1. Volume 12, Issue 5, Pages 1239-1250 (November 2003)
Active and Inactive Orientations of the Transmembrane and Cytosolic Domains of the Erythropoietin Receptor Dimer 
Nadine Seubert, Yohan Royer, Judith Staerk, Katharina F Kubatzky, Virginie Moucadel, Shyam Krishnakumar, Steven O Smith, Stefan N Constantinescu 
Molecular Cell 
Volume 12, Issue 5, Pages (November 2003)
DOI: /S (03)

2. Figure 1 Design of Coiled-Coil EpoR Fusion Proteins
(A) The coiled-coil dimerization domain of Put3 replaced the extracellular domain of the EpoR and was fused using PCR overlap extension to consecutive residues of the EpoR TM domain (cc-EpoR); fusion to the first TM residue is denoted cc-EpoR-0. The constructs contain the EpoR signal sequence followed by the hemagglutinin (HA) tag and an α helix capping sequence (GNP) followed by the Put3 coiled coil.
(B) α-helical prediction of the precise positions of amino acid residues in the TM and JM domains of the EpoR. Positions a and d form the interface in coiled coils. Shown are the predicted α-helical positions of TM residue S238 and JM hydrophobic motif residues L253, I257, and W258 as well as a summary of the proliferation activity of the fusion proteins in Ba/F3 cells (see Figure 2).
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
One EpoR Dimeric Conformation Is Associated with Induction of Biologic Effects
(A) Proliferation in the absence of cytokines of IL3-dependent Ba/F3 cells expressing the wild-type EpoR cells after infection of retroviruses coding for the coiled-coil-EpoR (cc-EpoR) fusion proteins.
(B) Proliferation in the absence of cytokines of parental IL3-dependent Ba/F3 cells after infection with the seven cc-EpoR fusion proteins and sorting for equal GFP levels. Shown are the total number of cells after 4 days incubation in medium without cytokines.
(C) Total cell levels of the seven cc-EpoR fusion proteins in Ba/F3 cells infected to equal GFP levels, as revealed by Western blotting with anti-HA antibodies.
(D) HA staining for cell surface expression of cc-EpoR-0, cc-EpoR-I, cc-EpoR-II, cc-EpoR-III, cc-EpoR-IV, cc-EpoR-V, and cc-EpoR-VI in cells sorted for equal GFP expression. Resorting for HA expression in the case of cc-EpoR-II resulted in high surface expression levels but no induction of biologic effects. HA fluorescence is expressed as percent of HA staining for the wild-type HA-EpoR, which was considered 100%.
(E) CFU-E differentiation of day 12.5 fetal liver cells transduced with retroviruses coding for cc-EpoR-II, cc-EpoR-III, and cc-EpoR-VI fusion proteins. Shown are the numbers of CFU-E colonies per 200,000 nucleated fetal liver cells as averages of three independent experiments ± standard deviation. The efficiency of retroviral infection was 13.8%–16%. Addition of 3 U/ml recombinant Epo resulted in 1, colonies/200,000 fetal liver cells. Shown is the effect of Epo after normalization to an average of 15% infection efficiency.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 Orientation-Dependent Signaling of EpoR Dimers
(A and B) Activation of JAK2 (A) and STAT5 (B) tyrosine phosphorylation by fusion proteins cc-EpoR-III and cc-EpoR-VI. Tyrosine phosporylation of JAK2 was determined in Ba/F3 cells expressing either wild-type EpoR or a cc-EpoR construct at equal GFP levels after cell sorting. Wild-type EpoR was stimulated with 100 U/ml of Epo. Immunoprecipitations with anti-JAK2 or anti-STAT5 antibodies were analyzed by immunoblotting with anti-phosphotyrosine (pY) 4G10 antibody. Equal amounts of total protein were ensured by blotting with antibodies against JAK2 and STAT5, respectively.
