Poster Presentation 2019 Hunter Cell Biology Meeting

THE MOLECULAR INTERACTION OF SRC FAMILY KINASES WITH THE GROWTH HORMONE RECEPTOR AND ITS ROLE IN LIFESPAN EXTENSION AND LIVER REGENERATION (#115)

Yash Chhabra 1 , Pernille Seiffert 2 , Helena Steinocher 2 , Rachel Gormal 3 , Helle Bielefeldt-Ohmann 4 , Mayumi Ishikawa 5 , Manuel Fernandez-Rojas 6 , Louise Nikolasjen 2 , Frederic Meunier 3 , Birthe Kragelund 2 , Michael Waters 5 , Andrew Brooks 1
  1. University of Queensland Diamantina Institute, Woolloongabba, QUEENSLAND, Australia
  2. Department of Biology, University of Copenhagen, Copenhagen, Denmark
  3. Queensland Brain Institute, The University of Queensland, St Lucia, Queensland, Australia
  4. School of Veterinary Science, The University of Queensland, Brisbane, Queensland, Australia
  5. Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland, Australia
  6. Madrid Institute for Advanced Studies (IMDEA) in Food, Madrid, Spain

Emerging evidence suggests that GHR can signal through pathways additional to JAK2 tyrosine kinase, as has been reported in patients with GH resistance that exhibit normal JAK2-mediated STAT5 but impaired ERK activation. One such pathway involves the Src Family Kinases (SFK). The signalling choice between JAK2 and LYN kinase (a SFK member) by GHR can be modulated by realigning the receptor lower cytokine module in its extracellular domain. Existence of GHR-mediated SFK signalling in vivo has been demonstrated in a targeted Box1 motif mutant mouse model allowing complete abrogation of GHR-mediated JAK2/STAT signalling, but the mechanism of activation and its downstream effects remain elusive. In order to determine the structure-function attributes of GHR-mediated LYN activation, we have used co-immunoprecipitation to define the region in the GHR intracellular domain that interacts with LYN and vice versa and complemented this with interaction analysis by NMR. Using super resolution microscopy to track single molecule movement on the cell surface, we have identified differences in GHR mobility owing to JAK2 vs. LYN binding. Our results indicate that GHR binding to LYN is constitutive, independent of the Box1 motif and unconventional with the interaction site residing in well conserved region between the Box1 and Box2 motif of GHR in the potential lipid interacting domain. We found that cellular levels of JAK2 and LYN compete for binding to GHR and the relative amounts of JAK2 or LYN confers altered signalling. In contrast to JAK2, LYN caused a striking decline in GHR expression with differential ERK1/2 activation kinetics. Hepatic microarray in animal models designed to alter the balance of GHR-mediated signalling, has revealed cohort of genes regulated primarily by GHR-mediated SFK signalling. This study provides insight into the mechanism by which GH-stimulated LYN-ERK signalling is regulated and its physiological relevance in extending lifespan, and clinical relevance to liver regeneration, in Box1 mutant mouse models.