Pharmacology 3A: Principles of Pharmacology Receptor Tyrosine Kinases

College of Medical, Veterinary & Life Sciences

Pharmacology 3A (Session 2024-25)

Block 1 : Principles of Pharmacology Receptor Tyrosine Kinases Prof. Simon Kennedy simon.kennedy@glasgow.ac.ukILOS ... At the end of this class, you will be able to:

1. Understand what receptor tyrosine kinases (RTKs) and other kinase-linked receptors are;
2. Describe how RTKs are activated and induce cell signalling/biological responses;
3. Relate RTKs to diseases such as cancer and hypertension.A Key Reference

This gives a simple overview of RTKs and what they do: Wintheiser G.A. & Siberstein P. (2022). Physiology, Tyrosine Kinase Receptors. Link: https://pubmed.ncbi.nlm.nih.gov/30860767/Further reading- Rang & Dale, Chapter 3

The four main types of receptor

Type 1: Ligand-gated ion channels

Location Membrane Effector Ion channel Coupling Direct Examples Nicotinic acetylcholine receptor, GABAA receptor Structure Oligomeric assembly of subunits surrounding central pore

Type 2: G protein-coupled receptors

Location Membrane Effector Channel or enzyme Coupling G protein or arrestin Examples Muscarinic acetylcholine receptor, adrenoceptors Structure Monomeric or oligomeric assembly of subunits comprising seven transmembrane helices with intracellular G protein-coupling domain

Type 3: Receptor kinases

Location Membrane Effector Protein kinases Coupling Direct Examples Insulin, growth factors, cytokine receptors Structure Single transmembrane helix linking extracellular receptor domain to intracellular kinase domain

Type 4: Nuclear receptors

Location Intracellular Effector Gene transcription Coupling Via DNA Examples Steroid receptors Structure Monomeric structure with receptor- and DNA- binding domains

RTKs vs. other receptor types

1.Ligand-gated ion channels (ionotropic receptors) 2.G-protein-coupled receptors (metabotropic) 3.Kinase-linked receptors 4.Nuclear receptors lons lons R R E R/E G + (G + or O « NUCLEUS Hyperpolarisation or depolarisation Change in excitability Protein phosphorylation R Gene transcription Gene transcription Ca2+ release Other Protein synthesis Protein synthesis Cellular effects Cellular effects Time scale Milliseconds Seconds Hours Hours Examples Cytokine receptors Nicotinic ACh receptor Muscarinic ACh receptor Oestrogen receptor ----- + or O Second messengers Protein phosphorylation Cellular effects Cellular effects Rang et al: Rang & Dale's Pharmacology, 7e Copyright @ 2011 by Churchill Livingstone, an imprint of Elsevier Ltd. All rights reserved.

Kinase linked Receptors: key points

• Very different in structure from either ligand-gated channels or the GPCRs.
• Mediate the actions of a wide variety of protein mediators, including growth factors and cytokines and some hormones such as insulin and leptin.
• Receptors are large proteins (up to 1000 residues) with a single membrane-spanning helical region.
• Structure and size of the extracellular and intracellular domain varies.
• Over 100 such receptors have been cloned.
• They play a major role in controlling cell division, growth, differentiation, inflammation, tissue repair, apoptosis and immune responses.

Kinase-linked Receptors: types

• Receptor tyrosine kinases (RTKs). These receptors incorporate a tyrosine kinase moiety in the intracellular region. Examples- epidermal growth factor (EGF) and nerve growth factor (NGF), and also the group of Toll-like receptors that recognise bacterial lipopolysaccarides. The insulin receptor also belongs to the RTK class, although it has a more complex dimeric structure.
• Serine/threonine kinases. This smaller class is similar in structure to RTKs but phosphorylate serine and/or threonine residues rather than tyrosine. The main example is the receptor for transforming growth factor (TGF).
• Cytokine receptors. Lack intrinsic enzyme activity. Once activated by ligand they activate a variety of cytosolic tyrosine kinases eg Jak. Ligands include cytokines such as interferons and colony-stimulating factors involved in immunological responses.

Receptor Tyrosine Kinases: key points

• Activation of RTKs is usually associated with long term changes in cell function, where gene expression is required (eg: proliferation and differentiation).
• Examples of RTKs and functions regulated by them: - EGF (proliferation): Vascular remodelling/Cancer - VEGF (angiogenesis): Cancer - Insulin (glucose homeostasis): Diabetes

Receptor Tyrosine Kinases - Structure

Extracellular domain @ Ligand-binding domain immunoglobulin- like domain cysteine- rich domain - Single hydrophobic transmembrane a-helix Tyrosine kinase enzyme intrinsic part of the receptor L kinase insert region EGF receptor insulin receptor, IGF-1 receptor NGF receptor FGF receptor VEGF receptor PDGF receptor, M-CSF receptor

Receptor Tyrosine Kinase (Activation): Dimerisation

Low or no kinase activity

1 receptor tyrosine kinase 5 EXTRACELLULAR CYTOSOL tyrosine kinase domain INACTIVE Activation lip (unphosphorylated / blocking conformation)

Ligand binding promotes receptor dimerisation

2 1. signal dimer binds receptor tyrosine kinase EXTRACELLULAR CYTOSOL tyrosine kinase domain INACTIVE 2. kinase activity is stimulated

Tyrosines are phosphorylated

3 1. signal dimer binds receptor tyrosine kinase EXTRACELLULAR CYTOSOL tyrosine kinase domain p p p p p p INACTIVE ACTIVE 2. kinase activity is stimulated 3. tyrosines are phosphorylated Receptor dimerization causes a conformational change. This results in: Transphosphorylation of intracellular tyrosine residues.

