Understanding the Growth Hormone Receptor-βTrCP interactions: Molecular Tools for controlling Growth Hormone Sensitivity

Abstract

Growth hormone (GH) signaling plays major roles in the organism and, therefore, the sensitivity of the cells to GH and the signaling duration are under a tight control. GH receptor (GHR) endocytosis is one of the main mechanisms that determine the amount of GHRs at the cell surface, and allow the cells to regulate GH signaling. In this thesis we characterized the interaction between GHR and βTrCP, an essential factor that mediates ubiquitination events necessary for GHR endocytosis, and explored the potential of this interaction as a tool for manipulating the sensitivity of the cells to GH. We showed that βTrCP binds two motifs in GHR: the previously described unconventional UbE motif, and the downstream DSGRTS sequence, that corresponds to a consensus degron (DSGxxS) present in other βTrCP substrates. Interestingly, we found that while the UbE motif is necessary for both basal and GH-induced endocytosis, the DSGRTS sequence is only necessary for basal endocytosis. Therefore, besides the UbE motif, the DSGRTS has a crucial importance in the regulation of the cellular levels of GHR, and consequently the responsiveness of the cells to GH. Furthermore, we characterized in detail the interaction between βTrCP and the UbE motif. We showed that βTrCP ubiquitinates GHR in vitro in an UbE motif-dependent manner. The amino acid residues in βTrCP and UbE motif necessary for the interaction were determined and used together with NMR data, in the computation modeling of the interaction. This revealed that the interaction βTrCP-UbE motif is unconventional. Additionally, we found that the affinity of the βTrCP-UbE motif interaction can be regulated by phosphorylation of the Ser323 in the UbE motif. This suggests that Ser323 phosphorylation could be a mechanism of increasing the rate of GHR endocytosis, in response to certain stimuli, such as GH or other stressors. Cachexia, condition occurring in severely ill patients and characterized by extensive muscle loss, is frequently associated with a GH resistance phenotype. We proved that tumor-bearing mice suffering from cachexia have reduced amount of GHRs in the liver. This is potentially the mechanism leading to the GH resistance phenotype in cachexia patients. The unconventional interaction βTrCP –GHR is a potential target for discovery of drugs that, by interfering with it, inhibit GHR endocytosis, and as such, increase the number of GHRs at the cell surface and consequently improve GH sensitivity of the cells. Cachexia patients would therefore benefit from such a drug. Based on this, we developed a robust screening route to find potential lead compounds for future therapeutic development against cachexia. The findings of this thesis represent a significant progress in the understanding of the molecular mechanisms regulating GHR endocytosis, with focus on the translation of this knowledge into drug discovery, for the benefit of patients with chronic diseases suffering from cachexia.