Receptors for Thyrotropin-releasing Hormone in Prolactin-producing Rat Pituitary Cells in Culture

Abstract

Abstract A hypothalamic tripeptide, thyrotropin-releasing hormone (TRH), causes an increase in the rate of synthesis of prolactin by GH3 cells, a clonal strain of rat pituitary cells grown in culture. This cell system has been used to study the initial interaction between TRH and prolactin-producing cells. Tritiated TRH binds to a limited number of sites on GH3 cells, but TRH does not bind to control cell strains which do not respond biologically to the neurohormone. Half-maximum binding of TRH to GH3 cells occurs at a TRH concentration of 11 nm, while a half-maximum biological response to TRH, an increase in prolactin production, is obtained at a concentration of 2 nm. A maximum of 1.1 pmoles of TRH is bound per mg of GH3 cell protein, equivalent to 130,000 TRH receptors per cell. Initially the binding of TRH to GH3 cells is reversible, but it becomes irreversible over the ensuing 24-hour period. Binding of TRH to receptors has been studied in GH3 cell homogenates using a filter assay technique. At 0°, the dissociation constant of the TRH-GH3 receptor complex is 25 nm, and there is 0.75 pmole of TRH receptors per mg of GH3 cell protein. The halftime for the dissociation of the TRH-GH3 receptor complex is 2 to 3 hours. The binding of TRH to the GH3 receptor has a pH optimum of 7.5, does not require the presence of divalent cations, and is inhibited by high concentrations of calcium ion. We conclude that the action of TRH on GH3 cells involves an initial binding of the tripeptide to specific receptors.