Cellular Signaling Mechanisms Receptor Tyrosine Kinases Cytokine Receptors Nuclear Receptors

1. Cellular Signaling Mechanisms Receptor Tyrosine Kinases Cytokine Receptors Nuclear Receptors Death Receptors Phar 735/590 Winter 2006 Mark Leid Office: Pharmacy 407 Tel: 737-5809 E mail: Mark.Leid@oregonstate.edu

2. Objective • To understand the structural and mechanistic basis for cellular signaling involving: • Receptor Tyrosine Kinases (e.g., insulin receptor) • Cytokine receptors (e.g., interleukin receptors) • Nuclear receptors (e.g., steroid hormone receptors) • Death receptors (receptors mediating apoptosis) • This material will form the cornerstone for understanding the pharmacological basis of therapeutics with regard to agents acting in the above pathways. …so keep your mind open.

3. Funkadelic: Free Your Mind… (1971; Westbound Records) The purpose of this handout is to free your hand and mind so that you may participate in class more fully.

4. Signaling on an Intermediate Time Scale I. Receptor tyrosine kinases (RTKs) • Examples • Insulin Receptor • Insulin-like GFRs • Platelet-derived GFR • Epidermal GFR • Fibroblast GFR • RTKs span plasma membrane only once • Receptor harbors intrinsic tyrosine kinase activity that is activate by GF binding • The signaling pathway involves phosphorylation of cytoplasmic substrates by carboxyl terminus of RTK • Phosphosphorylation alters substrate's activity • Required for growth and development • Involved in metabolic and mitogenic processes • Implicated in pathological processes

5. RTK Superfamily

6. Regulation of glucose homeostasis • Insulin production: B cells of islets of Langerhans (60-80% of cells there are B cells). • Species differences in islet architecture • In general, four peptides with hormonal activity are secreted by the islet cells: • Insulin (beta or B cells; stimulates glucose uptake) • Glucagon (A cells; stimulates glycogenolysis and gluconeogenesis primarily in liver, both of which increase BS) • Somatostatin (D cells; negatively regulates A and B cell secretions) • Pancreatic polypeptide (F cells) • Insulin secretion is stimulated by glucose (alters the ATP/ADP ratio in a cell, blocks ATP-sensitive K+ channels (Kir6.2/SUR1), depolarizes the cell, opens voltage-gated Ca++ channels causing exocytosis).

7. ATP-sensitive K+ channel (Kir6.2/SUR1)

8. Y Y I I I ATP Y Y * * PY YP * * Intermediate Signaling--RTKs F. Signaling cascade • Growth factor binding to extracellular domain leading to; • Conformational change in protein resulting in; • Activation of the tyrosine kinase on the cytoplasmic face of the receptor, leading to; • Receptor autophosphorylation. Tyrosine Kinase

9. Sample Question Which of the following best describes the mechanism of action of Glipizide (Glucotrol, a sulfonylurea) in the treatment of non-insulin-dependent diabetes mellitus? A. Glipizide opens ATP-dependent K+ channels in pancreatic  cells and thereby enhances insulin secretion. Glipizide blocks ATP-dependent K+ channels in pancreatic  cells and thereby enhances insulin secretion. Glipizide directly increases expression of GLUT4, the insulin-sensitive glucose transporter in target tissues. D. Glipizide blocks absorption of carbohydrates from GI tract. E. Glipizide activates insulin receptors in target tissues

10. ATP Y I I I I YP YP PY PY YP YP PY PY YP YP * * * * * * * * Substrate with altered activity Intermediate Signaling--RTKs G. Signaling cascade 5. Cytoplasmic substrates bind to phosphotyrosine on RTK and are phosphorylated by the activated tyrosine kinase, which; 6. Alters the activity of the substrates (positively or negatively) 7. Signal terminated by receptor internalization

11. RTK-Mediated Transcriptional Regulation Glucose Insulin Secretion IR Activation Substrate Phosphorylation GLUT4-mediated glucose uptake

