Mechanism of hormone action

1. Mechanism of hormone action

2. Hormones • Three types • Proteins • Glycoproteins • Small pepstides • Large proteins • Lipids • Cholesterol derivatives • Eicosanoids • Amino acid derivatives

3. Hormones • Innate by themselves • Require mediation • Receptors • Binding sites for a hormone • Very specific

4. Hormone receptors • Two types • Transmembrane • Intracellular/nuclear • Proteins regardless of the type • Interaction between a hormone and a receptor • Initial step of hormone action

5. Transmembrane receptors • Protein hormones • Unable to pass through the plasma membrane • Size • Charges • Receptors must be located on the plasma membrane • Extracellular domain for interaction with hormone • Intracellular signaling system

6. Types of transmembrane receptors • Receptors with multiple transmembrane domains • Seven trans-membrane domain receptor • No intrinsic enzymatic activity (C-terminus) • Associated with intracellular proteins involved in signaling • G-proteins • Modification of extracellular domain (hormone binding site, N-terminus) • Glycosylation • Crucial for hormone binding

8. Trans-membrane domains (7) • Alpha-helix • Hydrophobic amino acids • Loops • Connect alpha helices • May be linked by disulfide bridges (extracellular loop 1 and 2)

9. Intracellular/cytoplasmic domain • Palmitoylation of some cysteine residues • Attachment of fatty acids • Fourth loop • Site for phosphorylation

10. General structure of seven trans-membrane receptor • Variations • Amino acid sequences • Variable length of N-terminus • Affects binding of ligand/hormone

12. Intracellular signaling • Generated when a hormone interacts with extracellular domain of the receptor • Conformational change within the trans-membrane helices • Exchange of GDP to GTP on the alpha-subunit of G-protein complex • Activation of Ga subunit • Dissociation of activated Ga from G-protein complex (bg)

14. Second messengers • Cyclic nucleotides (cAMP and cGMP) • cAMP • Widely used secondary messenger • Generated by adenyl cyclase • Activated by activated Ga subunit of G-protein complex • Activation of cyclic nucleotide-dependent protein kinases • Protein kinase A (cAMP)

15. Secondary messengers • Amplification of hormonal signals • Binding of hormone to the receptor • Activation of adenyl cyclase by activated Ga • Activation of protein kinase A by cAMP • Rapid clearance and inactivation • Phosphodiesterases • Inhibited by methylxanthines (caffeine, theophylline, and theobromine) • Phosphoprotein phosphatases

17. How do we know that cAMP is a secondary messenger? • Changes in production of cAMP after hormonal treatment • Correlation between amount of cAMP being produced and cellular response to the hormone • Inhibition of phosphodiesterase activity • Presence of ligand but no effects • Treatment with cAMP analogues/agonists • Similar response to that of hormone

18. Types of G-protein complex • Ga subunit (20 different types) • Gs (stimulatory Ga) • Gi (inhibitory Ga) • Go (associated with orphan receptors in neurons) • Gt (transducin found in retina, activates cGMP-specific phosphodiesterases) • bg complex • 4 or more

20. Identification of specific G-protein complex associated with particular receptor • Structurally similar to each other • Use of pertusis toxin (bacterial toxin) • Uncoupling of G-protein complex from the receptor • Gi is very susceptible

21. G-protein complex coupled with secondary messenger system other than cyclic nucleotides • Generated through phospholipid metabolism • Inositol triphosphate (IP3) • Diacylglycerol (DAG) • Arachidonic acid • Activation of phospholipase C (PLC) by activated Ga

22. IP3 • Water-soluble • Stimulate release of Ca • DAG • Binds to protein kinase C • Activated by elevated Ca

23. Medical importance • 65 % of prescription drugs target G-protein coupled receptors • Variety of ligands

24. Other protein hormone receptors • Transmembrane receptors with intrinsic tyrosine kinase activity • Receptor tyrosine kinase • Receptors for insulin and many growth factors • Transmembrane receptors with associated tyrosine kinases • Cytokine receptors • Receptors for growth hormone and prolactin • No intrinsic kinase activity • Interaction between receptor and hormone causes recruitment and activation of tyrosine kinases associated with receptor

25. Receptor tyrosine kinase • Approximately 100 receptor tyrosine kinases in human • Highly conserved • Domains • Extracellular • Hormone binding site • Transmembrane • Intracellular/cytoplasmic • Tyrosine kinase activity

26. 16 subfamilies • Based on extracellular domain • Variation on extracellular domain • Interaction with variety of factors • EGF, PDGF, and insulin

27. Activation of receptor • Dimerization • Dimeric ligand (two subunits) • Each subunit binds to a receptor • Two binding sites within a hormone • One hormone interacts with two receptors

28. Activation of receptor • Pre-existence as a dimer • Receptor is a dimer • Activated through interaction with ligand

29. Activation of receptor • Conformational changes in the kinase domain • Accessible to the substrate • Autophosphorylation of tyrosine residues (3 in insulin receptor) • Activation loop • Triggers conformational changes • ATP binding • Interaction with intracellular proteins • Phosphorylation of other proteins