Volume 7, Issue 2, Pages 377-385 (February 2001) The Erythropoietin Receptor Cytosolic Juxtamembrane Domain Contains an Essential, Precisely Oriented, Hydrophobic Motif  Stefan N Constantinescu, Lily Jun-shen Huang, Hyung-song Nam, Harvey F Lodish  Molecular Cell  Volume 7, Issue 2, Pages 377-385 (February 2001) DOI: 10.1016/S1097-2765(01)00185-X

Figure 1 Identity of Three Hydrophobic Residues in the EpoR Cytosolic Juxtamembrane Segment Is Crucial for Biologic Activity (A) Alanine-scanning mutagenesis of the EpoR cytosolic juxtamembrane domain that links the transmembrane domain (TMD) with box 1. Residues H249R250R251 were changed in group to AAA, while residues T252, L253, Q254, Q255, K256, I257, and W258 were each mutated to alanine. Activity is defined as induction of proliferation in Epo (1 U/ml) of Ba/F3 cells infected with bicistronic retroviruses. The activity induced by wild-type EpoR is defined as 100%. Activities below 40% were denoted +/−; between 40%–50%, +; between 50%–75%, ++; between 75%–90%, +++; and >90%, ++++. (B) Dose-dependent proliferation in Epo of Ba/F3 cells expressing the indicated EpoR mutants. IL3- dependent Ba/F3 cells growing in IL3 were infected with bicistronic retroviruses expressing wild-type (Wt) or different EpoR mutants and green fluorescence protein (GFP) and then sorted for similar GFP expression. Cells were grown in IL3 for 3 days, washed in medium with no growth factor to deplete IL3, and then incubated in culture medium supplemented with Epo at the indicated Epo concentrations. Duplicate cell samples were counted after 3 days in Epo. (C) Activation of JAK2 by Epo in Ba/F3 cells expressing the Wt EpoR and the indicated EpoR mutants. Cells were starved of growth factors for 4 hr in RPMI containing 1% bovine serum albumin and then stimulated with 100 U/ml Epo for 5 min. Cells were lysed in NP40 buffer, immunoprecipitated with JAK2 antibodies, and analyzed by Western blotting with anti-phosphotyrosine antibodies (upper panel) or with anti-JAK2 antibodies (lower panel) after stripping the membranes as described Molecular Cell 2001 7, 377-385DOI: (10.1016/S1097-2765(01)00185-X)

Figure 2 Precise Orientation of the EpoR Cytosolic Juxtamembrane Segment (A) Secondary structure prediction of the EpoR transmembrane and juxtamembrane domains. The secondary structure is predicted according to the algorithm of Rost and Sander (1993) and Rost et al. (1994). H, helix; E, extended (sheet); blank, other (loop). The reliability index of prediction is shown on a scale of 0–9, with 9 the highest, as described (Rost et al., 1994). (B) α-helical wheel projection of the register twist introduced by adding extra alanine residues. Each residue would add 109° rotation, with insertion of 3A residues bringing the register close to the original position. (C) Alanine insertion mutagenesis of the EpoR cytosolic juxtamembrane domain. Extra alanine residues (1A, 2A, 3A, or 4A) were inserted after R251. Activity is defined as in Figure 1A Molecular Cell 2001 7, 377-385DOI: (10.1016/S1097-2765(01)00185-X)

Figure 3 Expression and Biologic Activity of Alanine Insertion Mutants of EpoR (A) Similar expression of green fluorescence protein (GFP) by Ba/F3 cells infected with bicistronic viruses coding for wild-type (Wt) or mutant EpoRs and GFP and sorted for the top 1% GFP. (B) Western blot analysis of the cells in (A) using antibodies against the carboxyl terminus of EpoR. (C) Proliferation in 0.1 U/ml Epo of sorted Ba/F3 cells expressing the indicated wild-type (Wt) or mutant EpoRs. Four replicate cell samples were used for each cell line. Cells were depleted of IL3 by extensive washings with RPMI 1640 and then incubated in medium containing Epo for 4 days. (D) Epo dose-dependent proliferation of Ba/F3 cells expressing Wt EpoR, EpoR 1A, or EpoR 3A. Duplicate samples were used for each point. Cells were counted after 3 days' growth in Epo Molecular Cell 2001 7, 377-385DOI: (10.1016/S1097-2765(01)00185-X)

Figure 4 Activation of Signaling by Mutant Epo Receptors Cells sorted for GFP fluorescence expressing the Wt EpoR or mutants EpoR 1A or EpoR 3A were depleted of IL3 for 4 hr and then stimulated with 100 U/ml Epo for 7 min. Cells were lysed and immunoprecipitated with antibodies specific for JAK2 (A) and the EpoR (B) and then analyzed by Western blot using anti-phosphotyrosine antibodies (A, upper panel; B) or antibodies against JAK2 (A, lower panel). Tyrosine- phosphorylated forms of JAK2 and EpoR are denoted JAK2-P-Tyr and EpoR-P-Tyr, respectively Molecular Cell 2001 7, 377-385DOI: (10.1016/S1097-2765(01)00185-X)

Figure 5 Intragenic Complementation of the Defective EpoR 1A by Inserting Two Additional Ala Residues Upstream of the Juxtamembrane Domain or Downstream of R251 (A) Two additional Ala residues were inserted before the EpoR 1A transmembrane domain or at several positions in the transmembrane domain. In Wt T242 Ins AA, two additional Ala residues were inserted after position T242 of the Wt EpoR. (B) Two additional Ala residues were inserted systematically at each position after R251 until box 1 and after residue S266, which is located after box 1. Activity is assayed in Ba/F3 cells as described in Figure 2C Molecular Cell 2001 7, 377-385DOI: (10.1016/S1097-2765(01)00185-X)

Figure 6 Alignment of Nine Members of the Cytokine Receptor Superfamily The conserved Pro residues of box 1 as well as the conserved hydrophobic motif are shown in bold Molecular Cell 2001 7, 377-385DOI: (10.1016/S1097-2765(01)00185-X)

Figure 7 A Model of Epo-Induced Dimerization of the EpoR, JAK2 Transphosphorylation, and Tyrosine Phosphorylation of the EpoR Cytosolic Domain The transmembrane and part of the juxtamembrane domain are proposed to be α-helical. A key hydrophobic residue, L253 (red), is shown to be oriented on a right-handed α helix (A). Upon activation of the Wt EpoR by Epo, tyrosine phosphorylation of JAK2 and EpoR occur (-P). JAK2 transphosphorylates the partner EpoR in the complex (see Discussion). When the key hydrophobic residue L253 is mutated to Ala (blue), neither Epo-induced JAK2 activation nor EpoR phosphorylation occurs (B). Insertion of one extra Ala residue in the juxtamembrane cytosolic domain changes the register of L253 (red) and of the downstream I257 and W258 (not shown), which results in a change in JAK2 positioning on the EpoR relative to the membrane-spanning domain. Epo induces JAK2 transphosphorylation, but there is no detectable tyrosine phosphorylation of the EpoR cytosolic domain (C). Insertion of three extra Ala residues (EpoR 3A) extends the α helix and reorients L253 (red) to the same register as Wt EpoR. Normal activation of JAK2 and tyrosine phosphorylation of EpoR cytosolic domain occur (D) Molecular Cell 2001 7, 377-385DOI: (10.1016/S1097-2765(01)00185-X)