Nuclear progestin receptors in human uterus

The characteristics and transfer of human uterine progestin receptors from cytosol to nucleus were studied in a cell-free system using [3H]progesterone ([3H]P), [3H]norethindrone ([3H]NET), and [3H]norethindrone acetate ([3H]NETA) as progestin receptor tags. Nuclear receptor binding was observed only in the presence of uterine cytosol. Various prior treatments of uterine cytosol, including warming to 20 C, increasing the salt concentration to 150 mM KCl, or dilution of the cytosol protein concentration to 3 mg/ml, increased the affinity of the receptor for the cell nucleus and thus, facilitated more translocation. However, there was no detectable difference in the receptor sedimentation value after activation. The binding of salt-extractable (400 mM KCl) nuclear sites to [3H]P, [3H]NET, and [3H]NETA was characterized by distinct commonality in their physiochemical properties, as indexed by their coelution during gel chromatography (mol wt, 72,000), their similar sedimentation rates (4.2S), and their similar isoelectric profiles (isoelectric pH 5.3--5.6). The concentrations of specific binding sites were similar for the different 3H-labeled progestins used. However, in general, the number of binding sites was higher (3780--4480 molecules/cell) in proliferative phase tissues and significantly lower (1790--1920 molecules/cell) in secretory phase tissues. Furthermore, nuclear binding was progestin specific and of high affinity (Kd of NET, similar to or approximately 0.7--0.8 nM; Kd of P, similar to or approximately 0.9--1.2 nM; Kd of NETA, similar to or approximately 6.7--7.1 nM). Despite the similarities in the molecular properties, the nuclear bindings of NET and NETA could be distinguished from that of P by their lower dissociation rates (t 1/2 of NET, similar to or approximately 6.3 h; t 1/2 of NETA, similar to or approximately 5.5 h; t 1/2 of P, similar to or approximately 1.2 h). Moreover, NET and NETA, unlike P, did not bind to corticosteroid-binding globulin. These observations indicate that the various progestins mediate their action via a common receptor, which translocates from cytosol to nucleus. Further, because of the distinct advantages, NET could be conveniently used as a receptor tag to evaluate the progestin receptor.