Centro Infantil Boldrini, Campinas-SP, Brasil
IL7Rα mutations described in T- cell acute lymphoblastic leukemia patients are characterized by insertion of unpaired cysteine in the extracellular juxtamembrane region. The mutant cysteine contributes to aberrant homodimerization of IL7Rα chains, Jak1/Stat5 constitutive signaling and induction of cell proliferation and survival, as previously reported. However, cysteine insertions in the wild type IL7Rα not always lead to signaling, even when dimerization occur. We now aim to better understand the mechanisms of dimerization and signaling of the mutant IL7Rα using site directed mutagenesis and IL7R functional assays. Considering cysteine alignment with IL7Rα intracellular regions involved in signal transduction, we constructed 13 different artificial mutants but only 3 triggered constitutive signaling. 241insCA showed the strongest signaling results and conferred proliferative advantage to BaF3 cells. In the other hand, 10 clinical mutants with cysteine in disparate positions were found functional in terms of constitutive signaling. Dimer projections of clinical mutant transmembrane sequences, using PREDDIMER, suggested alternatively the presence of a dimerization motif based on serine, SxxSxxS, aligned with the cysteine. However, substitutions of two or each of the three serines by alanine or isoleucine in the clinical mutant did not abrogate the constitutive signaling. Interestingly, prolines are the second most common amino acid inserted in IL7Rα clinical mutants. Proline substitution by alanine resulted in decreased constitutive JAK/STAT activation. Reintroduction of proline in different positions, either N- or C-terminal to cysteine restored the receptor signaling. In summary, our results suggest that besides cysteine alignment and possible presence of transmembrane dimerization motifs, other conditions have to be better investigated. Cysteine neighboring amino acids like prolines and their structural characteristics may be important to the dynamics of dimerization and signaling of mutant IL7Rα.