A/Prof. Ken Rodgers School of Life Sciences

Lecture Content

  • To describe the structure and activity of receptors and ion channels
  • To describe the various forms of receptor-effector linkages
  • Receptor classification
  • Other drug targets such as enzymes and transporters

References

  • Rang HP, Dale MM, Ritter JM & Flower RJ, Henderson G (2016) Pharmacology, 8th Edition, Churchill Livingstone, Sydney
    • Chapter 3. How drugs act: molecular aspects

Targets for drug action

Targets for drug action 1

Paul Ehrlich: ‘drug action must be explicable in terms of conventional interactions between drugs and tissues’

Targets for drug action 2

  • Receptors
    • 1. Ligand-gated ion channels (ionotropic receptors)
    • 2. G-protein coupled receptors (metabotropic receptors)
    • 3. Kinase-linked receptors
    • 4. Nuclear receptors (intracellular receptors)
  • Ion channels
    • Voltage-gated ion channels
  • Enzymes
  • Transporters (carriers)
    • Symports and antiports

Drug targets: receptors

Receptor Structure

  • Receptors: An intracellular or cell-surface protein with which a drug or a signalling molecule interacts, to initiate a chain of biochemical events in the cell or organism
  • Receptors are proteins, glycoproteins or lipoproteins – these proteins provide a complex 3D shape
  • Four receptor SUPERFAMILIES are known

Ligand-Gated Ion Channels

Receptor Super Families

Ion channels

  • Gateways in the cell membrane that allow the passage of particular ions

Ligand-gated: trigger is agonist binding

Voltage-gated: trigger is a change in transmembrane potential

Structure of ligand-gated ion channels

Structure of ligand gated ion channels

Ionotropic receptors

  • Ligand-gated ion channel
    • Location: membrane
    • Effector: ion channel
    • Coupling: direct
    • Examples:
      • Nicotinic acetylcholine (ACh)
      • γ-Aminobutyric acid (GABA)
      • Excitatory amino acids (eg. NMDA, aspartate)
      • Glycine
    • Timescale: extremely rapid cell activation with a time scale of milliseconds
    • (note binding to receptor site is known as orthosteric binding as opposed to allosteric binding)

Ionotropic receptors

  • Allows ions to cross membrane thereby causing:
    • membrane polarisation (Cl–, K+)
    • membrane depolarization (Ca2+, Na+)
    • muscle contraction (nicotinic AChR of skeletal muscle)
    • signal transduction (via Ca2+ mobilisation or influx)

Ionotropic receptors

  • Example – Nicotinic AChR
  • 288 kDa heteropentamer with 5 subunits (α, γ, α, β, δ, each 50 to 58 kDa) plus associated non-selective cation channel (for +ve charged ions)
  • Each subunit contains 4 membrane spanning α-helixes to form pore ~0.7 nm in diameter
  • 2 ACh molecules must bind to 2 α subunits for full activation conformational change to open ion channel (twisting of α subunits )
  • Allows passage of Na+, K+, Ca2+ but at normal resting potentials, Na+ is major ion

Nicotinic ACh receptor

Nicotinic ACh receptor

Summary: ligand-gated ion channels

  • sometimes called ionotropic receptors
  • involved mainly in fast synaptic transmission
  • several structural families exist, the commonest being heteromeric assemblies of four or five subunits, with transmembrane helices arranged around a central aqueous channel
  • ligand binding and channel opening occur on a millisecond timescale.
  • examples: nicotinic acetylcholine, GABA type A (GABAA) and 5-hydroxytryptamine type 3 (5-HT3) receptors.

Other ligand-gated ion channels

Receptor heterogeneity within families gives rise to subfamilies of different receptors in different tissues (eg nAChR in different regions of the brain

G-Protein coupled receptors

Types of receptor-effector linkage

receptor effector linkage

The importance of G-proteins

G-proteins

Structure of G protein coupled receptors

eg. muscarinic acetylcholine receptor

G-protein coupled receptors

G-protein coupled receptors

  • G–Protein Coupled Receptors (GPCRs) or ‘metabotropic’ receptors
  • Location: membrane
  • Effector: channel or enzyme
  • Coupling: G-protein

(affinity for guanyl nucleotides (GDP/GTP)

  • Examples:
    • Muscarinic ACh
    • Adrenoceptors
  • Timescale: slow cell activation with a time scale of seconds

Structure of muscarinic AcH

  • Seven transmembrane helix (heptahelical) structure

Family A: monoamine, neuropeptide and chemokine receptors

Family B: calcitonin and glucagon receptors

Family C: glutamate and GABA receptors

G-protein coupled receptors 2

G-protein coupled receptors 3

  • Amplification of signal: one receptor can activate many G-proteins
  • Active G-proteins can cause effector enzymes to produce many intracellular second messengers
  • Principal second messengers:
    • 1. Cyclic adenosine monophosphate (cAMP)
    • 2. Ca2+
    • 3. phosphoinositides (eg IP3 and DAG)

