1. Signal Transduction March 2009 Advanced Biochemistry Course First lecture

2. Plasma Membrane Structure and Function plasma membraneseparates the internal environment of the cell from its surroundings The plasma membrane separates the internal environment of the cell from its surroundings The plasma membrane is aphospholipid bilayerwithembedded proteins The plasma membrane is a phospholipid bilayer with embedded proteins. fluid consistency and amosaicpattern of embedded proteins. The plasma membrane has a fluid consistency and a mosaic pattern of embedded proteins.

3. Protein dynamics in the lipid bilayer 1) Proteins can move laterally in the plane of the membrane (capping) 2) Proteins can rotate around an axis vertical to the plan of the membrane (channels) 3) Proteins cannot tumble through the plan of the membrane

4. Lateral Rotational Thumbling

5. Signal Transduction Endocrinic- “ (Insulin; adrenalin) Paracrinic -transduction (histamin;prostaglandins) Autocrinic - “ (TGF/ IGF) Synaptic - transmission (neurotransmitters)

6. Models of Cell-Cell signaling (D)

7. Receptors Membrane receptors Cytosolic receptors 1. G-coupled receptors 2. Channel/receptors 3. Enzyme/receptors 1.Steroid receptors 2.Vitamin D 3. Retinoic acid

8. Cell surface receptors

9. Gramicidin A in lipid bilayer and water Antibiotic peptide Forms a pore in the cell wall of a bacteria and lets out monovalent cations (K+, Na+). [Membrane potential disappears and bacteria dies!] 15 amino acids, helical Channel is formed by a head- to-head dimer

10. 3.5 nm 4 nm Glycophorin

11. The Free Energy for Transferring a Helix of 20 Residues from the Membrane to Water Hydropathy plot

12.  -adrenergic receptor Rhodopsin Membrane topology

13. One of the largest families of membrane proteins Common structural architecture Extracellular N-terminal domain  Extracellular N-terminal domain Glycosylated Ligand recognition Intracellular C-terminal domain  Intracellular C-terminal domain Contains several putative phosphorylation sites Involved in desensitisation/internalisation 7 membrane spanning domains  7 membrane spanning domains Couple to G-proteins  signal transduction Divided into subfamilies based on sequence homology definition definition G-protein-coupled receptors

14. Current estimation ~1000 GPCRs in human genome Current estimation ~1000 GPCRs in human genome How many GPCRs are encoded by the human genome? Orphan GPCRs First estimation based on comparison with C. elegans First estimation based on comparison with C. elegans 19.100 genes ~1000 GPCRs ~5% of genome HuGo ~1800 GPCRs ~700 olfactory, gustatory and chemokinine receptors 300-400 transmitter GPCRs ~ 300-400 transmitter GPCRs 27.000 genes

15. 210 GPCRs bind un known natural ligands 160 orphan GPCRs 160 orphan GPCRs remain to be characterised 400 transmitter GPCRs ~ 400 transmitter GPCRs Orphan GPCRs

16. Rationale for oGPCR characterisation A. GPCRs are good drug targets 50% of subscription drugs interact with GPCRs Why is the pharmaceutical industry interested in oGPCRs? Hypertension Stomach ulcers Migraine Allergies B. GPCRs in disease states Disease states associated with GPCR mutations Rhodopsin receptor retinitis pigmentosa Vasopressin V2 nephrogenic diabetes Glucag on diabetes, hypertension GRF-receptor dwarfism Asp(60)Gly (60)

17. GPCR subfamilies Largest family Conserved DRY motif (i2) Conserved cysteines  -S-S- Family A: Rhodopsine-like Family B: Secretine-like Large N-terminal domain Several well conserved cysteine residues High Mr hormone ligands Family C: Metabotropic glutamate Long N-terminal domain N-terminus sufficient for ligand binding

18. RR* S-F-L-L-R-N Protease activated receptor (PAR)

19. Lipid head groups Polar/negatively charged Chains hydrophobic Lipid head groups The positioning of the the 7TMR in the membrane

21. Extracellular Cytoplasmic COOH - -NH2 i1 i2 i3 e1 e2 e3 TM1 TM2TM3TM4TM5TM6TM7 D R Y G-protein-coupled receptors -S-S-

22. How does the ligand activates the receptor in a selective way? A two state model is commonly used to characterize this activation Fluorescence spectroscopy was used to characterize the diversity of conformational states of the  2AR and its mode of activation

23. AGONIST-INVERSE AGONIST - ANTAGONIST agonist, antagonist and inverse agonist. ● Drug effects can be classified into three major phenotypes: agonist, antagonist and inverse agonist. ● Agonistinverse agonist ● Agonist and inverse agonist effects are associated with receptor activation and inactivation, respectively Antagonism ● Antagonism implies that a drug produces no effect when administered alone but blocks the effects of agonists and inverse agonists.

