Purification and Properties of Progesterone Receptors from Chick Oviduct (original) (raw)

Abstract

Steroid hormone receptor proteins have been the subject of intensive study for more than 10 years. This research has clearly implicated these molecules in the process of steroid hormone-induced cell development and differentiation. These studies have also shown that receptor proteins are structurally complex, undergoing changes in aggregation, conformation, and binding activities before and during their interaction with nuclear acceptor sites.lB2 The importance of these properties is apparent from the extensive similarities among all steroid receptor proteins studied, regardless of source or steroid-binding specificity. Work in this laboratory has centered on the purification and characterization of progesterone receptors in chick oviduct. This research has been performed with two goals in mind. First, we have studied these proteins to gain information on the relationship between their structure and in vivo mechanism of action. Second, we have sought to purify these proteins to investigate their effect on gene expression in vitro. Chick oviduct progesterone receptor contains two 4s components, designated A and B, which can be resolved by chromatography on a variety of ion-exchange These two components, which are present in approximately equal amounts in crude oviduct cytoplasmic extracts,s are not interconvertable. They have similar hormone-binding sites with respect to equilibrium and kinetic binding constants and hormonal stereospecificity.3 Although both receptor forms are taken up by oviduct nuclei in v i t r~,~'~ they have distinct specificities for binding to nuclear components. The receptor A component binds only to DNA, whereas the B component binds only to chro-Furthermore, B-component binding to chromatin is target tissue ~p e c i f i c .~J~ These properties of the progesterone receptor led us to p r o p o~e~~'~~~~ that both chromatin-and DNA-binding forms might be necessary for receptor function in vivo. We have recently shown in an in vitro system that steroid hormones regulate gene expression in the chick oviduct at the level of messenger RNA transcription.I3 In our hypothetical model, the chromatin-binding B protein would function to specify regions in the chromatin adjacent to hormonally regulated genes. This specifier activity would then direct the A protein to its site of action, where it would bind to the DNA and thus modify transcription by RNA polymerases. This model would suggest that aggregated forms of the receptor that contain

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  27. R. J. B. KING: You said that the intact 6s receptor will not bind to DNA, and yet it will promote RNA chain initiation in the chromatin assays. Is there a discrepancy here, or do you have an explanation? DR. SCHRADER: We think that there is an obligatory dissociation of the two subunits at a gene site that the receptor regulates. Dr. O'Malley will describe that model in considerable detail during his talk. We think that the initial interaction in- volves the intact AB dimer recognizing a chromatin acceptor site via the B