Lecture 08 (Chapter 13)

Signal Transduction

Signal TransductionSignal Transduction

• Signal-transducing receptors are frequently plasma membrane proteins that bind specific extracellular molecules• Growth factors• Hormones• Neurotransmitters

• Binding transmits signal to cell interior which initiates response (e.g., enzyme cascades)

3 Major Classes of Signal-transducing receptors• Ion channel linked receptors• Protein linked receptors• Enzyme linked receptors

Extracellular recognition domain, transmembrane domain, intracellular domain

Complete structures (with all domains) are not available for these receptors due to problems with crystallization, and large size which prevents study by NMR

Signal TransductionSignal Transduction

• Signal-transducing receptors are frequently plasma membrane proteins that bind specific extracellular molecules

• Growth factors• Hormones• Neurotransmitters

http://pharmaceuticalintelligence.com/tag/signal-transduction-pathways/

G proteinsG proteins• G proteins, (guanine nucleotide-binding proteins), are a family of protein receptors

that act as molecular switches inside cells. • Receive input signal from outside of the cell.• Signal is transmitted across membrane into the cytosol, and amplified. • These signal mediate several important physiological responses related to vision,

smell, and stress response.

http://en.wikipedia.org/wiki/G_protein

(1) ligand activates the G protein-coupled receptor. (2) Induces a conformational change in the G-protein allowing it to function as a guanine nucleotide exchange factor (GEF) that exchanges GTP for GDP (4) - thus turning the GPCR "on". The GTP (or GDP) is bound to the Gα subunit in the traditional view of heterotrimeric GPCR activation. (5)This exchange triggers the dissociation of the Gα subunit (which is bound to GTP) from the Gβγ dimer and the receptor as a whole.

As long as ligand remains bound to GPCR, production of Gα-GTP molecules will continue (signal amplification).

Structure of G ProteinStructure of G Protein

http://swift.cmbi.ru.nl/gv/GPCR/rvdkant/GPCR/Introduction.html

• Most G proteins are heterotrimers with Gα (45 kDa), Gβ (35 kDa) and Gγ (8 kDa) subunits.

• G protein dissociates into the Gα subunit (which is bound to GTP) and the Gβγ subunits, which both transmit the signal to different effector proteins.

• The human genome contains code for over 1000 of these receptors, which can affect a multitude of physiological processes.

Physiological processes mediated by G proteinsStimulus Receptor Effector Physiological responseEpinephrine Beta-adrenergic receptor adenylate cyclase glycogen breakdownLight rhodopsin c-GMP phosphodiesterase visual excitationIgE-antigen complexes mast cell IgE receptor phospholipase C histamine secretion in all

allergic reactionsAcetylcholine muscarinic receptor K channel slowing of pacemaker

activity that controls heartbeat

Structural basis of signal transduction by G ProteinsStructural basis of signal transduction by G Proteins

Introduction to Protein Structure, Branden & Tooze, 1999

• G1 - P-loop binds the beta phosphate of GDP and GTP• G2 – (Switch I), T35 binds the terminal phosphate (γ-phosphate) of GTP and the divalent Magnesium ion present in the

active site, but makes no contacts with GDP• G3 – (Switch II), has the DXXGQ motif, where D57 is specific for guanine binding rather than adenine and Glutamine-61

activates a catalytic water molecule for hydrolysis of GTP to GDP.• G4 - LVGNKxDL motif, provides specific interaction to guanine• G5 - SAK consensus sequence, A146 is specific for guanine rather than adenine• The switch motifs, G2 and G3 move upon activation by GTP. This conformation change mediates the basic functionality

as a molecular switch protein. This GTP bound state of Ras is On-state and GDP bound state is the Off-state.

• Ras (abbreviation for Rat sarcoma) proteins structurally similar to Gα subunit of G proteins

• Contains a six-stranded beta sheet and 5 alpha helices

• G domain (166 amino acids) which binds guanosine nucleotides.

