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G- Protein Coupled Receptors

GPCRs are the most dynamic and most abundant all the receptors. The G protein-coupled receptor (GPCR) superfamily comprises the largest and most diverse group of proteins in mammals. GPCRs are responsible for every aspect of human biology from vision, taste, sense of smell, sympathetic and parasympathetic nervous functions, metabolism, and immune regulation to reproduction. GPCRs interact with a number of ligands ranging from photons, ions, amino acids, odorants, pheromones, eicosanoids, neurotransmitters, peptides, proteins, and hormones.
 Nevertheless, for the majority of GPCRs, the identity of their natural ligands is still unknown, hence remain orphan receptors.
The simple dogma that underpins much of our current understanding of GPCRs, namely,

one GPCR gene− one GPCR protein− one functional GPCR− one G protein −one response

is showing distinct signs of wear.

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G- Protein Coupled Receptors

  1. 1. G-Protein-Coupled Receptors Dr. Prashant Shukla Junior Resident Dept of Pharmacology
  2. 2. 2 Contents  Introduction  Historical Background  GPCR - Basics  GPCR as Targets for Drug Designing  GPCR associated Diseases  Concept of Orphan GPCR  Future Prospects & Conclusions
  3. 3. Introduction 3 Receptors are the sensing elements in the system of chemical communications that coordinates the function of all the different cells in the body. The chemical messengers can be hormones, transmitters and other mediators.
  4. 4. 4 Types of receptors
  5. 5.  The G protein-coupled receptor (GPCR) superfamily comprises the largest and most diverse group of proteins in mammals.  Synonym: “seven-transmembrane” (7- TM), “serpentine” receptors, heptahelical receptors, serpentine receptor, and G protein–linked receptors (GPLR). 5 GPCRs
  6. 6. GPCRs  It is involved in information transfer (signal transduction) from outside the cell to the cellular interior.  GPCRs are responsible for every aspect of human biology from vision, taste, sense of smell, sympathetic and parasympathetic nervous functions, metabolism, and immune regulation to reproduction.  ~45% of all pharmaceutical drugs are 6
  7. 7. Receptors associated with GPCRs 1. GABAB Receptors  GABABR1 and GABABR2 2. Taste Receptors  T1R3 and T1R2 3. Adrenergic Receptors  Three subfamilies (α 1, α 2 and β )  Family A (rhodopsin-like) GPCRs 4. Opioid Receptors  Three cloned subtypes: δ, қ and μ 7
  8. 8. Receptors associated with GPCRs 5. Somatostatin Receptors  Five subtypes (SSTR1-5) 6. Purinergic Receptors  Neurotransmitters in the CNS, CVS, immune system, and other tissues i.e. adenosine and ATP 7. Olfactory Receptors  Represent the largest family of GPCRs, with >300 members 8. Vasopressin, Oxytocin and Other Receptors 8
  9. 9. The importance of GPCRs 1. Number (C.elegans 1100; H. sapiens, ~1000; D. melanogaster, 160; reflects number of olfactory receptor genes in worm [~1000] and mammal [several hundreds]), a few % of genome; 300-400 non-olfactory GPCRs) 2. Diversity (mostly small molecule ligands) 3. Evolutionarily conserved yeast to man (yeast Ga 45% identical to mammalian Gia) 4. Pharmaceutical importance: ~500 known molecular targets of drugs, 60% of these are cell surface receptors, 75% of these are GPCRs (GPCRs = ~45% of all known drug targets) 9
  10. 10. Historical background 10
  11. 11.  Robert Lefkowitz and Brian Kobilka: the 2012 Nobel Prize in Chemistry for groundbreaking discoveries that reveal the inner workings of an important family of receptors: G-protein–coupled receptors. 11 Historical background
  12. 12. 12 Common Experimental Tools used to Study GPCRs
  13. 13. GPCR- basics 13  Structure  Classification  Signal molecules/ Ligands  Physiological role  G proteins  Mechanism of action
  14. 14. GPCR- Basic structure CYTOSOL EXTRACELLULA R 14 Extracellular loops Intracellular loops Plasma membrane
  15. 15. 15 GPCR- Basic structure
