Mechanism of activation of the cystein-mutant and wild type IL7R receptors

Abstract

IL-7Ra is a transmembrane receptor that lacks kinase activity. Signal transduction by the IL-7R± is dependent on the associated protein tyrosine kinase JAK1 (Janus kinase 1), anchored to the intracellular domain of the receptor. IL-7R± forms a heterodimer with the gamma chain transmembrane receptor, which brings JAK3. Signaling occurs only in presence of IL-7, when JAK1 and JAK3 trans-phosphorylate each other leading to STAT activation. It has been hypothesized that upon IL7 binding, the receptor alpha and gamma chains approximate to a distance of 30Å enabling JAK1/JAK3 trans-phosphorylation and signal transduction. However, the mechanism of IL7R signaling, i.e. alignement and movement of interacting IL-7Ra and gamma chains is still largely unknown. Unlike the wild type receptor, leukemia-associated cysteine mutants of the IL-7Ra form homodimers by intermolecular disulfide bridges and signal constitutively in absence of IL-7, gamma chain, or JAK3. This simplified version of the IL-7 receptor can help understand the structural and dynamical features governing IL7R signaling. Moreover, it could lead to the development of new anti-leukemia drugs. Recently, Brooks and colleagues (2014) described in details the dimerization and signaling processes of the Growth Hormone Receptor (GHR- another member of the class I cytokine receptors). They showed that the homodimeric receptor chains display a clothespin-like movement upon GH binding, leading to the sliding apart of their anchored JAK2 subunits, so releasing their mutual repression and allowing their activation and signaling. In leukemic IL7R mutants some cysteine positions are preferred and they are grouped at the same side of the a-helix in Helical Wheel predictions indicating an interaction interface between the two TM a-helices of the homodimer. By cystein scaning experiments we found that simple insertions of cysteine in the transmembrane (TM) sequence of IL-7Ra are not enough to confer constitutive signaling, even though homodimerization occurred. This indicates that homodimerization per se is not enough for signaling to occur. Cysteine insertion has to be finely positioned so that interacting a-helix chains are correctly aligned. We experimentally excluded the presence of a TM serine dimerization motif but a full alanine scanning is being conducted in order to investigate hidden TM dimerization motifs. Additionally, we demonstrated the importance of the proline near the mutant cysteine. Proline is the second most common inserted residue in the IL7Ra natural mutants. Substitution of proline abrogates IL7R constitutive signaling and re-insertion of proline, in different positions near cysteine, rescues the signaling capacity. By FRET experiments we corroborated that intracellular domains of productive interacting chains approximate to a distance of 45Å and found evidence that lipid rafts are important in this process. We still do not know how JAK1 molecules move and are activated upon receptor homidimerization. We now propose to use tools, in vitro models and insights from the GHR, to extend our analysis on the functioning of the mutant and wild type IL7R (AU)