Lecture 2: Receptor Mechanisms

FM2003 | PT1445 | PT2201 Lecture 2 Dr Ashley Allshire Dept. Pharmacology & Therapeutics a.allshire@ucc.ie Receptor mechanisms Signalling mechanisms as drug targets Agonists bind to same site as ligand, producing same kind of signal, or bind to another site on receptor, producing no signal themselves but enhancing the signal of endogenous ligand (allosteric) Antagonists bind to same site as ligand, diminishing or blocking the signal competitively, or bind to an allosteric site on the receptor, diminishing signal produced by endogenous ligand, or cross the membrane & intercept the signal downstream of the receptor. Lecture 2 2 Drug action at cell level R&D chapter 3 Endogenous ligand Receptor Drug (agonist) Altered cell function signal transduction Receptor proteins  belong to one of 4 types that differ structurally & functionally  3 of these receptor groups are situated in the cell (plasma) membrane; the fourth is intracellular (cytosol/nucleus)  all include a ligand-binding domain & an effector domain Lecture 2 3 Lecture 2 4 R&D fig. 3.2 Receptor classes 1 Ion channels  4-5 subunits ligand Cell membrane  ligand-gated  conduct Na+, K+, Cl- or Ca2+  alter cell membrane potential or intracellular ionic composition  fast communication (s-ms) Examples Nicotinic acetylcholine receptor (nAChR) GABAA receptor Glycine receptor Glutamate receptors 5-HT3 receptor P2x purine receptor Lecture 2 5 GABAA nAChR Na+ acetylcholine Side view a b d Top view a b d a - - - Na+ - a g 0.65 nm pore (cation selective) Lecture 2 6 2 G protein-coupled receptors N receptor G protein effector cell membrane a C GTP bg GDP second messenger(s) Receptors linked to effectors (adenylate cyclase/phospholipase C/ion channels) that change intracellular level of second messenger(s), via GTP-binding proteins (G proteins) Lecture 2 7 GPCR general mechanism ligand effector ion channel (e.g. K+) membrane hyperpolarization receptor effector enzyme a GDP b g GDP second messenger(s) GTP → intracellular effects Lecture 2 8 G-protein coupled receptors N Ligand-binding domain cont.  Single polypeptide chain (800-1,100 aa residues), spanning membrane 7 times: a-Helices most highly conserved  Third intracellular loop couples to G protein & determines G-protein selectivity. C G-protein coupling domain  Smaller ligands bind within helix cluster, larger ligands at extracellular surface. e.g. muscarinic acetylcholine receptor (mAChR) GABAB receptor 5-HT adrenoceptors (serotonin) receptors (except 5-HT3) histamine H2, eicosanoid, many peptide & purine R dopamine receptors (thrombin & other protease receptors) opioid receptors chemoreceptors Lecture 2 9 Variation on a theme – protease activated receptors Thrombin receptor Receptor activated by protease cleavage of N-terminus domain, revealing a ‘tethered agonist’: thrombin (a serine protease) tethered agonist thrombin receptor phosphorylated active inactive desensitized Lecture 2 10 G proteins  Subtypes either activate (Gs, Gq) or inhibit (Gi, G0) specific effectors  Signalling takes seconds to minutes  Scope for signal amplification  Second messengers generated in turn activate protein kinases that phosphorylate target proteins cAMP-dependent protein kinases (PKA) cGMP-dependent protein kinases (PKG) Ca2+- & Ca/calmodulin-dependent protein kinases (PKC) Diacylglycerol-dependent PKC etc. Lecture 2 11 Effectors (= ‘effectors’ on previous slides) (= end targets that mediate cell response(s)) R&D fig. 3.14 Lecture 2 12 Second messengers  cAMP synthesized from ATP by adenylate cyclase activates PKA that phosphorylates target proteins broken down by phosphodiesterases  cGMP formed from GTP by guanylate cyclase, (etc.)  diacylglycerol & IP3 (inositol tris phosphate) formed from phosphoinositides by phospholipase C IP3 triggers Ca2+ release from ER DAG co-activates PKC  Ca2+ enters across cell membrane or released from intracellular stores, via Ca channels activates PKC that phosphorylates target proteins re-sequestered into ER or pumped out of cell Lecture 2 13 3 Receptors with direct kinase activity Receptors for polypeptide hormones regulating cell proliferation & differentiation e.g. epidermal growth factor (EGF) insulin-like growth factor (IGF-1) nerve growth factor (NGF) platelet-derived growth factor (PDGF) These receptors are monomers - single polypeptide chain comprising a(n) ◼ extracellular domain containing the ligand-binding site ◼ single a-helix crossing the membrane ◼ intracellular domain with catalytic activity tyrosine kinase serine/threonine kinase Lecture 2 14 Kinase-linked receptors continued ligand out cell membrane in protein  ATP tyr-phosphorylated protein ADP Tyrosine kinase receptors regulate growth, differentiation, development e.g. receptors for insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), etc.  Serine/threonine kinase receptors e.g. tumour growth factor (TGF) b receptor Lecture 2 15 Receptor tyrosine kinase (RTK) ligand cell membrane P P P P RAS P activation GDP Raf phosphorylation altered gene transcription GTP MEK phosphorylation various MAP kinase transcription Grb2 phosphorylation factors nucleus Lecture 2 16 Cell response to cytokines Cytokines are soluble proteins that regulate cell growth, differentiation & function. They include  interferons a b g formed in & secreted by virus-infected cells bind to membrane receptors ➔  cell growth  expression of antiviral proteins  interleukins 2-7, 9-12, 15 Lecture 2 17 A variation on the RTK theme Cytokine receptor ligand P P P P Stat P P Stat altered gene transcription Jak Jak Lecture 2 18 4 N Intracellular receptors Ligand-binding domain lipid-soluble ligand transcriptionactivating domain C hsp90 hsp90 Activated receptor binds response elements in DNA  Process takes minutes to hours  Therapeutic consequences DNA-binding domain alters transcription of specific genes Lag in response, e.g. slow onset in anti-inflammatory effect of glucocorticoids Effects can persist long after agonist concentration is reduced to zero 19 Intracellular receptors continued These mediate cell response to  Steroids (glucocorticoids, mineralocorticoids, sex steroids) & vitamin D  Thyroid hormone  Inducers of drug metabolism, acting via the Ah receptor e.g. barbiturates, TCDD (tetrachlorodibenzodioxan) Lecture 2 20 Glucocorticoid receptor glucocorticoid-binding protein GBP G glucocorticoid G hsp70 GR IPGR hsp90 glucocorticoid receptor altered gene transcription glucocorticoid response element GRE GRE Lecture 2 cytosol nucleus 21 Intracellular receptors, continued PPARs (peroxisome proliferator-activated receptors) Bind fatty acids & FA metabolites (prostaglandins, polyunsaturated FAs) → regulate genes for lipid / carbohydrate metabolism/transport  PPARa role in dyslipidaemias, coronary heart disease, obesity agonists include fibrates, plasticizers  PPARg1/2 roles in diabetes, atherosclerosis, cancer agonists include thiazolidinediones (type 2 diabetes) antagonists include the plasticizer bisphenol A  PPARd Lecture 2 22 Summary  Cell response to ligand or drug is via any of 4 general signalling pathways that integrate extracellular chemical (& physical) information.  Receptors are located in the cell membrane or intracellularly.  Signalling rate ranges from rapid (ligand-gated ion channels) to slow (intracellular receptors).  Specificity of signalling depends on target enzymes/ion channels expressed in a particular cell type, resulting in appropriate response.  Receptor number & activity may vary over time. 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