  16. 16. GPCR: Classification Based on Sequence homology and functional similarity ◦ Class A (or 1) (Rhodopsin-like) ◦ Class B (or 2) (Secretin receptor family) ◦ Class C (or 3) (Metabotropic glutamate/ pheromone) ◦ Class D (or 4) (Fungal mating pheromone receptors) ◦ Class E (or 5) (Cyclic AMP receptors) ◦ Class F (or 6) (Frizzled/Smoothened) 16
  17. 17. Based on phylogenetic origin: The GRAFS classification system has been proposed 1. Glutamate 2. Rhodopsin 3. Adhesion 4. Frizzled/Taste 5. Secretin 17 GPCR: Classification
  18. 18. 18 GPCR Tree
  19. 19. Signal molecules/ Ligands of GPCRs GPCRs interact with a number of ligands ranging from photons, ions, amino acids, odorants, pheromones, eicosanoids, neurotransmitters, peptides, proteins, and hormones. Nevertheless, for the majority of GPCRs, the identity of their natural ligands is still unknown, hence remain orphan receptors. 19
  20. 20. Signal molecules 20 Biogenic amines: Adrenaline, noradrenaline, dopamine, 5-HT, histamine, acetylcholine Amino acids and ions: Glutamate, Ca2+, GABA Lipids : PAF, prostaglandins, leukotrienes, anandamine
  21. 21. 21 Peptides / proteins : GnRH, angiotensin, bradykinin, thrombin, bombesin, glucagon, calcitonin, vasoactive intestinal peptides, PTH, FSH, LH, TSH Nucleotides : Adenosine nucleotides, adenine nucleotides, uridine nucleotides Others : Light, odorants, pheromones, opiates Signal molecules…
  22. 22. Physiological roles 22 1. Visual sense: Rhodopsin 2. Sense of smell: Olfactory receptor 3. Behavioral and mood regulation: Serotonin, dopamine, GABA and glutamate 4. Immune system activity and inflammation: Chemokine receptors, histamine receptors 5. ANS transmission: β adrenergic receptors 6. Apoptosis
  23. 23. Structure of G Protein G proteins, also known as guanine nucleotide-binding proteins, involved in transmitting signals and function as molecular switches. Their activity is regulated by factors that control their ability to bind to and hydrolyze GTP to GDP. When they bind GTP, they are 'on', and, when they bind GDP, they are 'off '. 23
  24. 24.  G protein complexes are made up of  20 alpha (α)  6 beta (β)  12 gamma (γ) subunits.  Beta and gamma subunits can form a stable dimeric complex referred to as the beta-gamma complex. α subunit β subunit γ subunit 24
  25. 25. Types of G Proteins 25
  26. 26. G protein cycle 26 Basal state Activated state
  27. 27. GPC Receptors G Protein Receptors Signaling Pathway GS Beta adrenergic receptors, glucagon, histamine, serotonin Increase CAMP Excitatory effects Gi Alpha2 adrenergic receptors, mAchR, opioid, serotonin Decrease CAMP Cardiac K+ channel open- decrease heart rate Gq mAchR, H1, α1, Vasopressin type 1, 5HT1C PLC- IP3 , DAG Increase Cytoplasmic Ca Gt Rhodopsin and colour opsins in retinal rod and cone cells Increase cGMP phosphodiesterase. Decrease cGMP 27
  28. 28. G Protein Mediated Pathways Secondary messenger Systems Involved In Signal Transduction: Adenylate cyclase cAMP mediated pathway  Phospholipase mediated pathway GPCR s can also directly activate the ion channels 28
  29. 29. cAMP Mediated Pathway The cAMP-dependent pathway, also known as the adenylyl cyclase pathway, is a G protein-coupled receptor triggered signaling cascade used in cell communication.  Gs cAMP Dependent Pathway  Gi cAMP Dependent Pathway 29
  30. 30. GTP GDP  GDP GTP  ATP cAMP Cell response AT Protein kinase ADP P Inactive protein Active protein hormone Adenylate cyclase Signaling System AC RS Inhibitor Ri   CYTOSOL EXTRACELLULA R 30 Gs cAMP Dependent Pathway
  31. 31. Gi cAMP Dependent Pathway 31
  32. 32. 32 CYTOSOL EXTRACELLULA R Gq Protein Coupled Receptor
  33. 33. 33 Gt PCR: involved in photo transduction. Gt Protein Coupled Receptor
  34. 34. Signal Amplification through G proteins 34
  35. 35. Regulation of GPCRs Turning GPCRs Off  A cell must also be able to stop responding to protect overstimulation  High activation of a receptor leads to a reduced ability to be stimulated in the future (desensitization)  Can also significantly limit therapeutic usefulness of many receptor agonists. 35
