Dimerization between hERα and -β is inhibited by PKA. COS-1 cell extracts were immunoprecipitated with B10 (A) or M2 (B) after transient transfection with HEG0, HEG0^236A, HEG0^236E, and/or hERβ1, as well as pSG5-PKA (as appropriate), and [³⁵S]methionine labeling in the presence or absence of ligands as described in Fig. 6. (C) Immunoblotting with the B10 and M2 monoclonal antibodies, used together, served to control for the presence of HEG0, HEG0^236A, HEG0^236E, and hERβ1 in the appropriate cell lysates. The molecular size markers are shown (in kilodaltons) on the right.

Inhibition of dimerization by PKA is prevented by 17β-estradiol and 4-hydroxytamoxifen but not by ICI 182,780. COS-1 cells were transiently transfected with HEG0, HEG19, HEG0^236A, HEG19^236A, HEG0^236E, and HEG19^236A and pSG5-PKA as shown. The cells were labelled for 1 h with [³⁵S]methionine in the presence or absence of E2, OHT, or ICI (final concentrations, 10⁻⁷ M). The extracts were divided in two and immunoprecipitated with B10 (upper panels) or F3 (lower panels) as shown. Triangles indicate the position of a nonspecific band (2). The molecular size markers are shown (in kilodaltons) on the right.

DNA binding by hERα is inhibited by phosphorylation of serine 236. (A to D) COS-1 cells were transiently transfected with pSG5, HEG0, or HEG0^236A. E2, OHT, and ICI were added 2 h prior to harvesting. (A and C) An expression plasmid encoding the catalytic subunit of PKA (63) was transfected, along with the pSG5, HEG0, or HEG0^236A. (B and D) Carrier (NH₄OH) or 8-Br-cAMP was added at the same time as E2, OHT, or ICI. (C and D) The PKA inhibitor H89 was also added at the same time as E2, OHT, or ICI. Immunoblotting with B10 was performed to control for levels of receptor protein (A to D, lower panels).

Loss of hERα DNA binding is due to inhibition of dimerization. (A) Gel shifts were performed with extracts prepared from COS-1 cells transfected with HEG0 alone (lane 2) or together with wild-type HEG19 or serine 236 mutants (lanes 3 to 6) and HEG19 alone (lane 7) or together with serine 236 mutants of HEG0 (lanes 8 to 10). (B) HEG0, HEG19, HEG0^236E, and HEG19^236E were synthesised in vitro either separately or together in the presence of [³⁵S]methionine. The in vitro translation products were divided in two and immunoprecipitated with the B10 monoclonal antibody (lanes 1 to 7). Immunoprecipitation with the F3 (lanes 8 to 14) monoclonal antibody was used to show that HEG0, HEG19, and the 236E mutants are present in the appropriate in vitro translations. (C) HEG0, HEG0^236E, and hERβ1 were synthesized in vitro either separately or together in the presence of [³⁵S]methionine. The in vitro translation products were divided in two and immunoprecipitated with B10 (lanes 1 to 5) or M2 (lanes 6 to 10) monoclonal antibodies.

Schematic representation of hERα. (A) Regions A to F, as initially described by Krust et al. (49) are depicted, the numbers below referring to amino acid positions of the boundaries for the different regions. Also shown are the positions of the epitopes for monoclonal antibodies B10 and F3. The portion of hERα coding region contained within HEG0, HE15, and HEG19 are also shown. (B) The amino acids comprising the core DBD and their involvement in DNA binding, as determined crystallographically, is represented. The amino acids marked in boldface participate in the hydrophobic core. Asterisks mark the residues which interact with base pairs; those making phosphate contacts (squares) or participating in the dimer interface (circles) are also indicated. Closed and open circles and squares denote direct interactions and interactions via ordered water molecules, respectively (75). The position of serine 236 is marked by an arrow.

