Binding of [3H]progesterone to the human progesterone receptor: differences between individual and mixed isoforms

0013.7227/96/$03.00/O Endocrinology Copyright G 1996 by The Endomne Vol. 137, No 6 Prrnted in U S.A. Soaety Binding of [3H]Progesterone to the Human Progesterone Receptor: Differences between Individual and Mixed Isoforms* P. CARBAJO, K. CHRISTENSEN, D. P. EDWARDS, AND D. F. SKAFAR Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan the Department of Pathology and Program in Molecular Biology, University of Colorado Sciences Center (K.C., D.P.E.), Denver, Colorado 80262 ABSTRACT The human progesterone receptor (hPR) exists as two isoforms, hPR-A and 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 expressed hPR-A and hPR-B and asked whether the progesterone-binding mechanisms are the same or different for the two forms of hPR and for their mixture. We investigated 1) the cooperativity of binding [“HIprogesterone to the receptor, as measured by the Hill coefficient (n,); and 2) the dissociation rate of [“Hlprogesterone from the receptor. To compare the effects of dimerization, these ligand-binding properties were measured over a range of receptor concentrations. Binding of [“Hlprogesterone to hPR-A was positively cooperative at all concentrations used; the limiting value for the Hill coefficient was 1.47 2 0.11 at high receptor concentrations (5-19 nM) and 1.31 + 0.06 at low receptor concentrations (l-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 2 0.25 X lo-” min-l , respectively. By contrast, the hPR-B concentration had a marked effect on positive cooperative binding and the dissociation rate of progesterone. At high hPR-B concentrations (3-5 nM), the limiting Hill coefficient was 1.49 2 0.11, which is indicative ofmoderately strong positive cooperativity, whereas at lower hPR-B concentrations (l-3 IIM), the Hill coefficient 48201; Health and was reduced to 1.1, which is essentially noncooperative. The [“Hlprogesterone dissociation rate was 4.52 i- 0.44 X lo-” min ’ at the higher concentrations of hPR-B and was increased to 1.6 2 0.11 x lo-” min-’ at the lower concentrations. Thus, over the same concentration range where hPR-A exhibited no significant change in positive cooperativity or the dissociation rate, these progesteronebinding 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 the concentration of receptor. However, the hPR-AB mixture differed from hPR-B alone in that the mixture required a greater receptor concentration (7-10 us.. 3-5 nM) to exhibit positive cooperativity and the increased dissociation rate. These results show, first, that each hPR isoform displays different [“HIprogesterone-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 only at the N-terminus, yet positive cooperativity and changes in the dissociation rate constant are indicative of conformational changes affecting hormone binding, these results also strongly suggest that the N-terminus may directly or indirectly interact with the C-terminal ligand-binding domain. (Endocrinology 137: 23392346, 1996) T HE HUMAN progesterone receptor (hPR), a ligand-activated transcription factor, exists in two isoforms, designated A and B (1,2). The only structural difference between the two forms lies in the additional 164 amino acids present at the amino-terminal end of hPR-B that are absent in hPR-A (3). Recent reports demonstrate a functional difference between the A and B forms. hPR-A can act as a transcriptional repressor of hPR-B (4). The effect is hormone dependent, and cell and promoter specific. The hPR-A and hPR-B isoforms also respond differently to progesterone antagonists; antagonist-occupied hPR-B can activate transcription, whereas antagonist-occupied hPR-A cannot (5). Furthermore, coexpression of hPR-A can block transcription activation by antagonist-occupied hPR-B. There is also evidence that the two receptor isoforms may influence transcription through independent mechanisms (6, 7). Because of these observed differences in the abilities of the isoforms to activate transcription, fluctuations in the levels of A and B isoforms may influence the cellular response to progesterone and its antagonists. For example, the chicken oviduct exhibits seasonal variation in the level of A receptors (8). The decreased levels of A receptors in the winter correlate with decreased nuclear binding to chromatin it7 ZJ~ZKJ and in vitro. In murine mammary tissue, the levels of PR-A and PR-B forms and its corresponding messenger RNAs vary according to the developmental state of the animal: nulliparous, pregnant, lactating, or lactational involution (9, 10). During the human menstrual cycle, the levels of total progesterone and estrogen receptors are highest during the late proliferative phase and are significantly lower during the late secretory phase (11). The ratio of the A and B forms in the uterine endometrium also changes during the menstrual cycle (12). The mechanism of action of the steroid hormone receptors includes several steps, starting with ligand binding and the associated conformational changes in the protein, dimeriza- Received January 5, 1996. Address all correspondence and requests for reprints to: Dr. D. F. Skafar, Department of Physiology, Wayne State University School of Medicine, 540 East Canfield, Detroit, Michigan 48201. E-mail: . * This work was supported by NSF Grants IBN-9104857 and IBN9407376 (to D.F.S.) and the Wayne State University Minority Faculty Research Fund (to D.F.S.). 2339 LIGAND-BINDING 2340 PROPERTIES tion, phosphorylation, DNA binding, and interactions with other transcription factors (13, 14). It is important to determine the step(s) in the mechanism of action of steroid receptors that differs between the A and B forms to understand the basis for the observed differences in transactivation. In particular, as hormone binding is the biological signal, the question arises whether hormone binding and steps associated with it, such as conformation changes in the receptor, are different between the two forms. Dimerization of the hPR, besides being a fundamental property of the protein, has a potential role as a regulatory step. For the hPR, three distinct dimeric complexes, composed of AA, AB, and BB, occur when complexed with the progesterone response element (15, 16) and in solution (15). It is, therefore, critical to understand the conditions under which receptors form dimers and how dimerization affects their biochemical properties and biological activity. Studies of the binding mechanism and kin (...truncated)