  36. 36. 36 Desensitization mechanisms include 1. “down-regulation” or reduction of receptor number 2. “sequestration” or apparent shielding of the receptors from interacting ligands 3. “uncoupling” from G-proteins. Regulation of GPCRs
  37. 37.  Homologous desensitization: The activation dependent regulation of receptors.  Heterologous desensitization: Receptor activation-independent regulation of receptors. 37 Regulation of GPCRs
  38. 38. 38
  39. 39. Homologous desensitization  The activated state of GPCRs serves not only as an activator of G proteins, but also as the substrate for GPCR kinases (GRKs). 39
  40. 40. 40
  41. 41. 41 Homologous desensitization
  42. 42.  Based on feedback regulation of receptors by the second-messenger- regulated kinases.  Eg. Upon stimulation, β- receptors leads to ↑ cAMP, which activates PKA. PKA can then phosphorylate the β- receptors themselves, even those particular receptor proteins that were not activated by the current stimulation. These PKA- phosphorylated receptors are less able to mount a response. 42 Heterologous desensitization
  43. 43. Receptor Drugs and some key indications AT1 angiotensin II receptor Antagonists e.g. losartan in treatment of HT or CHF α1A-c receptor Antagonists e.g. tamsulosin to treat disorders asso. with enlarged prostate β1- receptor Antagonists e.g. propranolol, atenolol, metoprolol, carvedilol to treat essential HT or CHF β2- receptor Agonists e.g. terbutaline, salbutamol, formoterol for treatment of COPD or Bronchial asthma D2 receptor Antagonists e.g. Haloperidol & clozapine to treat schizophrenia Agonists e.g. levodopa for Parkinsonism 43 GPCR as drug targets
  44. 44. 44 Receptor Drugs and some key indications D3 receptor Antagonists e.g. haloperidol in schizophrenia 5-HT2A receptor Antagonists e.g. clozapine for schizophrenia. Indirect agonists e.g. fluvoxamine for depression 5-HT2C receptor Antagonists e.g. clozapine for schizophrenia CCR5 Associated with progression of AIDS e.g. Aplaviroc and maraviroc M3 Antagonists e.g. Atropine to dilate pupil; Scopolamine for motion sickness Neuropeptide S receptor Asthma susceptibility e.g. Neuromedin and neurotensin Associated with bleeding diathesis e.g. GPCR as drug targets…
  45. 45. 45 GPCR as drug targets…
  46. 46. Diseases associated with G- proteins Abnormal G protein signalling can result by 1. Bacterial toxins (Cholera and pertussis) 2. Gene mutations  Loss of function mutations  Gain of function mutations 3. Altered GPCR folding 46
  47. 47. Mutations in GPCR Mutations in genes encoding are an important cause of human disease Help to define critical structure-function relationships Two types –  Loss-of-function : Block signalling in response to the corresponding agonist(s)  Hormone resistance, mimicking hormone deficiency  Gain-of-function : Lead to constitutive, agonist-independent activation of signaling  Mimic states of hormone excess 47
  48. 48. Cone opsins Colour blindness X-linked, AR Rhodopsin Retinitis pigmentosa AD; AR V2 vasopressin Diabetes insipidus X-Linked ACTH Familial ACTH resistance AR LH ♂ pseudohermaphrodite AR TSH Cong. hypothyroidism AR TRH Central hypothyroidism AR Diseases caused by Loss of function Mutation 48
  49. 49. 49 FSH Hypergonadotropic Ovarian failure AR Ca2+ sensing Hypocalciuric hypercalcaemia AD Ca2+ sensing Neonatal hyperthyroidism AR GHRH G H deficiency AR GnRH Central hypogonadism AR Endothelin-B Hirschsprung disease Complex Melanocortin 4 Extreme obesity Co-dominant PTH/PTHrP Chondrodysplasia AR Diseases caused by Loss of function Mutation
  50. 50. 50 Rhodopsin Congenital night blindness AD LH Familial ♂ precocious puberty AD LH Sporadic Leydig cells tumours Somatic TSH Familial non-autoimmune hyperthyroidism AD TSH Sporadic hyperfunctional thyroid adenomas Somatic Ca2+ sensing Familial hypocalcaemia AD PTH/PTHrP Jansen metaphyseal chondrodysplasia AD Diseases caused by Gain of function Mutation
