Heterotetrameric structure of the human progesterone receptor ( steroid receptors / photoafinity labeling / chemical cross-linking )

Nonactivated progesterone receptors in extracts of human T47D mammary carcinoma cells were investigated. Chemical cross-linking with dimethyl suberimidate resulted in complete stabilization of the A and B receptors with an average molecular mass of 340 kDa. For analyzing the subunit structure, we concentrated on the larger B receptor, which was separated from the A form by immunoaffnimty chromatography. Progressive cross-linking of the photoaffmity-labeled receptor resulted in patterns oflabeled bands in SDS gels, which are indicative of a heterotetrameric structure. It consists of one receptor polypeptide in association with two 90-kDa subunits and one polypeptide of -60 kDa. The completely cross-linked B receptor has a molecular mass of -390 kDa. To identify the subunits, the oligomeric B receptor was cross-linked with a cleavable bisimidate, highly purified by immunoaffinity chromatography, and analyzed by gel electrophoresis and immunoblotting. The receptor polypeptide has a mass of 116.5 kDa. The 90-kDa band was identified as the heat shock protein hsp90 and was roughly twice as intense as the receptor polypeptide. By use of specific antibodies, we identified the fourth receptor subunit as a 59-kDa protein (p59); we did not obtain any evidence for the heat shock protein hsp7O being a receptor component. We suggest an analogous heterotetrameric structure for the nonactivated A receptor. The progesterone receptor is a member of the steroid hormone receptor family; it is exceptional in the sense that it is expressed in vertebrates in two forms, A and B (1, 2). These steroid-binding polypeptides are identical except that the amino-terminal region is missing from the A polypeptide (3). Both polypeptides appear to be synthesized as individual entities (4, 5) but may be functionally distinct (5, 6). Progesterone receptors can be recovered in extracts of target cells in a nonactivated large molecular mass state that is unable to interact with DNA but binds the hormonal ligand (7). Although A and B receptor polypeptides are known to occur in independent large molecular mass forms (8-10), the structures of these are uncertain. Reports in the literature describe various protein species of largely differing size, which have been found in association with purified progesterone receptors of different vertebrate species (11-15). However, copurification of proteins with a given receptor poses a problem: it is very difficult to distinguish between a bona fide structural component and a persistent contaminant of the purification protocol. Chemical cross-linking between protein subunits prior to extensive purification provides a means to avoid such confusion. We used this approach here to obtain definitive information on the subunit structure of progesterone receptors in terms of stoichiometry and identity of the individual components. For technical reasons, we largely concentrated on the large molecular mass B receptor of human T47D mammary carcinoma cells. We unequivocally demonstrate that it has a heterotetrameric subunit