Relevance of the low and high affinity thyrotropin-binding sites of human thyroid membranes to the stimulation of adenylate cyclase.
TSH is known to interact on thyroid membranes with two classes of binding sites that differ in affinity and capacity. To assess the relevance of the class of TSH-binding sites characterized by low affinity and high capacity to the stimulation of adenylate cyclase, we studied the interactions of desialylated hCG (as-hCG) and its beta-subunit (as-hCG beta) with human thyroid membranes. In low ionic strength buffer, pH 7.8, where both classes of sites are operant, as-hCG fully inhibited and as-hCG beta partially inhibited [125I] bovine (b) TSH binding. Scatchard analysis of the [125I]bTSH binding inhibition curve in the presence of 1.0 X 10(-5) M as-hCG beta clearly indicated that as-hCG beta interacted only with the low affinity class of binding sites, leaving the high affinity class unaffected. In the presence of 140 mM NaCl, [125I]bTSH interacted predominantly with the high affinity class of binding sites; as-hCG fully inhibited [125I]bTSH binding to this class of sites, whereas as-hCG beta displayed essentially no interaction. Scatchard analysis of [125I]as-hCG beta binding to human thyroid membranes in low ionic strength buffer revealed a single apparent class of sites with low affinity (Kd = approximately 1.0 X 10(-6) M) and high capacity (Q = approximately 300 pmol/mg membrane protein). The bTSH preparation (Thytropar) showed a 10-fold greater binding inhibition potency at these sites than either the as-hCG or the as-hCG beta preparation, in keeping with the inference that as-hCG beta interacts with the low affinity class of TSH-binding sites. At a concentration more than 3 times that necessary to inhibit TSH binding to the low affinity class of sites, the as-hCG beta molecule neither stimulated adenylate cyclase nor inhibited the ability of TSH to do so. In contrast, the as-hCG molecule, which interacts with both classes of TSH-binding sites, fully inhibited TSH stimulation of adenylate cyclase. We conclude that the low affinity class of TSH-binding sites is not the class of sites through which TSH stimulates adenylate cyclase, and that this role is best ascribed to the high affinity class of TSH-binding sites.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011989:Receptors, Thyrotropin; D013961:Thyroid Gland / Q000378:metabolism*; D013972:Thyrotropin / Q000378:metabolism*
D006063:Chorionic Gonadotropin; D011956:Receptors, Cell Surface; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000262:Adenylyl Cyclases