  51. 51. Mis-folded GPCRs Point mutations resulting in protein sequence variations may result in production of mis-folded and disease-causing proteins  Retain proper function but end up in parts of cell where function is inappropriate, or even deleterious, to cell function. 51
  52. 52. Disease/ Abnormality GPCR Pharmacoperones Retinitis pigmentosa Rhodopsi n 9-cis-retinal, 11-cis-retinal, 11-cis- 7-ring retinal Nephrogenic diabetes insipidus V2R SR121463 (satavaptan), SR49059 (relcovaptan), VPA-985, YM087, OPC41061 (tolvaptan), OPC31260 Hypogonadotropic hypogonadism GnRHR Indoles, quinolones, erythromycin-derived macrolides Familial hypocalciuric hypercalcemia Ca2+ sensing NPS R-568 Diseases due to GPCR misfolding 52
  53. 53. POLYMORPHISMS OF GPCR  Variations in GPCR gene sequence can have important consequences beyond causing Mendelian diseases  As more polymorphisms are discovered more examples of variations in GPCR gene sequence will be found 53
  54. 54. POLYMORPHISMS.... Challenges Ahead Whether such differences are important in individual variation in drug response (pharmacogenomics) Whether they could confer susceptibility to disease. 54
  55. 55. Allosteric Modulators of G- protein •Bind receptor domains topographically distinct from orthosteric site, altering biological activity of orthosteric ligand by changing its binding affinity, functional efficacy or both. • Potential for engendering greater GPCR subtype-selectivity • Challenge for detecting /validating allosteric behaviors • Contribute to physical or pathophysiological processes. 55
  56. 56. ORPHAN GPCRs  Lack their pharmacological identities  Pre-genome era: Most GPCRs were found by sequence similarity using nucleic acid- based homology screening approaches  After genome sequencing: 150 Orphan GPCRs using bio-informatic analysis  First Orphan GPCR was G21, later found to be 5HT1A receptor in 1988 Focus of intense research effort, both in academia and in industry 56
  57. 57. 57 GPCR types No. of members Orphan receptors Glutamate- class GPCRs 22 Two third (15) Rhodopsin- class GPCRs 701 63 Adhesion- class GPCRs 33 Majority Frizzled/ taste GPCRs 36 (11 frizzled and 25 taste) None among frizzled ; Most taste Secretin-class GPCRs 15 None
  58. 58. The de-Orphanization of GPCRs  Evolutionarily conserved and thus are expected to be active  Reverse Pharmacological Approaches based on receptor reactivity & receptor binding are applied  Isolating natural ligand provides a first hint of function, structural cues for lead design  Once de-orphanised, GPCRs can be used for designing new drugs. 58
  59. 59. Tools for de-orphanization High -throughput screening  GPCR over expressing cells  Ligand libraries: chemicals, serum, peptides  Finding a robust marker: Measure receptor binding or Receptor reactivity  Finding an endogenous ligand 59
  60. 60. Search available orphans that have an effect on a specific therapeutic area Analyze Orphans using: 1. Laser capture micro-dissection to determine the localization 2. Microarray to compare the level of transcript expression Screening against Compound Libraries Identify compound hits and optimize for pre-clinical and, if successful, clinical trials 60
  61. 61. Issues of Orphan GPCR research  Deorphanization is a risky, lengthy and demanding endeavour  GPCRs exist not only as monomers but as dimers or higher oligomers  Concentration of transmitters in their natural environment. 61
  62. 62. GPCR Screening  Cell-based screens performed with calcium- sensitive or membrane-potential-sensitive dyes  Gs- and Gi-coupled GPCRs are assayed via cAMP determinations using either a cell- based real time cAMP assay or other validated cAMP assay platform  All screens include positive controls and a comprehensive report. 62
  63. 63. Recent developments  Ligand-induced selective signaling (LiSS): It states that different ligands selectively recruit different intracellular signaling proteins to produce different phenotypic effects in cells .  Terry Kenakin proposed this concept and is rapidly becoming a generic theme for GPCRs.  This phenomenon is referred to by different groups using a variety of terms such as: “functional selectivity,” “biased agonism,” “ligand-selective agonism,” “agonist-directed trafficking of signaling,” or “agonist-receptor 63
