Transmembrane orientation and extracellular juxtamembrane residues of GP130 are critical for signal transduction

Abstract Glycoprotein 130 (GP130) is an archetypal class I cytokine receptor that serves as a shared signalling subunit for a group of cytokines known as interleukin 6 (IL-6)-type cytokines. GP130 does not have an intrinsic kinase activity and relies on Janus kinases (JAK) 1, 2, or Tyrosine kinase 2 (TYK2) for phosphorylation of tyrosine residues on its cytoplasmic domain for signal transduction. In this study we investigated the molecular mechanism of GP130 activation. We utilized GP130 constructs that were held in varying states of activation by replacing the entire extracellular region of the receptor with a leucine zipper dimerisation domain and inserted alanine residues into the extracellular juxtamembrane linker sequence to induce rotation of the transmembrane domains. These chimeras differentially activated the associated JAK2 molecules which also correlated with receptor tyrosine phosphorylation status. Förster resonance energy transfer (FRET) microscopy in live cells was used to show that GP130 cytoplasmic domains were in close proximity in active chimeras compared to the inactive receptors, presumably to facilitate JAK protein activation. Coarse-grained molecular dynamics simulations provided a structural rationale for this register-dependent mechanism, showing that active chimeras preferentially maintain compact transmembrane packing and reduced membrane-proximal cytosolic spacing, whereas signalling-deficient constructs sample more heterogeneous transmembrane geometries and increased cytosolic separation. Finally, mutational analysis identified the negatively charged juxtamembrane residues E617 and E619 as critical for signal transduction; charge reversal at these positions in the full-length receptor generated a dominant-negative GP130 variant devoid of signalling capacity.