Introduction ReceptEUR Network

Helical cytokines are polypeptide mediators with a common three-dimensional structure that elicit a variety of bioactivities through binding to cell surface receptors. Cytokine receptors are single transmembrane proteins that can form homo or heteromeric complexes functionally competent to orchestrate specific cellular programs. These receptors are devoid of enzymatic activity and are coupled to one or more of the four Janus tyrosine kinases (JAK1, JAK2, JAK3 and Tyk2). JAKs are shared among different receptors and signalling specificity is ensured through the formation of unique cytokine/receptor/JAK scaffolds that regulate the accessibility of substrates.
Cytokines and their cognate receptors play a crucial role in blood formation and are major orchestrators of inflammatory and immune defences. Defects in cytokine receptors or in the associated JAKs have been linked to human diseases, such as severe combined immunodeficiencies (SCID), congenital neutropenia, myeloproliferative diseases, leukaemia and others, for which satisfactory therapies do not exist today. The basic principles of cytokine action, i.e. binding to receptors and activation of JAK-mediated signalling pathways, have been uncovered during the last decade. However, recent studies have revealed a much more complex functional relationship between receptor complexes and JAKs. Members of this RTN proposal have contributed significantly to the discovery that JAKs can control intracellular traffic, stability and fate of cognate receptors and are not simple enzymatic switches, as originally thought. Today, the overall picture emerging from the ensemble of these studies is the following. Despite structural homologies and shared signalling effectors, a marked heterogeneity exists in terms of the intracellular routes of cytokine receptors and the action exerted by JAKs on such traffic.
Cytokines and their receptors also have been found to be central in the pathogenesis and pathophysiology of human disease. For instance, in Polycythemia vera (PV), a marked decrease in the level of the thrombopoietin receptor (Tpo-R) on the surface of platelets was described. This decrease in Tpo-R surface expression is due to impaired traffic, as Tpo-R is retained in the ER of megakaryocytes. The ICP has recently been involved in the identification and functional characterization of a mutant form of JAK2 (JAK2 V617F). The V617F substitution is located within a region that negatively regulates JAK2 catalytic activity, as shown by partner OS. The mutation is found on one or both alleles in approximately 90% of PV patients. Identical mutations are also found in Essential Thrombocythemia patients and in a minority of Idiopathic myelofibrosis patients. SC discovered that the JAK2 V617F mutant is constitutively activated and induces Stat activation and factor independence in cell lines. Importantly, JAK2 V617F impairs the traffic of Tpo-R, but not of Epo-R, to the plasma membrane. In another pathologic situation, research at the EMCR revealed that 20% of severe congenital neutropenia patients, while under G-CSF treatment, acquire mutations in G-CSF-R, resulting in a truncation of the membrane-distal cytosolic domain. This truncated G-CSF receptor is highly expressed on the cell surface and hampered in its intracellular routing, and transduces an altered signal that fails to control neutrophil differentiation. Importantly, these mutations are strongly associated with disease progression to acute myeloid leukaemia. Finally, severe combined immunodeficiency syndromes (SCID) represent another example where alterations of a cytokine receptor or a JAK are implicated, a topic that has been investigated extensively at U-TAMP and Q-BELF.
The identification and cloning of cytokines and polypeptide growth factors represent a break-through of modern biomedical science with major clinical benefits. For instance, Epo is now used worldwide for treating anaemia of kidney diseases, cancer and AIDS. Tpo is being tested for stimulating haematopoietic stem cell renewal and platelet formation, while type I IFN (alpha and beta) are used in various types of cancer (e.g., chronic myeloid leukaemia), viral hepatitis and multiple sclerosis. Other examples of clinical application of cytokines are the use of G-CSF in the treatment of severe neutropenia and for mobilizing haematopoietic stem cells for transplantation purposes and interleukin 7 for immune reconstitution after stem cell transplantation. In the area of Epo, Tpo and G-CSF treatment, a major challenge is to prolong ligand activity, to increase the interval between administrations and to reduce dose levels to minimize the risk of neutralizing antibody formation. Another challenge is to overcome Epo and G-CSF refractoriness in myelodysplasia. How the cognate receptors are involved in cytokine clearance is not understood, but prolonging their half-life or promoting their recycling are key aims of industry, which depend on better understanding of receptor traffic.

Overall Objectives
The major overall objective of the receptEUR network is to decipher the mechanisms by which JAKs affect the traffic and consequently the signalling outcomes of a number of cytokine receptors expressed in the haematopoietic and immune systems and uncover the functional consequences in normal and pathophysiological conditions. Moreover, basic findings revolving around the cytokine receptor-JAK scaffold will be exploited in order to ultimately modulate receptor levels, spatial configuration, ligand sensitivity and signalling duration towards therapeutic purposes. These aims will require a long-term and steady sharing of expertise, knowledge, reagents and technologies among the members of the RTN and their close collaborators.

Specific project objectives
(A) To apply a systematic analysis to dissect the molecular basis of receptor traffic, assembly, internalization and sorting and the impact thereof on signalling for selected receptors relevant to the (patho)physiological models.

(B) To identify novel partners for receptors and JAKs during intracellular routing, assembly on the cell-surface and after internalization. With MAPPIT screens, searches for interactors of receptors and JAKs and proteins involved in receptor traffic will be performed. These newly isolated proteins can then be used as baits to identify signalling networks.

(C) To elucidate the molecular defects at the level of cytokine receptor traffic in haematopoietic disorders, i.e., leukemia, severe combined neutropenia, Polycythemia Vera, myeloproliferative disease, severe combined immunodeficiency, rheumatoid arthritis.

(D) To create animal models and to use animal-derived primary cells in which cytokine receptor traffic and assembly as well as cytokine receptor related diseases can be studied.

(E) To obtain assays amenable for screening of small molecule modulators of cytokine receptor traffic and inhibitors of mutant JAK2. Assays will be established for cell-surface assembly, activation and internalization of cytokine receptors (especially the receptors for protein drugs such as Epo, G-CSF, interferons) and for non-ATP-dependent inhibition of JAKs and of their effects on receptor trafficking.