12. Sample Question • A. Inducing translocation of GLUT-4, the insulin-sensitive glucose transporter, to the plasma membrane • B. Blocking ATP-dependent K+ channels in pancreatic  cells and thereby enhances its own secretion • C. Activating MAP kinase • D. Inducing insulin receptor internalization • E. Inducing insulin receptor translocation to the nucleus Insulin stimulates glucose uptake in sensitive tissues by:

13. RTK Signaling Pathways

14. T Cells B Cells NK Cells CLP HSC RBC Megakary. Mono-Mac Neutro Mast Eosinophil CMP Intermediate Signaling II. Cytokine Receptors • Family members • Interleukins • Interferons • Erythropoietin • GM-CSF • TNFa • Leptin • Growth Hormone • Play key roles in immune system and hematopoiesis

15. Cytokines: mediators of immune system signaling

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17. Common Myeloid Precursor IL1, IL3, IL6, SCF, GM-CSF, IL12 CFU-GEMM IL3, SCF, GM-CSF, IL5, IL6, IL11, IL12, LIF, Epo, IFNg, IL4, Inhibin, TGFb CFU-GM (CFU-C) IL3, GM-CSF, G-CSF IL1, IL3, IL4, IL5, IL6, IL9, IL11, IL12, TGFb , SCF, LIF, bFGF, M-CSF, RA, Chemokines, AcSDKP, pEEDCK, TNFa/b, Inhibin, Activin A, RA, IL4 Totipotent / Pluripotent Stem Cell IL1, IL, IL6, SCF, SCPF G-CSF, IL11, IL12, thymosin b4 MP1a, TGFb BFU-Meg IL3, GM-CSF Epo, Meg-CSF, IL1, IL6, IL7, IL11, IL12, SCF, LIF, bFGF, Endothelins, TGFb BFU-GEMM IL3, GM-CSF, EPO SCF, LIF, IL5, IL6, IL9, IL11, IL12, bFGF, IGF, E-CSF, M-CSF, Activin A, Chemokines, LIF, Epo, IFNg MIP, NRP, AcSDKP, RA, inhibin, TNFa/b, TGFb CFU-Eo IL3, IL5, GM-CSF, IL4, IL7 CFU-Bas IL3, IL5, GM-CSF, SCF CFU-MC SCF, MCOP, IL3 CFU-Meg IL3, GM-CSF Epo, Meg-CSF, IL1, IL6, IL7, IL11, SCF, LIF, bFGF, Endothelins, TGFb CFU-M IL3, GM-CSF, G-CSF IL4, IL6, IL12, IFNg, RA, IL4 CFU-G IL3, GM-CSF, G-CSF IL4, IL6, IL12, IFNg, RA, CFU-G inhibitory factor, IFNg, IL4 Myeloblast IL3, IL4, GM-CSF Myeloblast IL3, IL5, GM-CSF, IL4 CFU-E IL3, GM-CSF, EPO IL11, IGF, E-CSF, SCF, Activin A, IL5, IFNg, IP, NRP, AcSDKP, RA Monoblast IL3, GM-CSF, G-CSF, IL4 Eosinophilic Myelocyte IL3, IL5, GM-CSF, IL4 BasophilicMyelocyte IL3, IL4, GM-CSF Megakaryoblast IL3, GM-CSF Epo, Meg-CSF, IL1, IL6, IL7, IL11, SCF, LIF, bFGF Myeloblast IL3, GM-CSF, G-CSF, IL4 Promonocyte IL3, GM-CSF, M-CSF, IL4 Megakaryocyte TPO, IL6, Epo, Meg-CSF, IL1, IL6, IL7, IL11, SCF, LIF, bFGF Neutroophilic myelocyte GM-CSF, G-CSF, IL4 Proerythroblast EPO Monocyte GM-CSF, M-CSF, IL13 Mast Cell Erythrocyte Thrombocyte PMN Macrophage Eosinophil Basophil