  64. 64. It has important implications in specific drug development and in minimizing side effects. E.g. the effects of the two naturally occurring GnRHs, GnRH I and GnRH II, operating through the single GnRH type I receptor. GnRH I is much more potent in generating inositol phosphate than in its antiproliferative effects on certain cells, whereas GnRH II does not show much difference between these two effects. An extreme example is a GnRH antagonist, which has no intrinsic stimulation of inositol phosphate generation but has potent antiproliferative activity. It has been shown that the Tyr8 of GnRH II is the main determinant of selective antiproliferative effects and identified residues in the TM domains and ECLs of the 64
  65. 65.  The LiSS concept has now been demonstrated for many GPCRs and is creating a new level of sophistication, which challenges the dogma that ligand engagement of a GPCR consistently elicits a specific intracellular signal. Instead, it has become increasingly clear that the nature of the ligand and the dynamically changing intracellular environment alter the flavor of the signaling. Indeed, it appears that there is a new era of drug discovery on our doorstep, in which screening for novel ligands will not simply involve receptor binding and/or the most convenient high-throughput functional signal output but instead will screen for the appropriate intracellular signal, which reflects the desired phenotypic response of a cell for a disease state or pathophysiology. Equally, appropriate cells will have to be used to ensure an appropriate intracellular context. Although these challenges are substantial, we believe they will bear fruit in the longer term efforts of GPCR drug discovery in the spin-off benefits of reduced failure in the clinic through lack of specificity and off-target effects. 65
  66. 66. 66
  67. 67. GPCR signaling independent of G proteins  There are many ways in which GPCRs can signal independently of G proteins. So, a case has been made for abandoning the term “G protein-coupled receptors” and referring to them as “seven-transmembrane receptors.”  The first convincing evidence for the existence of GPCR-independent signaling came from the works of 67
  68. 68.  An example is angiotensin II at its AT1 receptor activating both β-arrestin and G proteins. When antagonists such as angiotensin II-receptor blockers (losartan and valsartan) engage the binding site, neither signal is propagated. However, another type of antagonist (SII) does not activate the G protein pathway but exclusively recruits β-arrestin and activates ERK. 68 GPCR signaling independent of G proteins…
  69. 69. Constitutively active receptors  G-protein-coupled receptors may also be constitutively (i.e. spontaneously) active in the absence of any agonist.  This was first shown for β-adrenoceptor.  Histamine H3 receptor also shows constitutive activity.  It means that inverse agonists can play a role here. 69
  70. 70. GPCRs and drug discovery  Regarded as “Drug Discovery Engines” of 21st Century  G protein-coupled receptors (GPCRs) represent 50-60% of the current drug targets.  The pace of GPCR-targeted new molecular entities (NMEs) approved by the USFDA in the recent years still remains to a level near its historical average, with five in 2010, five in 2011, seven in 2012, six in 2013, and eight in 2014. 70
  71. 71. Novel pancreatic β-cell GPCRs  About 20 GPCRs have been found in pancreatic β-cells, all of which can potentially stimulate or inhibit insulin secretion. The glucagon-like peptide 1 (GLP1) receptor is one of these. Insulin secretion is stimulated by glucose transport through the glucose transporter 2 into the β-cell.  Activation of GPCRs such as GLP1 can enhance the amount of intracellular calcium, for example through activation of Gαq/11 and subsequent generation of IP3 and release of Ca++ from intracellular stores, thereby potentiating glucose stimulation of insulin secretion.  Among the other GPCRs identified in β-cells are the newly discovered free fatty acid receptors, GPR40, 43, and 41. GPR40 couples to Gαq/11, so free fatty acid would enhance the calcium response of the β-cell to glucose and increase insulin secretion. Insulin responses to glucose are improved in mutant mice overexpressing the GPR40 receptor and in normal rats treated with GRP40 agonists . 71