Comparison of the transcriptional activities of hERα mutants on various estrogen-responsive reporter genes. (A to F) Transcriptional stimulation in COS-1 cells of six reporter genes by the wild-type human ERα (HEG0) and the serine 236 mutants HEG0^236A and HEG0^236E, together with pSG5 or pSG5-PKA, in the presence or absence of E2 (10 nM), OHT (100 nM), and ICI (100 nM) as indicated. The results are displayed in the form of a bar chart. The average values of at least three independent experiments (±10%) are given in each case. Transcriptional activation was determined with ERE-TATA-CAT (A), ERE-3-TATA-CAT (B), ERE-tk-CAT (C), ERE-3-tk-CAT (D), vit-tk-CAT (E), and ERE-G-CAT (F). For each reporter gene the level of transactivation by HEG0 in the presence of E2 was taken as 100%. In panel G COS-1 cells were cotransfected with the reporter gene 17M-ERE-TATA-CAT, HEG0, HEG0^236A, or HEG0^236E, together with either pSG5 or pSG5-PKA and GAL-VP16, as indicated. Ligands were added as described above. The results are displayed in the form of a bar chart. The average values of at least three independent experiments (±10%) are given in each case. The level of transactivation by GAL-VP16 was taken as 100%.

Comparison of the in vitro DNA binding by wild-type hERα and mutants of serine 236. (A) COS-1 cells were transiently transfected with pSG5 (lane 1), HEG0 (lane 2), mutants of HEG0 (lanes 3 to 5), HE15 and its mutants (lanes 6 to 10), or HEG19 and its mutants (lanes 10 to 13) in the presence of 10⁻⁷ M E2. Lysates were prepared in HS buffer, and gel shifts were performed as described in Materials and Methods. (B) COS-1 cells were transiently transfected with pSG5 (lane 1), HEG0 (lanes 2 to 5), HEG0^236A (lanes 6 to 9), or HEG0^236E (lanes 10 to 13). Carrier (ethanol) or the ligands E2, OHT, or ICI (prepared in ethanol) were added to a final concentration of 10⁻⁷ M 2 h before harvesting. Western blot analysis of the extracts was performed with monoclonal antibodies B10 (panel A, lanes 1 to 9; panel B) or F3 (panel A, lanes 10 to 13) to control for the levels of receptor proteins.

Serine 236 is phosphorylated by PKA. (A) WCEs of COS-1 cells transiently transfected with HEG0, HEG19, or HE15 were immunoprecipitated with F3 (lanes 1 and 2) and B10 (lane 3) monoclonal antibodies, phosphorylated with PKA, and analyzed by SDS-PAGE and autoradiography. The solid triangle marks the position of an irrelevant 68-kDa polypeptide observed with the F3 monoclonal antibody and previously described (2). Another triangle at 40 kDa marks the presumed position of the catalytic subunit of PKA used for in vitro phosphorylation. (B) Phosphorylation of HE15 and HE15^236A by PKA and PKC. Immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose, immunoprobed with B10, and revealed by the alkaline phosphatase method to determine the levels of HE15 and HE15^236A (lower panel). The nitrocellulose membrane was autoradiographed for the phosphorylation signal (upper panel). The filled triangle shows the position of the catalytic subunit of PKA. The positions of molecular size markers (in kilodaltons) are shown. (C and D) Two-dimensional phosphopeptide maps of HE15 and HE15^236A, respectively, following in vitro phosphorylation by PKA as in panel B. (D) The empty circles highlight the spots present in the HE15 map but missing from PKA phosphorylation of HE15^236A. (E) COS-1 cells transiently transfected with pSG5 (lane 1), HEG0 (lanes 2, 3, and 6), or HEG0^236A (lanes 4, 5, and 7) in the absence (lanes 1, 2, 4, 6, and 7) or the presence (lanes 3 and 5) of pSG5-PKA were labeled with [³²P]orthophosphate. 8-Br-cAMP (100 nM) was added as appropriate (lanes 6 and 7). Immunoprecipitations were resolved by SDS-PAGE and autoradiographed (upper panel). Immunoblotting with monoclonal antibody B10 was used to control for hERα protein levels (lower panel).