24. Inverse agonist - a ligand that prefers the inactive form of the receptor Agonist- a ligand that activates the receptor Antagonist- a ligand that inhibits the receptor Partial agonist- a low affinity agonist r R* Inactive form Active form Two- State Model

25. r Rr R Agonist rA R*A Inverse Agonist active inactive Partial agonists and antagonists bind to both r and R states Receptor states and inverse agonists Activation in the absence of an agonist; over-expression (Two-State Model)

26. Energy landscape diagram describing a possible mechanism of GPCR activation by an agonist

27. Inverse agonist r full and inverse agonists rR partial agonists neutral antagonists r R passive antagonists 1) Inverse agonist (propranolol) binds to the r form of the receptor and the activity of the system is suppressed below its normal spontaneous state 2) In between full and inverse agonists are those agonists that bind to both r and R states. These are partial agonists. These are unable to achieve maximal stimulation even if all receptor binding sites are occupied Inverse agonism offers a potential of developing new drugs that attenuate the effect of mutant receptors that are constitutively active 3) The neutral antagonists (  -blockers such as pindolol) bind to both r and R conformations and are better regarded as passive antagonists They impede the binding of both agonists and inverse agonists. Therefore, pindolol affects the heart only during exercise and stress while propranolol also suppresses the resting heart rate

28. Activation of G-protein-coupled receptors Ligand Efficacy: The effect of different classes of drugs on a GPCR that has some detectable basal activity

29. FULL AGONIST PATRIAL AGONIST ANTAGONIST INVERSE AGONIST

30. Rhodopsin Dopamine

31. How can we determine the mechanism of activation? Three different methods are used, which could be applied to explore the mechanism of activation of 7TMR or any other receptors The focus will be on the adrenergic receptors

32. F R E T fluorescence resonance energy transfer A donor chromophore, in its electronic excited state, may transfer energy to an acceptor chromophore (in close proximity < 10nm) through Non-radiative dipole-dipole coupling When both chromophores are fluorescent, the term "fluorescence resonance energy transfer (FRET)" is often used instead, although the energy is not actually transferred by fluorescence

33. Fluorescence resonance energy transfer (FRET) The two fluorescent probes report in real time through a fast decrease in FRET the intra-molecular conformational rearrangements associated with receptor activation

34. Norepinephrine (NE) Agonist Inverse agonist Yohimbine

35. Swaminath, G. et al. J. Biol. Chem. 2004;279:686-691 Binding site for norepinephrine in the  2AR

36. Top-down view of hormone receptor with an adrenalin molecule

37. A multi-step agonist binding

38. good partial Agonist selective Agonist inverse Agonist antagonist weak partial agonist

39. Gether et al., J. Biol. Chem. 1998;273:17979 Arrangement of transmembrane domains of a prototypical G protein-coupled receptor as viewed from the extracellular surface of the membrane (based on the projection maps from two-dimensional crystals of rhodopsin) The Asp3-Arg3 pair at the cytoplasmic end of transmembrane domain 3 (TM3) is part of the highly conserved (D/E)RY motif found in beta2-AR and other rhodopsin-family GPCRs, whereas the Glu6at the cytoplasmic end of TM6 is highly conserved in amine and opsin receptors. The ionic link between the Asp3-Arg3pair and Glu6 is known as the ionic lock

40. Break ionic lock Activate rotamer toggle switch

41. The Ionic Link Asp3-Arg3 (D/R) The Asp3-Arg3 (D/R) pair at the cytoplasmic end of TM3 (D/E)RY transmembrane domain 3 (TM3) is part of the highly conserved (D/E)RY motif found in  2AR and other rhodopsin- family GPCRs, whereas the Glu6TM6 Glu6 at the cytoplasmic end of TM6 is highly conserved in amine and opsin receptors. The ionic link between the Asp3-Arg3Glu6 ionic lock Asp3-Arg3 pair and Glu6 is known as the ionic lock

42. How is the receptor activated ? ● Previous biophysical studies on the  2-AR suggest that agonist binding and activation occurs through at least one conformational intermediate, implying that at least one molecular switch is involved. different agonistspartial agonistsinduce ● These studies also show that structurally different agonists and partial agonists differ in their ability to induce specific conformational transitions.

43. Swaminath, G. et al. J. Biol. Chem. 2004;279:686-691 Cellular responses to catecholamines cAMP accumulation in HEK cells expressing  2AR The state that stabilizes the binding of the catechol ring alone (R1) is not sufficient to activate the G proteins

44. Fluorescence spectroscopy to monitor disruption of the ionic lock at the  AR

45. Fluorescence spectroscopy to monitor disruption of the ionic lock at the  AR Mutated A271C and binding of mBrBimane Mutated I135W Relies on the quenching of of bimane fluorescence by Trp at near contact distance in the 5-15 Å range The bimane-tryptophan technique