18. Totipotent / Pluripotent Stem Cell IL1, IL, IL6, SCF, SCPF G-CSF, IL11, IL12, thymosin b4 MP1a, TGFb Common Lympoid Precursor IL1, IL2, IL6, IL7 IL6, IL11, IL12, G-CSF, LIF, and SCF are required to maintain B cell potential Pro B cell IL1, IL2, IL3, IL4 IL5, IL6, IL7, IL10 LGL (Null cells) CD4-CD8- TCR- IL2, IL4, IL7, IL9, IL10, TSTGF, Thymic hormones CD4+CD8+ TCRablo Pre B cell IL3, IL4, IL7, SCF, IFNg Immature B cell IL1, IL4, IL5, IL6 IL2 IL5 IL7 IL12 IL2 IL7 IL12 Mature B cell IL1, IL4, IL6, 13 NK cells IL1, IL2, IL4, IL7, IL12, IL13, TNF T Helper CD4+CD8- TCRab+ IL10 T Suppressor CD4-CD8+ TCRab+ CD4-CD8- TCRgd+ IL10 Antibody-producing IgM secreting B cell Switched plasma cell Secreting non-IgM Isotype Switch Signals IL1, IL2, IL4, IL6, IL10, IL13 IFNg, TGFb Memory B cell (activated by rechallenge)

19. Cytokine Receptor-mediated Txn Regulation C. Cytokine receptors (CR) are very similar to RTKs, both in terms of overall structure and cytoplasmic substrates that are ultimately tyrosine phosphorylated. D. CRs do not possess intrinsic tyrosine kinase activity. E. Therefore, CRs must rely on a second or intermediary protein(s) that functions as a surrogate tyrosine kinase. F. The proteins that function as surrogate tyrosine kinases for CRs are the JAKs family of tyrosine kinases (Tyk1, Tyk2, Jak1, Jak2, and Jak3).

20. Cytokine Receptor-mediated Txn Regulation • G. Cytoplasmic substrates for JAKs are the STAT (Signal Transducers and Activators of Transcription; STAT1, 2,3, 4, 5a, 5b, 6) proteins • H. Following phosphorylation by JAKS kinases, STAT proteins: • Physically interact (homo- or heterodimers) • Translocate to nucleus as a complex • Bind to a specific DNA sequence • Activate transcription of the corresponding (target) gene

21. Jak/Stat Signaling: IL-6 Receptor Family

22. 5 Cytokine Receptor Activation and Signaling Pathway Receptor binds cytokine. Receptors dimerize, JAKs kinases associated with cytoplasmic tails interact, are activated, and phosphorylate STAT proteins (and also themselves and the receptor on tyrosines). STAT proteins dimerize STAT protein dimers translocate to the nucleus. In the nucleus, STAT dimers bind to specific DNA sequences (response elements; 5´-TTN5-6AA-3´) located in the promoter region of a target gene and regulate expression of that gene.

23. Cytokine Receptor IFNg IFNa/b IL2 IL3 IL4 IL6 IL10 IL12 JAK JAK1/2 JAK1/Tyk2 JAK1/3 JAK2 JAK1/3 JAK1 JAK1/Tyk2 JAK2/TYK2 STAT STAT1 STAT2 STAT5 STAT5 STAT6 STAT3 STAT3 STAT4 Specificity in Cytokine Signaling

24. 5 Cytokine Receptor Activation and Signaling Pathway • Cytokine target genes include those encoding other cytokines, growth factors, transcription factors • Other cytokine target genes are SOCS (suppressors of cytokine signaling) and PIAS (protein inhibitor of activated STAT) family members, which serve to down regulate the cytokine responsiveness of the cell. • Pseudosubstrates • Direct binding to/inhibition of STAT dimers • Covalent modifications that target STATs for degradation

25. Sample Question Cytokine receptors play a major role(s) in: I. Glucose homeostasis II. Immune system function III. Hematopoiesis A. I only B. III only C. I and II only D. II and III only E. I, II and III

