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. 2021 Apr 28;220(7):e202009179. doi: 10.1083/jcb.202009179

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© 2021 Venkatesan et al.

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Figure 2. — The C-domain of CRT_Del52 confers specificity for Mpl and forms disulfide-linked dimers. (A) Structure of CRT_WT (Protein Data Bank accession no. 6ENY, subunit G; Blees et al., 2017) with a globular domain (yellow, residues 18–203 from N-domain and 301–335 from C-domain), a P-domain (blue, residues 206–300), and an α-helical (residues 336–386) C-domain (red). The C-terminus of the C-domain (pink, residues 387–417) is not resolved in the structure. Sequences of CRT_Del52 C-domain truncation constructs are shown. The lower panel shows indicated C-domain sequences (line 1), secondary structure predictions performed using I-TASSER (Roy et al., 2010; lines 2 and 3; line 3 is the confidence score [conf. score] of the prediction), and the secondary structure based on 6eny or the CRT_Del52 model discussed in this study (line 4). The sequence of the mutant-specific C-tail used to produce the anti-CRT(C_mut) antibody is underlined, and cysteine residues are in large font. (B and D) IP with anti-Mpl antibody or control IP (no antibody marked as lysate + beads) of lysates from HEK293T cells (B) or Ba/F3-Mpl cells (D) expressing Mpl along with his-GB1–tagged C-domain constructs (B and D) or full-length CRT_Del52 (D), as indicated, and subsequent blots with the indicated antibodies. Data are representative of three independent experiments (from two separate transductions for D). (C and F) lysates from HEK293T cells expressing indicated CRT_Del52 C-domain constructs were separated by SDS-PAGE under reducing (12.5% gels) or nonreducing (4–20% gradient gels) conditions and immunoblotted with indicated antibodies. Data are representative of three (C) or one (F) independent experiments (see also panel B for relative expression levels of WT CRT vs. CRT_Del52 C-domain expression). (E) Proliferation of Ba/F3-Mpl cells expressing His and GB1-tagged C-domains or full-length untagged CRT_Del52 or lacking CRT_Del52 (Vec) as indicated in the absence of mouse IL-3. Data are averaged from two separate transductions of Ba/F3-Mpl cells with the relevant viruses in a total of four analyses. Ab, antibody; MW, molecular weight.