Types of receptors
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Receptor Pharmacology: Types of Receptors
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Types of receptors
- 1. TYPES OF RECEPTORS MODERATOR – DR. VANDANA TAYAL PRESENTED BY – DR. SAHIL KUMAR
- 2. OUTLINE Definition History Receptor Theories Criteria for classifying receptors Types of Receptors : • Ligand Gated Ion Channels • G-Protein Coupled Receptors • Enzyme Linked Receptors • Nuclear Receptors Summary
- 3. TARGETS FOR DRUG ACTION
- 4. What are receptors? Receptor is a cellular macromolecule, whose function is to recognize and respond to chemical signals
- 5. HISTORY 1878 – John Langley proposed 'receptive substance' 1905 - Receptive substance on surface of skeletal muscle mediate drug action. Different in different species Paul Ehrlich designated 'receptor‘ to be anchoring group of the protoplasmic molecule for the administered compound Envisaged molecules extending from cells that the body could use to distinguish and mount an immune response to foreign objects 1948 - Ahlquist showed the differential action of adrenaline & demonstrated its effects on two distinct receptor populations & the theory of receptor-mediated drug interactions gained acceptance
- 6. 1970s - Pharmacology entered a new phase following the development of receptor-labelling techniques Multiple subtypes of receptors now known, which has paved the way for clinically superior drugs
- 7. CRITERIA FOR CLASSIFYING RECEPTORS Pharmacological Criteria Tissue Distribution Ligand Binding Transducer Pathway Molecular Cloning
- 8. RECEPTOR THEORIES • Occupation theory (1937) • Rate theory (1961) • Two State Receptor Theory (1983)
- 9. TYPES OF RECEPTORS Ligand Gated Ion Channels G-Protein Coupled receptors Enzyme Linked receptors Nuclear receptors
- 10. RECEPTOR SUBTYPES & NOMENCLATURE IUPHAR- International Union of Basic and Applied Pharmacology
- 11. TYPE 1 : LIGAND GATED ION CHANNELS • Ionotropic Receptors • Typically receptors on which neurotransmitters act • Timescale : Milliseconds • Localization : Membrane • Effector : Ion Channel • Coupling : Direct • Examples : Nicotinic Ach Receptor, GABAA Receptor, Glutamate Receptor, Glycine receptor, 5 Hydroxytryptamine type 3 (5 – HT3)
- 13. MOLECULAR STRUCTURE Nicotinic Ach receptor studied in great detail Pentameric Assembly of 4 types of subunits α, β, γ and δ 4 membrane spanning α-helices inserted into membrane 2 Ach binding sites, both must bind Ach molecules for receptor activation Lining of central transmembrane pore formed by helical segments of each subunit (negatively charged AA). 5 helices sharply kinked inwards halfway, forming a constriction
- 14. GATING MECHANISM Ach molecules bind, twists the α subunits, kinked helices either straighten out or swing out of the way, opening channel pore Conductance produced by different Ach like agonists is the same whereas mean channel lifetime varies Mutation of a critical residue in helix changes channel from being cation selective (excitatory) to being anion selective (typical of receptors for inhibitory transmitters like GABA)
- 15. Most excitatory neurotransmitters cause Increase in Na+ and K+ permeability net inward current carried mainly by Na+ Depolarization of the membrane (probability to generate action potential) Speed of this response implies that coupling between the receptor and ionic channel is a direct one (no intermediate biochemical steps involved)
- 16. VOLTAGE OPERATED CHANNELS These channels open when the cell membrane is depolarised. They underlie the mechanism of membrane excitability Activation induced by membrane depolarisation is short lasting, even if the depolarisation is maintained The most important channels in this group are selective sodium, potassium or calcium channels
- 18. Ligand Gated receptors v/s VOCs • ROCs appear to assume only two states whereas VOCs undergo a third state called refractory (inactivated) state. • Voltage gated channels have no major endogenous modulator (like Ach)
- 19. TYPE 2 : G – PROTEIN – COUPLED RECEPTORS • Alfred Goodman Gilman & Martin Rodbell (1994) • Metabotropic or 7 – Transmembrane/ Heptahelical receptors • Largest family • Timescale : Seconds • Location : Membrane • Effector : Channel or Enzyme • Coupling : G- Protein • Examples : adrenoceptors, Muscarinic Ach, histamine, serotonin, opioid, cannabinoid, amine, peptide, prostanoid receptors
- 21. MOLECULAR STRUCTURE • Single polypeptide chain 1100 residues. 7 Transmembrane α-helices, an extracellular N-terminal domain and intracellular C-terminal domain • 3rd cytoplasmic loop couples to the G- Protein
