Resolution of high and low affinity progesterone receptors from human breast carcinoma T47D cells

Abstract

Each of four independent experimental approaches showed that human breast carcinoma T47D cells contain both high and low affinity progesterone receptors. (i) Equilibrium-specific [³H]progesterone binding to adherent cultured cells revealed dissociation constants (K_d) of 1.5 and 60 nM and 0.33 and 2.4×10⁶ sites/cell, respectively. Both the high and low affinity receptors were specific for progestins as demonstrated by steroid binding competition studies conducted at 5 and 50 nM [³H]progesterone. (ii) Equilibrium [³H]progesterone binding to the resolved soluble and particulate fractions from a cell homogenate sedimented at 40,000×g.min revealed K_d=1.4 nM high affinity binding sites exclusively in the supernatant fraction and K_d=24 nM low affinity sites exclusively in the particulate fraction. Extraction of the particulate fraction with a high ionic strength buffer solubilized the low affinity receptors stoichiometrically; but once solubilized, they displayed K_d=2.4 nM high affinity progesterone binding. Characterizations of ³H-ligand bound specifically to progesterone receptors in intact cells or resolved subcellular fractions revealed no [³H]progesterone metabolites that could account for the low affinity binding. (iii) Calculations based on the rate constants of [³H] progesterone association with or dissociation from adherent cells revealed the same dissociation constants for both high and low affinity binding as those determined by equilibrium measurements. (iv) Nonionic detergent extraction of cells incubated with a wide range of [³H]progesterone concentrations revealed high affinity progesterone binding to receptors in the detergent-soluble fraction and low affinity binding associated primarily with the particulate residue, consistent with the data on equilibrium progesterone binding to resolved cell homogenate fractions. The rate of extraction of the high affinity receptor-progesterone complex with nonionic detergent (t_½=1 min at 0°C) equaled the rate of extraction of a representative lysosomal enzyme, β-acetylglucosaminidase.