Intracellular proteins bind to phospho-tyrosine docking sites

4 1. signal dimer binds receptor tyrosine kinase 1 EXTRACELLULAR CYTOSOL tyrosine kinase domain p p P P P p P P p p p p INACTIVE ACTIVE ACTIVE 2. kinase activity is stimulated 3. tyrosines are phosphorylated 4. intracellular proteins bind to phospho-tyrosine docking sites Phosphorylated tyrosines function as docking sites for intracellular proteins which relay and amplify downstream signal

Receptor Tyrosine Kinase (Activation): Examples

Human EGF Receptor (HER)

EGF Exterior EGF EGF- binding domains EGF binding domain - Loop - important for a functional receptor dimer Membrane Figure 16-17a Molecular Cell Biology, Sixth Edition 2008 W. H. Freeman and Company HER1 HER2 HER3 HER4 Ligands are: EGF, HB-EGF, TGFa Homo or heterodimerisation No ligand Always active Dimerisation with HER1, HER3 and HER4 Facilitates EGF signalling Lacks functional kinase domain (weak) Only active when complexed with HER2 Ligands are: HB-EGF, neuregulins Heparin-binding-EGF-like growth factor is synthesized by monocytes and macrophages as a membrane-anchored mitogenic and ch glycoprotein.

PDGF dimer

EGF Exterior EGF EGF- binding domains Membrane Figure 16-17a Molecular Cell Biology, Sixth Edition 2008 W. H. Freeman and Company PDGF dimer PDGF P P PIP P P P P PDGF receptors PDGF exists as a preformed dimer, i.e. 2 receptor binding sites Thus the agonist directly cross- links 2 PDGF receptor chains

FGF binding

EGF Exterior EGF EGF- binding domains Membrane Figure 16-17a Molecular Cell Biology, Sixth Edition 2008 W. H. Freeman and Company FGF binds to the extracellular domain of the receptor and to heparan sulfate (essential for efficient activation of the receptor) PDGF dimer P P PIP P P P P PDGF receptors a FGFR FGF Heparan sulfate FGF FRS20 GRB2 FGFR kinase PDGFA very important hormone acts at an RTK!

The Discoverers of Insulin

FREDERICK GRANT BANTING 1891 - 1941 JOHN JAMES RICKARD MACLEOD CHARLES HERBERT BEST JAMES BERTRAM COLLIP 1876 - 1935 1899 - 1978 1892 - 1965A very important hormone acts at an RTK! Instead, it was Banting, who supported by Best, made the experiments that proved that an extract from the Langerhans' islands could keep dogs with diabetes alive for several months in the laboratory of Macleod. This extract, purified by Collip, saved the life of people dying by diabetes for the first time in January 1922. The discovery of insulin was made in 1921 and the Nobel prize was awarded to Banting and Macleod already 1923. The correct choice of laureates has, however, been debated. Banting shared his prize money with Best who helped him with the experiments and Macleod shared his part with Collip who purified the extract. The question arises, who was actually the person discovering insulin and did the right person get the prize? Ryden & Lindsten; Diabetes Res Clin Prac; 2021; 175; 108819.

Receptor Tyrosine Kinase (Activation): Insulin/IGF-I

Insulin/IGF-I are soluble monomeric polypeptides Receptors are expressed as disulphide bond-linked preformed heteromers Insulin/IGF-I bind to the alpha subunits to induce a conformational change that is transmitted to the beta subunits which then transphosphorylate each other Insulin and IGF Extracellular Alpha subunit (hormone binding domains) + fell Beta subunit (ATP-binding and tyrosine kinase domains) Cytoplasm

Receptor Tyrosine Kinases: Signalling

RTK (dimer) Small GTPases PI3K Phospholipids RTK (monomer) Docking/Adaptor Protein Small GTPases MAPKs GENE TRANSCRIPTION BIOLOGICAL EFFECTS

Receptor Tyrosine Kinases: Signalling Pathways

RTK (dimer) Small GTPases PI3K Phospholipids RTK (monomer) Docking/Adaptor Protein Small GTPases MAPKS GENE TRANSCRIPTION BIOLOGICAL EFFECTSDocking and adaptor proteins-intracellular events The phosphorylated tyrosine residues act as high affinity docking sites for intracellular proteins One important group is SH2 domain proteins which contain a 100AA recognition site Individual SH2 proteins bind selectively- making the response to receptor activation specific

Insulin Signalling

Insulin signalling P P P P P P P P P P P P P PTB IRS-1 P P SH2 P SH2 SH2

RTKs signalling: SH2 domain proteins

Many SH2 domain proteins are enzymes- kinases or phospholipases End result is activation or inhibition of transcription factors which migrate to the nucleus This can alter gene transcription An important transcription factor is NFkappaB Normally complexed with an inhibitor in the cytoplasm RTKs can phosphorylate the inhibitor to allow the NFkappaB to start moving Possible drug target? TLRs/Cytokine receptor IKKa/B P P IKB 1 1KB IKK Inhibitors eg MS-345541, IMD- 0354 and ML120B 1ΚΚα/β NFKB NF-KB Inhibitors eg DHMEQ NFKB NF-KB decoy oligonucleotides Inflammatory Gene