- 22. G-PROTEINS AND THEIR ROLE • Family of membrane-resident proteins whose function is to recognize activated GPCRs and pass on the message to the effector systems that generate a cellular response • Function of G-Protein: 3 subunits (α, β, γ) are anchored to the membrane through attached lipid residues Coupling of the α subunit to an agonist-occupied receptor causes bound GDP to exchange with intracellular GTP; α–GTP complex dissociates from receptor and from βγ complex
- 24. • Amplification : A single agonist–receptor complex can activate several G- protein molecules, each of these can remain associated with the effector enzyme for long enough to produce many molecules of product (Second messenger) • Four main classes of G-protein (Gs, Gi, Go and Gq) show selectivity with respect to both the receptors and the effectors with which they couple • Cholera toxin and pertussis toxin
- 26. Adenylate cyclase • catalyses formation of the intracellular messenger cAMP • cAMP activates various protein kinases that control cell function in many different ways by causing phosphorylation of various enzymes, carriers & other proteins Targets for G-Proteins
- 27. Phospholipase C/IP3/DAG • catalyzes the formation of IP3 and DAG from membrane phospholipid • IP3 increases free cytosolic Ca2+ (releasing Ca2+ from intracellular compartments) which initiates many events • DAG activates protein kinase C, which controls many cellular functions by phosphorylating proteins
- 28. Ion Channels appears to be general mechanism for controlling K⁺and Ca⁺⁺ channels by direct interaction between the βγ-subunit of G0 and the channel Phospholipase A2 (formation of arachidonic acid and eicosanoids) The Rho/Rho kinase system G12/13type G-protein. α subunit interacts with guanosine nucleotide exchange factor, which facilitates GDP–GTP exchange at another GTPase, Rho. On exchange Rho activated & activates Rho kinase - phosphorylates substrate proteins The MAP kinase system activated by cytokines and growth factors acting on kinase-linked receptors and by GPCR ligands. Controls processes involved in cell division, apoptosis and tissue regeneration
- 29. Protease Activated Receptors End of extracellular N-terminal tail of the receptor snipped off to expose 5-6 N-terminal residues that bind to receptor domains in extracellular loops, functioning as ‘tethered agonist’ A PAR molecule can be activated only once Inactivation by further proteolytic cleavage that frees tethered ligand, or by desensitization
- 31. TYPE 3 : KINASE LINKED AND RELATED RECEPTORS • Large, heterogenous group responding mainly to protein mediators. • Timescale : Hours • Location : Membrane • Effector : Protein Kinases • Coupling : Direct • Examples : Insulin, Growth Factors, Cytokine, ANF receptors
- 33. MOLECULAR STRUCTURE AND TYPES Large proteins - single chain ~ 1000 residues, single membrane-spanning helical region, with a large extracellular ligand-binding domain, and an intracellular domain of variable size and function Types – • Receptor tyrosine kinases (RTKs) • Serine/threonine kinases • Cytokine receptors. lack intrinsic enzyme activity. When occupied, they associate with and activate, a cytosolic tyrosine kinase, such as Jak (the Janus kinase)
- 34. SIGNAL TRANSDUCTION Generally involves dimerization autophosphorylation of tyrosine residues, act as acceptors for the SH2 domains of intracellular proteins Involved mainly in events controlling cell growth and differentiation, and act indirectly by regulating gene transcription Two important pathways are: – the Ras/Raf/ MAP kinase pathway - cell division, growth and differentiation. – the Jak/Stat pathway activated by many cytokines - controls the synthesis and release of many inflammatory mediators
- 38. TYPE 4 : NUCLEAR RECEPTORS • Regulate gene transcription. ?Misnomer • Timescale : Hours • Location : Intracellular • Effector : Gene transcription • Coupling : Via DNA • Examples : Steroid Hormones, Thyroid Hormones, Retinoic acid and Vitamin D receptors • This family includes a great many (40%) orphan receptors
- 40. The N-terminal domain harbours the AF1 site that binds to other cell specific transcription factors and modifies the binding or activity of the receptor itself The Core domain consists of the structure responsible for DNA recognition and binding. They bind to the hormone response elements located in genes to regulate them Hinge region in the molecule allows it to dimerise with other NRs and also to exhibit DNA binding C-terminal domain contains the ligand binding module and is specific to each class of receptor
