Lecture 27- Birge Mechanisms of action of peptide hormones Raymond B. Birge, PhD Learning Objectives •Review basic biology of Insulin/Glucagon in homeostasis •Review receptor classifications (emphasis on GPCRs versus Receptor tyrosine kinases) •Understand basic biological distinctions between Glucagon and Insulin •Understand basic signaling mechanisms between Glucagon and Insulin Signaling and Cellular Communication in Metazoans 600-700 million years ago; origin of multicellular organisms Examples of Hormones I. Peptides and Proteins (Growth factors) II. Fatty acid derivatives (Prostaglandins, Eicosanoids) III. Amino acid derivatives (Thyroxine, Epinephrine) IV. Steroids (Estrogen, Progesterone) Examples of second messengers I. cyclic nucleotides (cAMP, cGMP) II. Lipid products (DAG, phosphoinositol lipids) III. Ions, small molecules (Ca2+, NO, Na+, K+) IV. Modified proteins (phosphorylation, modular (domains) The plasma membrane partitions growth factors and signaling cascades Polar Non-polar Polar 1. Highly specific transmembrane receptor 2. Signal is changed and amplified 3. Cell responds with biological readout and downmodulates initial signals Types of growth factor/target cell interactions 1. Paracrine GF produced by distinct Insulin/ target cell Glucagon/NGF 2. Autocrine GF produced by receptor- EGF/ expressing cell tumors 3. Juxtacrine GF produced by distinct target cell but remains associated with target cell TGF-a 4. Holocrine GF has no signal sequence, released from target cell by unknown mechanism IL’s Paracrine/Endocrine Growth Factor Signaling Examples: Insulin, Glucagon Autocrine Growth Factors Examples: Oncogenic cells (ie Breast cancer cells Overproduce EGFR (Her-2) and EGF. Paracrine Growth factor Signaling Examples: Neurotrophic fcators (NGF, BDNF, GGF) Receptor Classification by Mechanism of Action G Protein-Coupled Receptor (GPCR): 7 transmembrane receptors (Glucagon, ACTH, TSH, LH, Calcitonin, PTH, Thrombin, Dopamine etc) Receptors with Intrinsic Enzymatic Activity (Type I or Type II): (Insulin, EGF, NGF, FGF, VEGF, NT-3, BDNF, etc) Receptors with Associated Enzymatic Activity: (Cytokines, EPO, TGF- , BMPs, Interferons) Receptors that Stimulate Intracellular Proteolysis: (IL-1, TNF- ) How are signals propagated through cells? 4 Distinct Elements: 1. Signal. Generation, processing, and release of growth factor 2. Signal Detection. Surface receptor/cytoplasmic receptor 3. Signal Transduction. (PM -> cytoplasm -> nucleus) 4. Alteration in gene expression. Biology of Insulin and Glucagon In maintenance of blood sugar Insulin: Secreted by beta cells of the pancreas in response to high blood sugar. In response to insulin, cells (muscle, red blood cells, and adipocytes) take up glucose from the blood. Glucagon: Secreted by alpha cells of the pancreas in response to low blood sugar. When blood glucose is high, no glucagon Is secreted When blood glucose is low, glucagon causes the liver to release stored glucose into the blood. 10-10 10-10 M 10-7 cAMP ATP PKA P phosphorylase kinase P phosphorylase P glycogen synthase 10-6 Signal; ie Insulin Kinetics of induction of immediate and delayed gene expression following growth factor stimulation Figure 6.2 The Biology of Cancer (© Garland Science 2007) Table 6.1 The Biology of Cancer (© Garland Science 2007) Figure 6.3 The Biology of Cancer (© Garland Science 2007) Receptor Tyrosine Kinases Receptor protein-tyrosine kinases transmit signals across the plasma membrane, from the cell exterior to the cytoplasm. In cells transformed by many oncogenic proteins-(ie, ErbB, EGFR, Bcr-Abl) there is dramatic increase in protein tyrosine phosphorylations PROTEIN KINASES Each kinase phosphorylates particular sequence motifs Phosphorylation is not restricted to one site on the polypeptide chain Proteins can be phosphorylated by more than one kinase (allows convergence of several signaling pathways) Phosphorylation can effect enzymatic activity * alter electrostatic interactions and alter equilibrium from one conformation to another * instrumental in forming new interactions (ie, H-bonding, SH2 domains) * expose domains or motifs buried in the protein structure (ie NLS and NES) Consensus amino acids can be used to identify kinase specificity Polypeptide Growth Factors activate Receptor Tyrosine kinases (RTK) by a common mechanism GF-induced activation of a Receptor Tyrosine Kinase. 1. Ligand-induced dimerization 2. “Auto-phosphorylation” of the RTK on the cytoplasmic or intracellular domain. 3. The dimeric state of the receptor is said to be “activated” Figure 6.8b The Biology of Cancer (© Garland Science 2007) The pocket of the SH2 domain is highly specific for phospho-tyrosine because of the bulky size of the tyrosine. SH2 specificity is achieved by amino acids directly C-terminal to the pTyr (particularly +1, +2, and +3). GF-induced activation of a Receptor Tyrosine Kinase-part 2. 4. Phosphorylation on tyrosine acts as a “magnet” to recruit cellular proteins with Src Homology-2 (SH2) domain. 5. One of these proteins is called Grb2 (Growth Factor Receptor Binding protein-clone-2), an intracellular adaptor protein with SH2 and SH3 domains. 6. Simultaneously, Grb2 binds the activated RTKAND SOS, an exchange factor for the G protein, Ras to provide a link in the signal transduction. GF-induced activation of a Receptor Tyrosine Kinase-part 3. 7. Because Grb2 recruits SOS to the plasma membrane, it meets up with its partner Ras, resulting in the conversion of Ras-GDP into Ras-GTP. 8. Ras-GTP is activated and binds more signaling proteins, leading to the activation of MAP kinase. 9. Activated MAP kinase is translocated into the nucleus to activate transcription factors. The Ras Cycle Common Ras mutants Ras-GDP (Inactive) Pi GDP V12 Ras= Always active i.e, constitutive activation of downstream pathways N17 Ras=Always inactive i.e, dominant negative for activation of downstream pathways GAP * GTP Ras-GTP (Active) V12,N17=Functionally-dead molecule ie, no effect in downstream pathways The MAP Kinase (Extracellular-Related Kinase ERK) Cascade Mitogens, Growth Factors GTPase Ras MAP4K RA F MAP3K MAPKKK MAP2K MAPK Rac, Cdc42 PAK MAPKK/MEK1 MAPK Cytokines, Cell Stress MEKK1 } Serine/Threonine Kinases JNKK/SEK JNK Transcriptional Regulation Cytosol Nucleus Many growth factor signals terminate via the activation of transcription factors Raf Localization of MAP K Summary and Take-Home Points 1. Identify differences between autocrine, paracrine, and endocrine signaling 2. Distinguish major differences between insulin and glucagon signaling, and differences in how receptors are activated (ie, RTK versus GPCR) 3. Understand RTK signal transduction mechanisms 4. Relationship between RTK activation and gene expression