1. Structural Basis for Ligand- Receptor Recognition and Dimerization Moosa Mohammadi Dept. of Pharmacology Medical Science Building, 4th Floor, Rooms 425, 431 mohammad@saturn.med.nyu.edu

2. In multicellular organisms, the decision a cell has to make whether to divide, to differentiate or to die is controlled by ligands (growth factors, hormones) that circulate outside of the cell. These ligands in order to transmit their signals must interact with cell surface receptors that possess enzymatic activity known as protein kinase activity. Nucleous Plasmamembrane Barrier Ligands Covalently linkedAssociated kinase

3. Single Transmembrane-Spanning Receptors Extracellular Intracellular Ligand Receptor

4. Mechanisms of Signal Transduction Extracellular Intracellular Conformational Change    GDPGTP

6. Receptor Dimerization Extracellular Intracellular Y Y Y Y Y Y

8. Early experiments suggesting that receptors undergo dimerization Truncated receptors lacking the cytoplasmic domain inhibit signaling. Transmembrane helices are interchangeable between different receptors. Antibodies against the cytoplasmic domain activate the kinase domain.

10. Antibody-mediated activation Extracellular Intracellular Receptor

11. Extracellular Intracellular Receptor Fab

13. Receptor Ras Raf MAPKK MAPK Sos Grb2 Growth factor Shc Jun Fos NUCLEUS

14. Phenotypes of patients with Apert syndrome which results from point mutation in FGF receptor

15. Growth Hormone (GH) Receptor Erythropoietin (EPO) Receptor Bone Morphogenic Protein (BMP) Receptor Vascular Endothelial Growth (VEGF) Receptor Nerve Growth Factor (NGF) Receptor Fibroblast Growth factor (FGF) Receptor Ligand-Receptor Systems

16. Large family of single-pass transmembrane receptors. Receptors bind polypeptide ligands: mediators of cell growth, differentiation and immune responses. Cytoplasmic domain does not contain intrinsic protein tyrosine kinase activity - associated with Jak tyrosine kinases. Cytokine Receptors

18. GH stimulates the growth and metabolism of muscle, bone and cartilage cells. GH is a member of the 4-helix bundle family. The active form of GH is a monomer. Stoichiometry of binding is 1:2 GH-GHR. Activation Through Binding of a Monomeric Ligand – Growth Hormone

19. Ribbon Diagram of Growth Hormone

20. AA CC DD BB Four-Helix-Bundle Structure

21. Four-Helix-Bundle is Stabilized by Hydrophobic Contacts Between the Four Helices

22. Ribbon Diagram of Extracellular Domain of Growth Hormone Receptor

23. Fibronectin Type III Domain is a Close Cousin of Immunoglobulin Superfamily Domains

27. Ribbon Diagram of the 1:2 Complex between GH and GHR

28. GH-GHR Complex GH-GHR contact I: 1230 Å 2 GHR-GHR contact: 500 Å 2 GH-GHR contact II: 900 Å 2

29. Details of GH-GHR Interactions

30. Ribbon Diagram of the 1:1 Complex Between GH and Prolactin Receptor

31. Hormone-Receptor Interactions Involving the Linker Region of the Receptor

32. Hormone-Receptor Interactions Involving the  F-  G Loop in the C-terminal Fibronectin Domain

33. Activation Through Binding of a Monomeric Ligand – Erythropoietin (EPO) EPO is a haematopoietic cytokine required for differentiation and proliferation of precursor cells into red blood cells. Like GH, EPO is monomeric and belongs to the 4-helix bundle family. EPO binds to its receptor (EPOR) with a stoichiometry of 1:2 EPO-EPOR.

34. Epo:EpoR Complex - Dependence on Receptor Orientation

35. RasMol Presentation of the Dimeric EPO-EPOR Structure

36. Growth Hormone Paradigm

37. Bone morphogenic proteins (BMPs) belong to the Transforming Growth Factor  (TGF  superfamily of ligands which includes TGF  activins/Inhibins and GDFs. BMPs regulate bone and cartilage formation in adults and are also involved in central steps in early embryonic development. BMPs are dimeric ligands and have the characteristic “Cystine Knot “ motif found in other members of TGF  family. The receptors for TGF  family of ligands are transmembrane receptors with intrinsic serine/threonine kinase activity. Activation of a Receptor Serine/Threonine Kinase – Bone Morphogenic Protein (BMP) Receptor

38. TGF  Receptors Extracellular Intracellular Ser/Thr kinase Cys-rich

39. Activation via Dimeric Ligand: BMP-BMPR Structure

40. RasMol Presentation of the Dimeric BMP-BMPR Structure

41. Large family of single-pass transmembrane receptors. Receptors are predominantly for growth factors but also for insulin. Cytoplasmic domain contains intrinsic protein tyrosine kinase activity. Receptor Tyrosine Kinases

42. Receptor Tyrosine Kinase Family

43. VEGF is a mitogen that is highly specific for endothelial cells. VEGF is a potent angiogenic factor involved in the development of the vascular system and also in tumor angiogenesis. VEGF is a covalent (disulfide-linked) dimer. Activation Through Binding of a Dimeric Ligand – Vascular Endothelial Growth Factor

44. VEGFR Family Ig-like

45. VEGF-Flt1 Dimer

46. Flt1-D2 VEGF

47. VEGF Interacting Residues Ig domain 2

48. RasMol Presentation of the Dimeric VEGF-FLT1 Structure

49. Model for Flt1 Dimerization

50. NGF is a member of a family of neurotrophins which also includes brain-derived neurotrophic factor (BDNF), NT-3, NT-4/5 and NT-6. NGF mediates neuronal differentiation and survival. These neurotrophins are non-covalent dimers, members of the cystine knot family. Activation Through Binding of a Dimeric Ligand – Neurotrophic Growth Factor

51. Trk (NGF Receptor) Family Cys-rich Leu-rich Ig-like

52. NGF-Trk Dimer Domain 5

53. Common and Specific Interaction Sites Common interface Specificity interface

54. Common and Specific Interaction Sites Specificity interface Common interface

55. Alignment of Neurotrophic Factors and Trk-D5 Common site Specificity site Ligands Receptors

56. RasMol Presentation of the Dimeric NGF-TRK Structure

57. Dimeric Ligands with “Cystine Knot” Motif (BMP-2)

58. Receptor Dimerization by Dimeric Ligands with “Cystine Knot” Motif

59. Growth Hormone Paradigm

60. Crystal Structure of Flt3 Ligand; Dimer of two 4-helix Bundels

61. RasMol Presentation of FLT3 Ligand; Dimer of two 4-helix Bundles

63. Dimerization by Flt3L Versus VEGF Flt3L-Flt3 (model)VEGF-Flt1 (partial model)