26. Slow/Persistent Signaling Pathways III. Nuclear Hormone Receptor Superfamily • Background • 48 members of the family in humans. • Play diverse roles in regulation of growth, development and homeostasis. • Based on importance in biology/medicine and the simple mechanism of regulation, NRs are one of the best studied and understood classes of TXN factors. • Soluble, non-membrane-associated proteins that function as ligand-dependent transcription factors

27. A/B C D E H2N - - COOH DNA LIGAND NUCLEAR RECEPTOR SUPERFAMILY STEROID HORMONE RECEPTORS VITAMIN D RECEPTOR ECDYSONE RECEPTOR OXYSTEROL RECEPTORS ORPHAN RECEPTORS XENOBIOTIC RECEPTORS RETINOIC ACID RECEPTORS THYROID HORMONE RECEPTORS PEROXISOME PROLIFERATOR- ACTIVATED RECEPTORS FATTY ACID RECEPTORS BILE ACID RECEPTORS ANDROSTANE RECEPTOR

28. GCNF HNF4 TR2, 4 NGFI FAMILY ROR FAMILY RVR FAMILY SF1 LRH-1 DAX-1 SHP TLX PNR A/B C D E H2N - - COOH DNA LIGAND ORPHAN NUCLEAR RECEPTORS COUP-TF FAMILY

29. Positive or Negative Regulation of Transcription HR HR Transcription (Gene Expression) Promoter Region Exon Exon Exon Exon Nuclear mRNA mRNA processing Mature mRNA Exon Exon Exon Exon Translation N Function Protein C DNA

30. Positive or Negative Regulation of Transcription HR HR Transcription (Gene Expression) Exon Exon Exon Exon Nuclear mRNA DNA Hormone Receptors Are Transcription Factors • Bind directly to DNA (promoter region of gene) • Regulate transcription in a hormone-dependent manner

31. HR HR Transcription (Gene Expression) Exon Exon Exon Exon Nuclear mRNA Positive or Negative Regulation of Transcription H H DNA Hormone Receptors Are Transcription Factors • Bind directly to DNA (promoter region of gene) • Regulate transcription in a hormone-dependent manner What are the mechanisms by which hormone receptors bind DNA and regulate transcription in a hormone-dependent manner?

32. DNA Binding by Nuclear Receptors Alpha helix sits in major groove of DNA

33. APO and HOLOLIGAND BINDING DOMAINS STRUCTURES AGONIST and ANTAGONIST CONFORMATIONS H9 H9 H9 H1 H8 H1 H1 H10 H8 H8 H4 H10 H10 H4 H4 H5 H7 H7 H5 H5 H12 H3 H7 H2 H11 H12 H3 H3 H6 H11 H11 H6 H6 H12 apo-RXR (Bourguet & al.,1995) ER / DiethylStilbestrol (Shiau & al., 1998) holo-ER/4 hydroxyTamoxifen (Shiau & al., 1998) AGONIST COACTIVATOR BINDING ANTAGONIST COREPRESSOR BINDING

34. Activation of NR LBD Bottomline Structurally unique drugs push receptors into unique conformations that have unique activities…

35. Chromosomal DNA is Highly Compacted

36. Transcriptional Repressors Condensation: Deacetylation, Methylation, Phosphorylation Transcriptional Activators Decondensation: Acetylation, Methylation, Remodeling, Proteolysis

37. Activation of Transcription • Receptor-DNA interaction (DNA binding) • Receptor-Drug interaction (Ligand binding) • Regulation of transcription involves recruitment to the template of: • Enzymes that covalently modify the protein components (histones) of the nucleosome that decrease their affinity for DNA. • "Nucleosome remodeling complexes" that push nucleosomes around and enhance the access of RNA polymerase II to the template. • Once the above have been recruited and have acted, proteases are then recruited that degrade the receptor, and most likely other proteins, to clear the template and enhance initiation of RNA polymerase II-mediated transcription.

