Cytokine receptors play a pivotal role in many cellular processes, including metabolism control, growth, neural stem cell activation, inflammatory responses, bone, and blood cell development. Cytokine receptors do not possess intrinsic kinase activity, however, they bind Janus kinases (JAKs) via a conserved intracellular proline rich Box1 motif and a less conserved Box2 region. The growth hormone receptor (GHR) and GP130 are archetypical members for short and tall class I cytokine receptors, respectively. GP130 has important roles in cancer and inflammation as illustrated in mouse models harbouring mutations in the SOCS (suppressors of cytokine signalling) binding region of GP130, resulting in hyper-active STAT1/3 signalling promoting gastric cancer incidences. Moreover, small in-frame activating deletions in domain 2 of GP130 have been identified in 60% of patients with inflammatory hepatocellular adenomas, making GP130 D2 mutants hepatic oncogenes. Although numerous studies have investigated the structure of the GP130 receptor extracellular complex, little is known about its activation mechanism and regulation of signal transduction.Elucidation of the structure of JAK2 bound to an archetypal cytokine receptor would greatly enhance the understanding of how these receptors activate their associated JAK2. A recombinant protein expression method was optimised for the GHRBox1-2 fused to JAK2FERM-SH2 in order to delineate the mechanism of GHR recognition by its cognate JAK partner. This GHRBox1-2-JAK2FERM-SH2 fusion protein resulted in a high purity monodispersed protein. Identity and appropriate folding of the purified protein was validated using mass spectrometry and circular dichroism spectroscopy, respectively. Although the purified GHR/JAK2 protein had acceptable purity, crystallisation trials did not results in sizable crystals suitable for X-ray crystallography. Purified protein was subjected to NMR studies by outcompeting GHR in the complex by 15N-labelled GHR-ICDmp. Despite low intensity NMR signal, it was clearly demonstrated that Box2 residing amino acids, including Val343, Glu344, and Leu348 along with Glu320 in GHR interbox region are involved in interactions with JAK2.Multiple cytokine receptors including GHR and GP130 have been shown to interact and activate the SFK member, LYN. GHR has been shown to bind to LYN and activate an alternative signalling pathway independent of JAK/STAT. In similar attempts, LYN and GP130 interaction was investigated using various GP130 mutants/truncations were generated and their association with LYN was analysed by co-immunoprecipitation. Recurrent non-specific binding and time constraints hampered the conclusion from these experiments. However, it was evident that GP130 expression was negatively regulated by LYN.To analyse the signal transducing orientation of GP130, the extracellular domain of the receptor was exchanged with the leucine zipper dimerisation domain of the c-JUN transcription factor to generate GP130 dimers on the cell surface of the pro-B cell line, Ba/F3. The effect of length, charge, and rotation of the GP130 extracellular juxtamembrane region was investigated resulting in identification of active and inactive receptor configurations. Firstly, it was demonstrated that negatively charged glutamic acid residues (Glu617&619) are important for appropriate receptor function and substitution of these residues to oppositely charged residues abrogates receptor activation. Variable rotations of the GP130 transmembrane and intracellular domains induced by three or four alanine insertions mimicking cytokine-induced activation led to differential activation of JAK/STAT and MAPK signalling pathways and generated distinct proliferative responses in Ba/F3 cells. Conversely, insertion of two alanine residues resulted in inactive receptor configuration that neither activated downstream signalling pathways nor conferred factor independent proliferation to its expressing cells. Active and inactive configurations of GP130 were further analysed by FRET (Förster resonance energy transfer) reporters to assess repositioning of the intracellular domains of GP130 with associated JAK1 molecules. FRET analysis in live cells showed that in the constitutively active chimeric GP130 receptors, the intracellular Box2 regions (FRET reporters placed following Box2 motifs) are in close proximity compared to an inactive chimeric GP130 receptor. In addition, the GP130 intracellular Box2 motifs were brought closer to JAK1 kinase domains in active GP130 receptors compared to inactive receptors. Based on these findings, a model was proposed where GP130 activation results in apposition of membrane proximal regions, aiding disruption of the JAK1 autoinhibitory interaction, and facilitating JAK1 transphosphorylation and activation. In this model, the JAK1 kinase domain is in close proximity of the GP130 cytoplasmic domain in the activated receptor presumably to phosphorylate tyrosine residues.Super resolution microscopy with single particle tracking photo-activated localization microscopy (spt-PALM) of GP130 inactive and active models (photoconvertible mEos2 fluorescent protein was fused to GP130 chimeras after Box2 motif) was performed to analyse diffusion properties of chimeric leucine zipper-GP130 receptors within cell membranes. Despite their divergent activation status, inactive and active models displayed similar mobility and equal ratio of mobile to immobile receptors. It was speculated that the lack of cytoplasmic region responsible for internalisation or recruitment of signalling molecules (e.g., STAT1 and STAT3) was responsible for similar movements by these receptors. Preliminary spt-PALM analysis of wild-type GP130 demonstrated that this receptor has a more pronounced bimodal mobility and moves slower on the membrane compared to chimeric receptors possibly due to the bulkier extracellular domain. Furthermore, it was illustrated that GP130 stimulation by the designer cytokine HiL-6 (Hyper-IL-6, IL-6 fused to the soluble IL-6Rα) increases receptor mobility and reduces the ratio of mobile to immobile receptors. This is the first study reporting the cell membrane mobility patterns of GP130 receptors and the effect of cytokine stimulation on those movements.In this study, it was demonstrated that similar to short cytokine receptors, relative orientation of the transmembrane domain of the GP130 plays an important role in signal transmission upon receptor activation.