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Structure-Function Properties of the Chicken Progesterone Receptor A Synthesized from Complementary Deoxyribonucleic Acid

Mary Anne Carson^*, Ming-Jer Tsai, Orla M. Conneely, Beth Lynn Maxwell, James H. Clark, Alan D. W. Dobson, Alex Elbrecht, David O. Toft, William T. Schrader and Bert W. O'Malley

Department of Cell Biology, Baylor College of Medicine Houston, Texas 77030
Department of Biochemistry and Molecular Biology, Mayo Medical School Rochester, Minnesota 55905

Address requests for reprints to: Bert W. O'Malley, Department of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030.

Abstract

The chicken progesterone receptor (PR) cDNA has been cloned and sequenced in our laboratory. Functional receptor A was synthesized from cDNA in two independent systems, by transient transfection of receptor-negative COS M6 cells and by in vitro transcription and translation. These receptors exhibited DNA and hormone binding properties similar to the native PR from oviduct. The ability of receptor to induce target gene transcription was measured by cotransfection of receptor-negative CV-1 cells with expression vectors containing the receptor A cDNA and a progesterone-inducible promotor linked to the chloramphenicol acetyl transferase (CAT) gene. In these assays, receptor A produced hormone-dependent induction of CAT activity. In order to define the functional domains of receptor A, expression constructs coding for C-terminal deletion proteins were prepared. Deletion of the C-terminus resulted in loss of hormone binding activity as well as a loss of CAT induction. However, when 290 amino acids were removed from the C-terminus, this severely truncated receptor protein produced hormone-independent target gene activation. Mutant receptor proteins which retained the highly conserved cysteinerich (C1) region were able to bind to DNA-cellulose, although removal of 290 amino acids from the Cterminus resulted in reduced affinity for DNA. Deletion of part or all of the C1 region resulted in loss of both DNA-binding and transcriptional activation capacities. These results confirm that C1 functions in DNA binding and transcriptional activation and that hormone binding activity can be localized to the Cterminal half of the protein.

FOOTNOTES

^* Supported by postdoctoral fellowship GM-10147.

Recipient of a postdoctoral fellowship from the Medical Research Council of Canada.

Received for publication August 13, 1987. Accepted for publication September 14, 1987.

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