38. N C Tail Fold THE HISTONE CODE Octamer • General structure of histones H4 H3 H2A H2B H3 • Modifications generally occur in the tails H2B H2A P P H4 M M A M A A M M A/M M A A A A M P A A A A A A = A = M = P

39. Sample Question Which of the following best describes the mechanism of action of Pioglitazone (Actos, a thiazolodinedione that activates the nuclear receptor PPAR) in the treatment of non-insulin-dependent diabetes mellitus? A. Pioglitazone opens ATP-dependent K+ channels in pancreatic  cells and thereby enhances insulin secretion. Pioglitazone blocks ATP-dependent K+ channels in pancreatic  cells and thereby enhances insulin secretion. Pioglitazone directly increases expression of GLUT4, the insulin-sensitive glucose transporter in target tissues. D. Pioglitazone blocks absorption of carbohydrates from GI tract. E. Pioglitazone binds to and activates insulin receptors in target tissues

40. Death Receptors • Death receptors are cell surface receptors that transmit apoptosis signals initiated by specific ligands, and can activate a caspase cascade within seconds of ligand binding resulting in a rapid cell death APO-2L (TRAIL) APO-1L (FasL/CD95L) Ligand APO-3L TNF Receptor Coupler Transducer Procaspase 8 Procaspase 8 Procaspase 8 Procaspase 8 Death Effector Domain Death Domain BID  Receptor  Intracellular death domain  Adaptor  Procaspase 8  Caspase 8  Caspase 3

41. Decoy receptors antagonize TRAIL-mediated induction of apoptosis • Decoy receptors that compete for binding of TRAIL with the DR4 and DR5 receptors. • The decoy receptors are called DcR1and DcR2 • Both of these receptors are capable of competing with DR4 or DR5 receptors for binding to the ligand (TRAIL) • However, ligation of these receptors does not initiate apoptosis since DcR1 does not possess a cytoplasmic domain, while DcR2 has a truncated death domain lacking 4 out of 6 amino acids essential for recruiting adaptor proteins.  

42. Death Receptor Signaling  Apoptosis • Receptor Intracellular death domain Adaptor Procaspase 8 Caspase 8 Bid Caspase 3 Mitochondrial Stability Cell & DNA Integrity

43. Multiple signaling pathways have evolved that allow multicellular organisms to respond to environmental signals • Rapid response: LGICs (TC, JI) • Intermediate response: GPCRs, RTKs, CRs (TF, ML) • Slow/Persistent Response: NRs, Death Receptors (ML) • A given cell receives hundreds, if not thousands, signals at any given time. • This cell must integrate these signals and come up with a response that is contextually appropriate. • There are multiple therapeutic targets (current and future) in each of these signaling pathways. • Death receptors represent an exciting new avenue to exploit in the Tx of proliferative disease. Summary

44. Sample Question Which of the following protein(s) is/are localized in the plasma membrane of mammalian cells? I. Insulin receptor II. Interleukin 6 receptor III. Estrogen receptor A. I only B. III only C. I and II only D. II and III only E. I, II and III

45. Sample Question Which of the following protein(s) is/are localized in the nucleus of mammalian cells and regulate(s) transcription DIRECTLY? I. Insulin receptor II. Interleukin 6 receptor III. Estrogen receptor A. I only B. III only C. I and II only D. II and III only E. I, II and III

46. Sample Question

47. Sample Question Which of the following would be most useful for induction of apoptosis in cancer cells that express Fas? A. Synthetic FAS agonists B. Synthetic FAS antagonists C. FLIP inhibitors D. Caspase 8 inhibitors E. Caspase 3 inhibitors

48. Sample Question Which of the following may underlie the resistance of some cancer cells to TRAIL-mediated apoptosis? I. Cancer cells that are sensitive to TRAIL-induced apoptosis express decoy receptors that compete with Death Receptors DR4 and DR5 for binding to TRAIL II. Cancer cells that are insensitive to TRAIL-mediated apoptosis express decoy receptors that compete with Death Receptors DR4 and DR5 for binding to TRAIL III. Cancer cells that are insensitive to TRAIL-mediated apoptosis express FLIP, which is an inhibitor of caspase 8 A. I only B. III only C. I and II only D. II and III only E. I, II, and III