46. Quenching during agonist binding to the site

47. TM 6 TM 3 TM6 TM6 E 268 TM3 TM3 D R Y Asp-Arg-Tyr A271C Ile135W

48. Iso Epi Sal Norepi Dop Cat HalLog[ligand] % maximal quenching Effect of ligand structure on the ionic lock

49. D130 R131 E268 Ionic Lock A135W H271C-Bimane Inactive Active DRYDRY

50. Ballesteros, J. A. et al. J. Biol. Chem. 2001;276:29171-29177 Molecular three-dimensional representations of the interaction of TM3 and TM6 at their cytoplasmic ends and the effects of 6.30 mutations

51. Jensen, A. D. et al. J. Biol. Chem. 2001;276:9279-9290 Proposed conformations of the inactive and active states of the  2AR

52. Fluorescence studies of receptor activation Cys265 These studies show that  2ARs labeled at Cys265 on the cytoplasmic end of TM6, adjacent to the G protein–coupling domain, is able to report conformational changes in the G protein–coupling domain. fluorophore These modifications alter the molecular environment around the fluorophore, which is translated to changes in fluorescence intensity and fluorescence lifetime. In these experiments, fluorescence lifetime analysis can detect discrete conformational states in a population of molecules, while fluorescence intensity measurements reflect their weighted average

53. tetramethyl rhodamine maleimide To monitor agonist induced conformational changes purified  receptors were labeled at Cys 265 (at the third intracellular loop) with tetramethyl rhodamine maleimide -Fluorescence intensity was followed as a function of time Norepi induced conformational changes are biphasic. The rapid change is similar for both dopamine and norepi Fluorescence Life-time spectroscopy ex 541nm and em at 571nm ex 541nm and em at 571nm

54. Swaminath, G. et al. J. Biol. Chem. 2005;280:22165-22171 Norepinephrine induces a biphasic conformational change in TMR-  2AR

55. Swaminath, G. et al. J. Biol. Chem. 2004;279:686-691 Conformational changes in TMR-  2AR in response to a panel of catecholamine-related ligands reveal the structural features of catecholamine ligands responsible for the rapid and slow components of the biphasic conformational change

56. Swaminath, G. et al. J. Biol. Chem. 2004;279:686 Norepinephrine induces a biphasic conformational change in TMR-  2AR Differences between the L- and D- enantiomers

57. Swaminath, G. et al. J. Biol. Chem. 2005;280:22165-22171 Catechol-induces conformational changes in TMR-  2AR in the presence of a saturating concentration of salbutamol

58. Swaminath, G. et al. J. Biol. Chem. 2005;280:22165-22171 Catechol (CAT) competes with isoproterenol, but not with salbutamol (SAL) or alprenolol, for binding to the  2AR

59. Break ionic lock Activate rotamer toggle switch

60. Swaminath, G. et al. J. Biol. Chem. 2005;280:22165-22171 Molecular model of the agonist binding pocket of the  2AR The model was generated using the crystal structure of rhodopsin as a template. TM segments involved in agonist binding are colored as follows: red, TM3; green, TM5; blue, TM6. A, the proposed binding site for isoproterenol. B, salbutamol docked into the upper (extracellular) region of the binding pocket. The aromatic ring of salbutamol interacts with Tyr-174, Phe-193, and Tyr-1995.38.

61. 3D structure of the  adrenergic receptor High-resolution structural information is essential for understanding molecular mechanisms of protein function; however, some limitations of crystallography must be recognized. In forming a crystal, a protein becomes locked in a single conformational state. This is a significant drawback when one considers the body of functional and biophysical evidence that GPCRs are conformationally complex and dynamic proteins. They do not behave as simple bimodal switches but adopt conformations that are specific for the bound ligand and the associated signaling partner (e.g. G proteins, arrestins). In the reported crystal structures, the conformation of the  2AR bound to carazolol is close to an inactive state. Carazolol is an inverse agonist, but suppresses not, very similar 50% of basal activity in the  2AR. Therefore, the  2AR structure might not represent a fully inactive receptor and could differ significantly from the unliganded receptor or one of the potential active states. Nature 450 (2007), pp. 383–387

63. B A

64. BRAIN regions affected by Parkinson’s disease

65. Pars compacta region of the substantia nigra in the neuronal brain appears dark Normal Parkinsonian

66. Dopaminergic Neurons Na +Tyrosine Ca ++ Receptor MAO  Dopamine Dopa Dopamine is converted to epinephrine TyrosineHydroxylase

67. Tyrosine Hydroxylase Dopa decarboxylase Dopamine-  -hydroxylase Norepinephrine- transmethylase Pathway for the synthesis of catecholamines

68. The receptor and effector are independent entities

69. GGGG   L Effector G-proteins Signal

70. Swaminath, G. et al. J. Biol. Chem. 2004;279:686-691 Norepinephrine (Norepi) induces a biphasic conformational change in TMR-  2AR

71. Swaminath, G. et al. J. Biol. Chem. 2004;279:686-691 Norepinephrine induces a biphasic conformational change in TMR-  2AR