BINDING OF [H-3] PROGESTERONE TO THE HUMAN PROGESTERONE-RECEPTOR - DIFFERENCES BETWEEN INDIVIDUAL AND MIXED ISOFORMS

The human progesterone receptor (hPR) exists as two isoforms, hPR-A an d hPR-B, which differ only in that hPR-A lacks 164 amino acids present at the amino-terminus of hPR-B. In this study we have separately expr essed hPR-A and hPR-B and asked whether the progesterone-binding mecha nisms are the same or different for the two forms of hPR and for their mixture. We investigated 1) the cooperativity of binding [H-3]progest erone to the receptor, as measured by the Hill coefficient (n(H)); and 2) the dissociation rate of [H-3]progesterone from the receptor. To c ompare the effects of dimerization, these ligand-binding properties we re measured over a range of receptor concentrations. Binding of [H-3]p rogesterone to hPR-A was positively cooperative at all concentrations used; the limiting value for the Hill coefficient was 1.47 +/- 0.11 at high receptor concentrations (5-19 nM) and 1.31 +/- 0.06 at low recep tor concentrations (1-4 nM). Similarly, little change was observed in the dissociation rate constant over the same concentration range; the values at high and low concentrations were 4.59 +/- 0.15 and 3.03 +/- 0.25 X 10(-3) min(-1), respectively. By contrast, the hPR-B concentrat ion had a marked effect on positive cooperative binding and the dissoc iation rate of progesterone. At high hPR-B concentrations (3-5 nM), th e limiting Hill coefficient was 1.49 +/- 0.11, which is indicative of moderately strong positive cooperativity, whereas at lower hPR-B conce ntrations (1-3 nM), the Hill coefficient was reduced to 1.1, which is essentially noncooperative. The [H-3]progesterone dissociation rate wa s 4.52 +/- 0.44 X 10(-3) min(-1) at the higher concentrations of hPR-B and was increased to 1.6 +/- 0.11 X 10(-3) min(-1) at the lower conce ntrations. Thus, over the same concentration range where hPR-A exhibit ed no significant change in positive cooperativity or the dissociation rate, these progesterone-binding properties were highly dependent on the concentration of hPR-B. When hPR-A and hPR-B were mixed, positive cooperative binding and the dissociation rate were more similar to hPR -B than to hPR-A, in that both binding parameters were dependent on th e concentration of receptor. However, the hPR-AB mixture differed from hPR-B alone in that the mixture required a greater receptor concentra tion (7-10 vs. 3-5 nM) to exhibit positive cooperativity and the incre ased dissociation rate. These results show, first, that each hPR isofo rm displays different [H-3]progesterone-binding properties, which are most prominent at low concentrations of receptor, and second, that one isoform can influence the other. As the two receptor forms differ onl y at the N-terminus, yet positive cooperativity and changes in the dis sociation rate constant are indicative of conformational changes affec ting hormone binding, these results also strongly suggest that the N-t erminus may directly or indirectly interact with the C-terminal ligand -binding domain.