Stan Bastiras

SIGNAL TRANSDUCTION BY THE GROWTH HORMONE RECEPTOR

Abstract It has been proposed that dimerization of identical receptor subunits by growth hormone (GH) is the mechanism of signal transduction across the cell membrane. We present here data with analogs of porcine GH (pGH), with GH receptors (GHR) mutated in the dimerization domain and with monoclonal antibodies to the GHR which indicate that dimerization is necessary but not sufficient for transduction. We also report nuclear uptake of GH both in vivo and in vitro, along with nuclear localization of the receptor and GH-binding protein (GHBP). This suggests that GH acts directly at the nucleus, and one possible target for this action is a rapid increase in transcription of C/EBP delta seen in 3T3-F442A cells in response to GH. This tyrosine kinase-dependent event may be an archetype for induction of other immediate early gene transcription factors which then interact to determine the programing of the subsequent transcriptional response to GH.

Efficient Recombinant Production of the 16 Amino Acid Peptide AOD9604

Introduction Chemical synthesis is currently seen as the method of choice for production of therapeutic peptides. Production through recombinant bacteria is a viable alternative but is not often pursued due to perceived problems with efficiency and proteolysis. BresaGen has developed a “toolkit” approach for production of peptides as insoluble fusion proteins in E coli. Segregation of peptides into insoluble inclusion bodies allows high expression rates and protects the peptides from proteolysis, while simplifying purification. A variety of fusion partners and cleavage mechanisms allow the system to be customized for the peptide in question, which is important in view of the widely-differing properties of different peptides with therapeutic potential. Certain post-translational modifications such as amidation at the carboxy terminal may also be incorporated. Recently, BresaGen has applied this approach to production of AOD9604, a 16 residue peptide being developed by Metabolic Pharmaceuticals (Melbourne, Australia) as a potential anti-obesity drug (Fig. 1). A version of AOD9604 produced by chemical synthesis is presently in Phase II clinical trials. However, in view of the extremely large potential market for this product and the need to keep production costs as low as possible, BresaGen were commissioned to develop a recombinant method for production of this peptide.