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thropoietin-EPOR complex is then internalized in a process dependent on Tyr-454, -456 and -504 and the p85 subunit of phosphatidylinositol 3 kinase, and targeted to the lysosomes for degradation (Figure 6A).30,31,35 The loss of these negative signaling regulatory and degradation domains due to EPOR truncation is usually thought to explain erythropoietin hypersensitivity in PFCP.6,7,14,27,29- 31,36,40 In the present study, we studied two extensive trun- cations, p.Glu399* and p.Glu425*, which lack seven of the eight conserved tyrosine residues (Table 1, Figure 6B) and all sequences that constitute the EPOR negative reg- ulatory domains. These mutants conferred greater ery- thropoietin hypersensitivity to Ba/F3 cells compared to EPOR WT. Thus, confirming previous reports, we showed that extensive truncations lacking all EPOR neg-
A
ative regulatory sites are sufficient in themselves to induce the PFCP phenotype.
Our results highlight that a different mechanism under- lies erythropoietin hypersensitivity due to frameshift EPOR mutations. The study of EPOR p.Gln434Profs*11 (EPOR FS) and EPOR p.Pro438Metfs*6 and their designed nonsense counterparts, EPOR p.Gln444* (EPOR STOP) and EPOR p.Pro434*, respectively, allowed us to discrimi- nate between the effects due to the truncations them- selves leading to the loss of SHP-1 and SOCS3 binding sites and those due to the appearance of a new cytoplas- mic tail. In accordance with the loss of negative regulatory sites and previous reports,9,14,27,29 EPOR FS and EPOR STOP Ba/F3 cells displayed persistent phosphorylation of STAT5 after erythropoietin removal, but only EPOR FS was able
BCD
Figure 6. Regulation of EPOR signaling and positions of the EPOR truncations. (A) Negative regulators of EPOR signaling and their binding sites in the C-terminal part of the receptor: negative signaling regulators (on the left) and internalization/degradation sites (on the right). The tyrosine (Y) number of the mature EPOR is also indicated in parentheses. (B) Proximal truncations due to EPOR p.Glu399* and p.Glu425* lack all negative regulation sites of the receptor. (C and D) More dis- tal truncations due to frameshift EPOR mutants and their designed counterparts retain the Tyr-426.
haematologica | 2018; 103(4)