this saturable process was indicated by the failure of native TSH to inhibit the binding of 125l-labelled bovine serum albumin by thyroid tissue, or to inhibit the binding of labelled TSH by several non-target tissues. Saturation kinetics indicated heterogeneous binding processes in thyroid tissue, with an affinity constant, K, for the higher affinity binding sites of the order of 109 M-1.Particulate fractions of thyroid tissue homogenate bound labelled TSH by a rapid, reversible, saturable process which displayed hormonal specificity, tissue specificity and homogeneous saturation kinetics. Binding affinity was in the range of K = 1-5 x 109 M-1, but depended upon conditions of incubation. Sodium chloride depressed binding affinity by reducing the association rate constant.Receptor binding provided a more sensitive criterion of damage in labelled TSH preparations than simple chromatographic or immunological tests, and revealed that considerable damage occurred during iodination by the chloramine-T method. A technique was developed using receptor binding to separate damaged label from reactive material. Labelled material recovered by dissociating hormone-receptor complexes in sodium thiocyanate, retained reactivity with receptors and unchanged chromatographic properties.Adenylate cyclase activity was assayed in homogenates using tritiated ATP substrate, and in incubated thyroid tissue slices by extraction of labelled cyclic-AMP after pre-labelling the tissue nucleotide pool by incubation with tritiated adenosine. The apparent affinity for TSH of the adenylate cyclase in homogenates and in intact tissue was in agreement with the binding affinity of receptors in homogenates and of the higher affinity receptors in tissue slices. The rate of activation of adenylate cyclase in homogenates corresponded with the rate of binding of labelled hormone.Human g-globulin containing long-acting thyroid stimulator (LATS) enhanced adenylate cyclase activity and inhibited the binding of labelled TSH in particulate fractions of guinea-pig thyroid homogenate. Studies of concentration dependence, time-course of effect on unoccupied receptors and on receptor-TSH complexes, and saturation kinetics of TSH binding in the presence of LATS, indicated that the action of this agent involved binding to unoccupied TSH receptors, probably at a site identical to the TSH-combining site.