  72. 72. GPCRs as new therapeutic targets for type 2 diabetes GPCRs that have received recent attention in the field of diabetes therapeutics include 1. Incretin receptors: GLP1R, GIPR (GPR119) 2. Free fatty acid receptor:FFAR1 (GPR40), FFAR4 (GPR120) 3. Bile acid receptor: GPBAR1 (TGR5) 72
  73. 73. Novel neuroendocrine GPCRs regulating reproduction  There have been a number of breakthroughs in neuroendocrinology in the last year.  After the seminal discovery that kisspeptin/GPR54 acts as a major whole-body sensor mediating diverse effects on the GnRH neuron described mutations in neurokinin B (NKB) and its receptor (TACR3), which give rise to hypogonadotropic hypogonadism and pubertal failure.  The discovery of NKB, dynorphin A, and GnIH as neuroendocrine regulators has provided new opportunities for research on novel GPCRs in fine tuning the hypothalamic-pituitary-gonadal axis and provides new pathways in which to interrogate feedback mechanisms and metabolic, photoperiod, 73
  74. 74. Role of H4 receptor in asthma H4 receptor was discovered with an orphan GPCR gene sequence followed by pharmacology characterization. Animal models suggested a role for the H4 receptor in mediating asthma and chronic pruritus associated with conditions such as atopic dermatitis. TheH4 antagonist JNJ 39758979 has recently been found to have efficacy in preclinical models of pruritus, dermatitis, asthma, and arthritis. Several other H4 antagonists have also been entered into clinical trials for these 74
  75. 75. Concept of pharmacopherones Pharmacoperones or Chaperone  Small nonpeptide molecules do scaffolding in order to promote correct folding.  Regulation of routing of cellular proteins will provide opportunity for novel drug development. 75
  76. 76. Permeate plasma membrane Enter cells Correct folding Allowing routing to plasma membrane How Chaperones Work ? 76 Bind selectively to misfolded proteins
  77. 77. Deorphanisation of GPR55  GPR55 has been recently deorphanized to be a receptor for lysophophatidylinositol. Other GPR55 ligands identified so far are neither cannabinoids nor bind to the cannabinoid CB1 and CB2 receptors.  GPR55 has been implicated in three therapeutic areas, including the regulation of energy intake and expenditure, resorption of bone, and agonist pro- carcinogensis. 77
  78. 78. The simple dogma that underpins much of our current understanding of GPCRs, namely, one GPCR gene− one GPCR protein− one functional GPCR− one G protein −one response is showing distinct signs of wear. 78 Future Prospects & Conclusions
  79. 79. Future Prospects & Conclusions  Ever expanding field of research  Concept is diverting from a linear signaling to increasingly complex signaling networks  Next generation platforms for studying internalisation and heterodimerisation is to adopt novel, universal β-arrestin recruitment assays for known and orphan GPCRs instead of second messenger signaling assays 79
  80. 80.  Further studies are needed to explore  Advances in novel forms  Methods to rescue function of misfolded or truncated GPCRs  Complexity demands collaborative approaches between persons of medicinal chemistry, analytical pharmacologists & bioinformatic experts 80 Future Prospects & Conclusions
  81. 81.  GPCRs were once considered highly tractable targets.  Current targets much lower success rates ◦ Low hanging fruit largely picked ◦ Lack of Hits ◦ Hits have high molecular weight  Poor PK/in vivo activity  Difficult to optimize 81 Future Prospects & Conclusions
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