- 41. CLASSIFICATION OF NUCLEAR RECEPTORS
- 44. CONTROL OF GENE TRANSCRIPTION Hormone Response Elements : Short (4-5 bp) sequences of DNA to which the NRs bind to modify gene transcription, present symmetrically in pairs. Recruits co-activators or co-repressors to modify gene expression through its AF1 and AF2 domains This receptor family responsible for the pharmacology of approximately 10% prescription drugs
- 45. SUMMARY
- 46. THANK YOU
Notas do Editor
- Many drugs exhibit structural specificity of action, i.e. specific chemical configuration is associated with a particular action, e.g. isopropyl substitution on the ethylamine side chain of sympathetic drugs produces compounds with marked cardiac and bronchial activity-most β adrenergic agonists and antagonists have this substitution. A 3 carbon internitrogen separation in the side chain of phenothiazines results in antidoparninergic-antipsychotic compounds, whereas 2 carbon separation produces anticholinergic antihistaminic compounds. Further, chiral drugs show stereospecificity in action, e.g. levanoradrenaline is 10 times more potent than dextro noradrenaline; d-propranolol is about 100 times less potent in blocking β receptors than the /-isomer, but both are equipotent local anaesthetics. Thus, the cell must have some mechanism to recognize a particular chemical configuration and three dimensional structure. It was calculated by Clark that adrenaline and acetylcholine produce their maximal effect on frog's heart by occupying only 1 /6000th of the cardiac cell surface thus, special regions of reactivity to such drugs must be present on the cell.
- Eg cardioselective B-blockers
- Ternary complex model – read up
- mRNA splicing : Single receptor can give rise to more than one receptor isoform. Depending on the location of the splice sites, splicing can result in inclusion or deletion of one or more mRNA coding regions, giving rise to long or short forms of the proteins which produces receptors with different binding characteristics and different signal transduction mechanism. mRNA editing involves substitution of one base in the mRNA for another and hence a small variation in AA sequence of the receptor. provides a uniform guideline for naming and classifying the results in the public domain. Provides global free access to characterization data for receptors, ion channels and drugs through the Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR)
- , conformational change occurs in extracellular part of the receptor, ….. Influx of about 107 ions/second occurs through a single channel under normal physiological conditions & the mean open time is 1-2 ms. Magnitude of single channel conductance confirms that permeation occurs through a physical pore through membrane because ion flow is too large for a carrier mechanism
- Not generally classified as ‘receptors’ because they are not the immediate targets of fast neurotransmitters.
- Drug-binding domains of voltage-gated sodium channels. The multiplicity of different binding sites and effects appears to be typical of many ion channels. DDT, dichlorodiphenyltrichloroethane (dicophane, a well-known insecticide); GPCR, G-protein-coupled receptor; PKA, protein kinase A; PKC, protein kinase C.
- G protein is “guanine nucleotide binding protein” The human genome includes genes encoding about 400 GPCRs (excluding odorant receptors), which constitute the commonest single class of targets for therapeutic drugs, and it is thought that many promising therapeutic drug targets of this type remain to be identified.
- First GPCR to be fully characterized was β-Adrenoceptor. Family A is largest. C smallest. X- Ray Crystallography and Fluorescence techniques help to obtain a clearer picture of mechanism of activation of GPCRs and designing new GPCR ligands 3 distinct families. Considerable sequence homology between members of one family but not between different families. They share the same heptahelical structure, differ in length of extracellular N-terminus and location of agonist binding domain. For small molecules, NA, the ligand-binding domain of class A receptors is buried in the cleft between the α-helical segments within the membrane. Peptide ligands bind more superficially to the extracellular domain
- interacts with a target protein (target 1). The βγ complex may also activate a target protein (target 2). GTPase activity of α subunit is increased when target protein is bound, leading to hydrolysis of the bound GTP to GDP, whereupon the α subunit reunites with βγ.