G-proteins: general scheme

G-protein coupled receptors 4

  • G-proteins
  • Involves intermediary G-proteins (Guanyl nucleotide-binding protein) present in receptor-membrane complex
  • G-proteins serve 3 roles
    • 1. G-proteins bind guanosine triphosphate (GTP) and GDP
      G-proteins exist in 2 states:
  • active form – GTP bound
  • inactive form – GDP bound
  • 2. G-proteins provide link between ligand-activated receptor and effector (enzyme/ion channel)
  • 3. G-Proteins have intrinsic GTPase activity which spontaneously hydrolyses bound GTP to bound GDP (switch themselves off)

The function of G-proteins

function of G-proteins

G-proteins offer specificity

  • Gi (‘i‘ for inhibitory) and Gs (‘s‘, for stimulatory) are heterotrimeric signal transduction complexes (αβγ)
  • α-subunit interacts with a specific receptor (ie muscarinic, dopamine, noradrenaline) and a specific enzyme (eg. adenylate cyclase)
  • On Gαi / Gαs activation, α subunit can dissociate from βγ
G-proteins specificity

Primary effectors for GPCRs 1

  • Targets for G-proteins
    • Adenylate cyclase/cAMP system
    • Phospholipase C/inositol phosphate system
    • Regulation of ion channels

Calcium vs. cAMP

Kinase-linked receptors

Types of receptor-effector linkage

receptor effector linkage

Structure of kinase-linked receptors

Structure of kinase linked receptors

Kinase-linked receptors 1

  • Location: membrane
  • Effector: protein kinases
  • Coupling: direct
  • Examples:
    • Insulin
    • Growth factors eg. Epidermal growth factor (EGF), nerve growth factor, platelet derived growth factor (PDGF)
    • Cytokine receptors eg. interferon-gamma (IFN-γ)

Kinase-linked receptors 2

  • Timescale: cell activation with a time scale of minutes to hours
  • These receptors consist of an extracellular hormone binding domain and a cytoplasmic enzyme domain
  • Enzyme is usually a protein tyrosine kinase, but can be a protein serine kinase, a protein threonine kinase or guanyl cyclase (activation of receptor by phosphorylation)

Kinase-linked receptors 2

  • Ligand binding
  • Conformational change in receptor causes inactive monomeric receptor molecules to bind (noncovalently) to one another to form active dimer
  • This brings together intracellular protein tyrosine kinase domains that become enzymatically active
  • Tyrosine (Y) residues in cytoplasmic domains become phosphorylated (by each other)
  • Enzymatic activity is activated to catalyse phosphorylation of substrate proteins
  • Cross phosphorylation intensifies or prolongs allosteric action of hormone

Kinase-linked receptors 3

Growth factor kinase

Insulin receptors

Insulin receptor

  • Insulin receptor is an exception – it exists as a dimer
  • Receptor is phosphorylated by cytosolic kinases
  • Phosphatidylinositol 3-hydroxy kinase {PI(3)K}, makes PIP2,PIP3
  • Grb2, Sos, activates Ras
  • Activation of PI-PLC

Insulin receptor

Insulin receptor

Nuclear receptors

Types of receptor-effector linkage

receptor linkage

Structure of nuclear receptors

Structure of nuclear receptors

Nuclear receptors 1

  • Location: intracellular
  • Effector: gene transcription
  • Coupling: via DNA
  • Examples:
    • eg.Testosterone
    • eg cortisol
  • Mineralocorticoids
    • eg aldosterone
  • Hormones and vitamins
    • eg. Vitamin D and thyroid hormone
    • Sex steroids
    • Glucocorticoids

Nuclear receptors 2

  • Timescale: cell activation with a time scale of hours
  • Agonist-receptor complex acting on DNA resulting in
    • 1. transcription and translation of mediator proteins or
    • 2. repression of expression of certain genes with inhibition of production of specific proteins

Nuclear receptors 3

Nuclear receptors

Glucocorticoid receptors

Glucocorticoid receptor

Glucocorticoid receptor

Receptor classification

  • Drugs are designed to bind to specific targets and these targets will only recognise certain drugs
  • No drugs are completely specific – range of targets and actions at those targets (basis of adverse reactions)
  • Drug receptors are receptors for endogenous mediators
  • Identification and Classification can be:
    • Based on effect of selective antagonists or representative agonists
    • Differences in nucleotide sequence – functional differences? (molecular biology methodology)
    • Orphan receptors exist

Drug targets

Drug targets: transporters

  • A transport protein can transport molecules across membranes (this may be required if they are not very lipid soluble)
  • Hydrolysis of ATP can provide the energy for transport of substances against their electrochemical gradient eg the sodium pump
  • In some cases the transport of organic molecules is coupled to the transport of ions (usually Na+), either in the same direction (symport) or in the opposite direction (antiport)

Drug targets: transporters

transporters

Examples of drug targets

Examples of drug targets

Lecture n* slides poll

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