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Characterization and Functional Properties of the A and B Forms of Human Progesterone Receptors Synthesized in a Baculovirus System

Department of Pathology and Program in Molecular Biology, University of Colorado Health Sciences Center Denver, Colorado 80262

Address requests for reprints to: Dr. Dean P. Edwards, University of Colorado Health Sciences Center, Department of Pathology (Campus Box B216) and Program in Molecular Biology, 4200 East Ninth Avenue, Denver, Colorado 80262.

Abstract

Human progesterone receptors (PR) were overexpressed in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus system. Recombinant viruses were constructed that produced either full-length A (94K) or B (120K) forms of human PR, and each was expressed as a functional protein. Steroid and DNA binding activities were found to be indistinguishable from that of endogenous human PR in T47D breast cancer cells. Moreover, as analyzed by gel-mobility shift, recombinant PR-A and PR-B each bound to specific progesterone response elements in a strictly hormone-dependent manner. Native receptors expressed in Sf9 cells also exhibited structural properties similar to that of endogenous PR. Cytosolic PR (PR-A or PR-B), prepared in low salt buffer, sedimented on density gradients as an 8S oligomeric complex that was converted largely to 4S by treatment with 0.4 M NaCl. Immune isolation of the 8S cytosol PR complex and analysis of protein composition revealed the presence of two specific copurifying proteins of approximately 90K and 70K. The 90-K component was identified immunologically as heat shock protein 90. The 70-K component was not identified but is likely to be the insect equivalent of heat shock protein 70. Immune isolation of PR from Sf9 cells metabolically labeled with [³²P_i], revealed that expressed PR was capable of being phosphorylated in insect cells. Hormone addition to Sf9 cells, however, did not stimulate the same increase in PR phosphorylation or upshift in mobility on sodium dodecyl sulfate gels that occurs with endogenous receptors in T47D cells. Thus some, but not all, phosphorylations occur with human PR expressed in Sf9 cells. These phosphorylation data, together with the fact that expressed PR required hormone for DNA binding, indicate that the hormonedependent phosphorylation step responsible for PR upshifts on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is not required for receptor binding to DNA. The baculovirus expression system, therefore, may prove valuable in dissecting the functional role(s) for both hormone-dependent and hormone-independent PR phosphorylation.

FOOTNOTES

This work was supported in part by USPHS Grants CA- 41386 and CA-46938, Cancer Center Core Grant P30 CA- 46934, the Lucille Markey Charitable Trust (to D.P.E.), and National Research Service Fellowship Award HD-07430 (to C.A.B.).

Received for publication August 6, 1991. Revision received September 10, 1991. Accepted for publication September 11, 1991.

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