- three subunits: α, β and γ. Guanine nucleotides bind to the α subunit, which has enzymic activity, conversion of GTP to GDP. The β and γ subunits remain together as a βγ complex. All three subunits are anchored to the membrane through a fatty acid chain, coupled to the G-protein through a reaction known as prenylation. In the ‘resting’ state, the G-protein exists as an unattached αβγ trimer, with GDP occupying the site on the α subunit. When a GPCR is activated by an agonist molecule, a conformational change occurs, involving the cytoplasmic domain of the receptor, causing it to acquire high affinity for αβγ. Association of αβγ with the receptor occurs within about 50 ms, causing the bound GDP to dissociate and to be replaced with GTP (GDP–GTP exchange), which in turn causes dissociation of the G-protein trimer, releasing α-GTP and βγ subunits; these are the ‘active’ forms of the G-protein, which diffuse in the membrane and can associate with various enzymes and ion channels, causing activation of the target. It was originally thought that only the α subunit had a signalling function, the βγ complex serving merely as a chaperone to keep the flighty α subunits out of range of the various effector proteins that they might otherwise excite. However, the βγ complexes actually make assignations of their own, and control effectors in much the same way as the α subunits Attachment of the α subunit to an effector molecule actually increases its GTPase activity, the magnitude of this increase being different for different types of effector.
- Cholera toxin and pertussis toxin : These toxins, which are enzymes, catalyse a conjugation reaction (ADP ribosylation) on the α subunit of G-proteins. Cholera toxin acts only on Gs, and it causes persistent activation. Many of the symptoms of cholera, such as the excessive secretion of fluid from the gastrointestinal epithelium, are due to the uncontrolled activation of adenylate cyclase that occurs. Pertussis toxin specifically blocks Gi and Go by preventing dissociation of the G-protein trimer.
- G 12/ 13 regulate cell processes through the use of Guanine Nucleotide Exchange Factors – they are not sensitive to pertussis toxin. Genes- GNA12, GNA13
- increased activity of voltage gated calcium channels in heart muscle cells. Phosphorylation of these channels increases the amount of Ca2+ entering the cell during the action potential, and thus increases the force of contraction of the heart In smooth muscle, cAMP-dependent protein kinase phosphorylates (thereby inactivating) another enzyme, myosin-light-chain kinase, which is required for contraction. This accounts for the smooth muscle relaxation Adenylyl cyclase can be activated directly by certain agents, including forskolin and fluoride ions 11 PDE subtypes exist; inhibited by theophylline & caffeine, Milrinone for CHF (PDE3), Rolipram for asthma (PDE 4) and Sidenafil (PDE 5)
- Receptor-mediated activation of phospholipase C results in the cleavage of phosphatidylinositol bisphosphate (PIP2), forming diacylglycerol (DAG) (which activates protein kinase C) and inositol trisphosphate (IP3) (which releases intracellular Ca2+). The role of inositol tetraphosphate (IP4), which is formed from IP3 and other inositol phosphates, is unclear, but it may facilitate Ca2+ entry through the plasma membrane. IP3 is inactivated by dephosphorylation to inositol. DAG is converted to phosphatidic acid, and these two products are used to regenerate PI and PIP2. many events, including contraction, secretion, enzyme activation and membrane hyperpolarization.
- Direct G-protein–channel interaction first shown for cardiac muscle, In cardiac muscle, mAChRs enhance K+ permeability (thus hyperpolarising the cells and inhibiting electrical activity). Similarly in neurons, many inhibitory drugs such as opioid analgesics reduce excitability by opening K+ channels or inhibiting Ca2+ channels. By enhancing hypoxia-induced pulm artery vasoconstriction, Rho kinase active. thought to be imp pathogenesis of pulmonary HT. Specific Rho kinase inhibitors e.g. fasudil. Rho–GDP, the resting form, is inactive, but when GDP–GTP exchange occurs, Rho is activated and controls smooth muscle contraction, proliferation, angiogenesis and synaptic remodeling.
- Activation of GPCRs normally by a diffusible agonist, One of the family of PARs, PAR-2 A PAR molecule can be activated only once, because the cleavage cannot be reversed, so continuous resynthesis of receptor protein is necessary. Inactivation occurs by a further proteolytic cleavage that frees the tethered ligand, or by desensitisation, involving phosphorylation, after which the receptor is internalised and degraded, to be replaced by newly synthesized protein. Examples? PAR-2 activated by a protease released from mast cells and expressed on sensory neurons (role in inflammatory pain).