(C) Constitutive nuclear translocation of activated STAT5 and STAT3 in cells expressing cc-EpoR-III and cc-EpoR-VI. STAT5 was detected by blotting with an anti-STAT5 antibody, while STAT3 was detected by blotting with an anti-phosphotyrosine-STAT3 (pY-STAT3) antibody.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 Orientation-Dependent Transcriptional Effects of EpoR Dimers
Ba/F3 cells expressing the wild-type (Wt) EpoR or the seven fusion proteins cc-EpoR at similar levels after sorting for GFP (sorted) or after selection in medium devoid of any cytokine (selected), were electroporated with the pLHRE-Luc (A), the pGl3bPpr2-Luc (B), or the pSRE-Luc (C and D) in order to measure transcriptional activity induced by STAT5, STAT3, and MAP kinase, respectively. pRL-TK was electroporated in each experiment for normalizing luciferase values. Cells expressing the wild-type EpoR were stimulated with 100 U/ml Epo (+) or mock stimulated (−). Cell lysates were prepared and assayed for luciferase activity. Results of one representative experiment performed in triplicate ± standard deviation are shown as luciferase ratio in relative light units (rlu). Similar results were obtained in three independent experiments. F8, F7Y464, and F7Y479 stand for EpoR cytosolic mutation where all eight Y residues were mutated to F, or where Y464 or Y479 was added back on the background of seven Y residues being mutated to F.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
The Effect of JAK2 Overexpression on Biologic Activity of cc-EpoR Fusion Proteins
(A) Total levels of cellular JAK2 in parental Ba/F3 cells and in Ba/F3 cells overexpressing JAK2, as detected by Western blotting with anti-JAK2 antibodies. A 1/5 dilution of cell lysates of JAK2 overexpression cells (JAK2 1/5) is shown for comparing JAK2 protein levels between parental (Ba/F3) and JAK2 overexpressing cells (JAK2). Ba/F3 cells were infected with bicistronic viruses coding for murine JAK2 and human CD4. Sorting for high cell surface CD4 levels resulted in selection for population of cells that stably overexpress JAK2.
(B) Proliferation of Ba/F3 overexpressing JAK2 after infection with viruses coding for the seven cc-EpoR fusion proteins. Shown are the total number of cells after 2 days incubation in medium without cytokines.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
cc-EpoR-IV Fusion Protein Mimics the Conformation of the Inactive EpoR Dimer
(A) Proliferation of Ba/F3 cells expressing the indicated cc-EpoR fusion proteins after infection with SFFV retrovirus coding for gp55-P.
(B) Cells expressing the wild-type EpoR or the cc-EpoR-IV fusion protein and rendered cytokine independent by infection with retrovirus coding for the gp55-P protein express high levels of viral envelope protein, as shown by Western blotting with an anti-gp55 antibody.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
Molecular Dynamics Simulations: Low-Energy Symmetric EpoR Conformations Corresponding to Active and Inactive Orientations
(A) Computational search for low-energy dimer structures of the EpoR TM region from L230 to S248. MD simulations were run on helix dimers having left-handed crossing angles and helix rotation angles (φ1 and φ2) from 0° to 360° for each helix. The figure plots the final φ1 and φ2 values for those structures that fall into a “cluster.” The two clusters (circled) with symmetric orientations correspond to cc-EpoR-IV (cluster 1) and cc-EpoR-III (cluster 2). The interhelical separation for this search was maintained at 10 Å.
(B) Helix interaction energies for TM dimer structures with left- and right-handed crossing angles. The average contribution of each residue to the dimer stability is shown for the energy-minimized average structures derived from clusters 1 and 2 (solid lines). The dashed lines show the interaction energies for the low-energy TM dimers with right-handed crossing angles having similar interfaces.
(C) Dimer structures of the EpoR TM region from L230 to S248 derived from the computational searches.
Molecular Cell  , DOI: ( /S (03) )