title: Characterization and functional properties of the A and B forms of human progesterone receptors synthesized in a baculovirus system.
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 [32Pi], 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 hormone-dependent 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.
authors.affiliation: Department of Pathology, University of Colorado Health Sciences Center, Denver 80262.
authors.forename: K
authors.initials: K
authors.lastname: Christensen
authors.forename: P A
authors.initials: PA
authors.lastname: Estes
authors.forename: S A
authors.initials: SA
authors.lastname: Oñate
authors.forename: C A
authors.initials: CA
authors.lastname: Beck
authors.forename: A
authors.initials: A
authors.lastname: DeMarzo
authors.forename: M
authors.initials: M
authors.lastname: Altmann
authors.forename: B A
authors.initials: BA
authors.lastname: Lieberman
authors.forename: J
authors.initials: J
authors.lastname: St John
authors.forename: S K
authors.initials: SK
authors.lastname: Nordeen
authors.forename: D P
authors.initials: DP
authors.lastname: Edwards
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D010957:Plasmids; D011397:Promegestone / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011980:Receptors, Progesterone; D011994:Recombinant Proteins; D004247:DNA; D011397:Promegestone