Regulation of GHR endocytosis: Understanding the GHR-βTrCP interaction

Upon binding of the Growth Hormone (GH) to the GH receptor (GHR) dimer, a multitude of signalling cascades are triggered that affect cell proliferation, differentiation, and metabolic pathways. The availability of GHR at the cell surface, and consequent responsiveness of cells to GH, can be modulated by tightly controlling the endocytosis of the receptor. The ubiquitin ligase SCF(βTrCP) is required for GHR endocytosis. βTrCP binds via its WD40 domain to the Ubiquitin-dependent Endocytosis (UbE) motif, DDSWVEFIELDIDD, present in the cytoplasmic tail of GHR. The goal of the present study is the molecular and functional characterization of the interaction between GHR and βTrCP. βTrCP interacts with its classical substrates (as IκB, and β-Catenin) via the degron, DSGxxS, when both serine residues are phosphorylated. Ongoing studies show that βTrCP acts on GHR predominantly via its UbE motif, although a DSGxxS motif downstream in the tail plays a role as well. We focus our research on the interaction via the UbE motif, of which the sequence differs substantially from the well characterized motif present in classical βTrCP substrates. In vitro binding assays showed a specific and direct interaction between the UbE motif of GHR and βTrCP. Purified SCFβTrCP is able to ubiquitinate GHR in vitro. Using NMR and binding studies we identified the amino acid residues that contribute to the UbE-βTrCP interaction. A special role for serine 323 is currently under investigation. The results so far show that UbE-βTrCP interaction is unconventional and specific and therefore it will be regarded as a potential drug target. by specifically disrupting this interaction, the endocytosis of GHR will be downregulated, the number of GHRs at the cell surface increased, and GH sensitivity improved. This drug might be used to treat conditions of decreased GH sensitivity such as cachexia, which occurs in cancer and AIDS patients, and it is characterized by extensive lost of muscle mass and decreased quality of life. Based on this principle, a robust drug screening was developed, which resulted in promising hits for future therapeutic development against cachexia.