The importance of the hydrophobic sequence in D1D2 domain for TpoR activation. (A) In silico analysis showing that the hydrophobic sequence in TpoR, EpoR, and GCSFR was performed with ExPASy ProtScale by using the scale Hphob. / Roseman. The presence of a hydrophobic patch in the extracellular domain is observed in TpoR and GCSFR but not EpoR, as shown in the graphs. Below the graphs are schematic representations of receptor domains comprising a hydrophobic signal peptide, ECD, hydrophobic transmembrane domain (TMD), and ICD. The red arrows indicate the hydrophobic cluster in ECD corresponding to the maximal hydrophobic peak in the TpoR and GCSFR sequences. Eventually, sequence alignment of the region surrounding hydrophobic cluster (bold residues) was performed with CLUSTAL Ω (1.2.1) multiple sequence alignment tool. Sequence of mutants TpoR 8A and EpoR LFFPL were also indicated. STAT5 transcriptional activity of CALR mutants when coexpressed with TpoR WT (B), TpoR 8A (C), EpoR WT (D), and the minimally changed EpoR LFFPL (E). Values shown represent the average of 3 independent experiments each done with 3 biological repeats ± standard error of the mean. ***P < .001. Statistical analysis (jmp pro12) was performed by using the nonparametric multiple comparisons Steel test with a control group (±1 µg/ml Eltrombopag; ±10 U/ml Epo; ±10 ng/ml Tpo).