Endocrinology 10 - JAK/STAT Signalling Today we are looking at how the GH signal transduction pathway for those physiological pathways occur Growth Hormone Synthesis - A peptide (191 amino acids long) - Molecular weight of 20kDa A. gene transcription Nucleus B. mRNA to RER C. translation on RER Rough ER D. post-translational processing E. packaging into Golgi secretory vesicles F. exocytosis - Gene expression in the nucleus and gene transcription mRNA is moved to the RER - And is translated there - Synthesised as a 217 amino acid pre-protein (191 + 27 aa single peptide) - This signal peptide anchors the pre-protein with thin the endoplasmic reticulum o So that peptidase (that is ER membrane bound) which is going to cleave that pre-protein at position 26-27) - Produces the mature peptide that in 101 on Inna Structure of the Growth Hormone: - Single polypeptide chain - Folds into a four alpha helical bundle o Glycine at position 120 which sits on the end of one of the helices o This amino acid is absolutely essential for GH function GH Receptor - Belongs to the cytosine Class 1 receptor - Extracellular domain; o 4 conserved cysteine residues (across the family is in the same place) o 5 peptide amino acid motif (WSXWS) - Transmembrane domain; hydrophobic region of the cell membrane - Intracellular domain; o No intrinsic kinase activity o Region that binds to a certain class kinase enzyme; JAK protein - GH belong to the homomeric receptor group o All components of that complex are the same molecule (all subunits are the same)
- Binding of GH to its receptor induces receptor dimerization o Two receptor molecules come together o Activation occurs - We need two because of the JAK proteins which sit associated with the intracellular tail - Once they are brought together, they will activate each other JAK Proteins - Janus Activated Kinases (JAK) - In the N-terminal region we have Janus homology domains . They allow JAK proteins to bind to the tail of the receptor - In the C-terminal, there is the kinase domain o Enzymatic activity - In between two, we have the pseudo-kinase domain o Regulates the activity of the kinase domain Tyrosine Kinases - Protein tyrosine kinases - catalyse the transfer of phosphate groups from ATP to tyrosine residues within protein substrates o By sticking this phosphate groups, it makes a conformational shape Affects the structure/stability/activity o Such as SH2 domains - Phosphorylating tyrosine's creates binding sites for other proteins JAK Phosphorylation - Experiment which shows us the phosphorylation event - 3T3 F442A (pre-adipose) cells - Exposed to hGH - Western blot of protein extracts - Antibody only recognises phosphorylated tyrosine residues STAT proteins SH2 domains: - Signal Transducer and Activator of Transcription - Found in wide range of signalling molecules. NH2 Coiled-coil transcriptional activation DNA-binding Linker SH2 Y COOH 1 C-terminal - SH2 domains allow proteins that contain them to bind to phosphorylated tyrosine residues on other proteins. binds to DNA response