- Homologous (agonist-specific) desensitisation involves phosphorylation of the activated receptor by a specific kinase (GPCR kinase, GRK). The phosphorylated receptor (P-R) then binds to arrestin, causing it to lose its ability to associate with a G-protein, and to undergo endocytosis, which removes the receptor from the membrane. Heterologous (cross-) desensitisation occurs as a result of phosphorylation of one type of receptor as a result of activation of kinases by another. PKA and PKC, protein kinase A and C, respectively.
- Incorporate a tyrosine kinase moiety in the intracellular region. E.g. receptors growth factors, such as epidermal growth factor and nerve growth factor, Toll-like receptors that recognize bacterial LPS and play an important role in the body’s reaction to infection, The insulin receptor (although it has a more complex dimeric structure). Similar in structure to RTKs but phosphorylate serine and/or threonine residues rather than tyrosine. E.g. receptor for transforming growth factor (TGF). Ligands for these receptors include cytokines such as interferons and colony-stimulating factors involved in immunological responses.
- Ras functions like a G-protein, and conveys the signal (by GDP/GTP exchange) from the SH2-domain protein, Grb, which is phosphorylated by the RTK. The last of these, mitogen-activated protein (MAP) kinase phosphorylates one or more transcription factors that initiate gene expression, resulting in a variety of cellular responses, including cell division. This three-tiered MAP kinase cascade forms part of many intracellular signalling pathways involved in a wide variety of disease processes, including malignancy, inflammation, neurodegeneration, atherosclerosis and much else.
- Dimerisation of these receptors occurs when the cytokine binds, and this attracts a cytosolic tyrosine kinase unit (Jak) to associate with, and phosphorylate, the receptor dimer. Jaks belong to a family of proteins, different members having specificity for different cytokine receptors. Among the targets for phosphorylation by Jak are a family of transcription factors (Stats). These are SH2-domain proteins that bind to the phosphotyrosine groups on the receptor–Jak complex, and are themselves phosphorylated. Thus activated, Stat migrates to the nucleus and activates gene expression
- • A few hormone receptors (e.g. atrial natriuretic factor) have a similar architecture and are linked to guanylyl cyclase.
- Central role of kinase cascades in signal transduction. Kinase cascades are activated by GPCRs, either directly or via different second messengers, by receptors that generate cGMP, or by kinase-linked receptors. The kinase cascades regulate various target proteins, which in turn produce a wide variety of short- and long-term effects.
- orphan receptors—receptors with no known well-defined ligands. RXR, a receptor cloned on the basis of its similarity with the vitamin A receptor and that was subsequently found to bind the vitamin A derivative 9-cis-retinoic acid. Over the intervening years, binding partners have been identified for many NRs (‘adopted orphans’; e.g. RXR) but the ligands of many others (’true orphans’) have yet to be identified, or perhaps do not exist as such.
- (activation function 1) At the molecular level, this comprises two Zn fingers—cysteine- (or cystine-/histidine-) rich loops in the amino acid chain that are held in a particular conformation by zinc ions.
- Class I : In the absence of their ligand, these NRs are predominantly located in the cytoplasm, complexed with heat shock and other proteins and possibly reversibly attached to the cytoskeleton. Liganded receptors generally form homodimers and translocate to the nucleus, where they can transactivate or transrepress genes by binding to ‘positive’ or ‘negative’ hormone response elements. Large numbers of genes can be regulated in this way by a single ligand Class II : LXR = Liver Oxysterol Receptor, FXR = Farsenoid (Bile acid) receptor, RXR = Retinoid receptor. Xenobiotic receptor = SXR. Bind to thiazolidinediones and Fibrates Hybrid Class = Retinoic Acid receptor (RAR)
- Ligand binding domain, DNA binding domain
- consensus sequence : a sequence of DNA having similar structure and function in different organisms. In nucleus, ligand-bound receptor recruits further proteins including co-activators or co-repressors to modify gene expression through its AF1 and AF2 domains. the enzymes that it regulates affect the pharmacokinetics of some 60% of all prescription drugs
- May bring about non-genomic actions by directly interacting with factors in the cytosol, or they may be covalently modified by phosphorylation or by protein–protein interactions with other transcription factors such that their function is altered.
- An example of the former is myasthenia gravis, a disease of the neuromuscular junction due to autoantibodies that inactivate nicotinic acetylcholine receptors.
- One of these involves the receptor for thyrotropin, producing continuous oversecretion of thyroid hormone; another involves the receptor for luteinizing hormone and results in precocious puberty.