• C terminal membrane targeting region (CAAX-COOH, also known as CAAX box)

• The G domain contains five G motifs that bind GDP/GTP directly

Ras-regulated signal pathwaysRas-regulated signal pathways

• Ras-regulated signal pathways control a number of processes, including:– Regulation of signal transduction processes

leading to cell multiplication and differentiation, cell adhesion, apoptosis, and cell migration

– Acting as molecular switches in response to protein tyrosine kinase receptors• Activated by GTP binding, and deactivated

by GDP binding.

• About 25% of all tumor cells have mutant Ras proteins that cannot be activated to hydrolyze GTP (deregulated)

• Remain bound to GTP and activated, thus leading to uncontrolled cell growth and decreased apoptosis

http://www.genecopoeia.com/product/search/pathway/h_rasPathway.php

G protein transducin and rhodopsinG protein transducin and rhodopsin

Rhodopsin•Light-sensitive receptor protein and biological pigment in photoreceptor cells of the retinal rod cells (convey ability to see under limited light).•Consists of the protein moiety opsin and a covalently bound cofactor, retinal. •Light energy induces conformational change in retinal, which activates the G protein transducin and subsequent cascade transforming light into neural signal via the optic nerve.

http://cherfan2010biology12assessment.wikispaces.com/The+Retina

Opsin Retinal

Rhodopsin

Transducin

GDP

Gtβ

Gtγ

Gtα

Opsin Retinal

RhodopsinRhodopsin

http://en.wikipedia.org/wiki/Rhodopsin; http://de.wikipedia.org/wiki/Rhodopsin#/media/File:1L9H_(Bovine_Rhodopsin)_2.png

• Rhodopsin consists of the protein moiety opsin and a reversibly covalently bound cofactor, retinal.

• Opsin is a bundle of seven transmembrane helices connected to each other by polypeptide loops.

• Opsin binds retinal (a photoreactive chromophore), which is located in a central pocket on the seventh helix at a lysine residue.

• The retinal lies horizontally with relation to the membrane. Each outer segment disc contains thousands of visual pigment molecules.

RetinalRetinal• Retinol is produced in the retina

from Vitamin A, from dietary beta-carotene.

• Retinol converted to retinal.• When lLight photon interacts

with retinal in photoreceptor cell, retinal isomerizes from the 11-cis to all-trans configuration.

• Retinal no longer fits into the opsin binding site.

• Opsin undergoes conformational change to metarhodopsin II.

• Metarhodopsin II is unstable and splits, yielding opsin and all-trans retinal.

• The opsin activates the regulatory protein transducin.

• 11-cis-retinal is restored and rhodopsin reassembled.

http://en.wikipedia.org/wiki/Rhodopsin

Visual Phototransduction via transducinVisual Phototransduction via transducin

http://webvision.med.utah.edu/book/part-ii-anatomy-and-physiology-of-the-retina/photoreceptors/

Light activation of opsin

Visual Phototransduction via transducinVisual Phototransduction via transducin

http://webvision.med.utah.edu/book/part-ii-anatomy-and-physiology-of-the-retina/photoreceptors/

Transducin, activated by opsin, dissociates from its bound GDP, binds GTP.

Gtα dissociates from Gtβ and Gtγ subunits, with the GTP still bound to Gtα

Visual Phototransduction via transducinVisual Phototransduction via transducin

http://webvision.med.utah.edu/book/part-ii-anatomy-and-physiology-of-the-retina/photoreceptors/

The Gtα-GTP complex activates phosphodiesterase (PDE).PDE breaks down cGMP to 5'-GMP. This lowers the concentration of cGMP and therefore the sodium channels close.

Visual Phototransduction via transducinVisual Phototransduction via transducin

http://webvision.med.utah.edu/book/part-ii-anatomy-and-physiology-of-the-retina/photoreceptors/

Closure of the sodium channels causes hyperpolarization of the cell due to the ongoing efflux of potassium ions.Hyperpolarization of the cell causes voltage-gated calcium channels to close.

Phototransduction AnimationPhototransduction Animation

http://sites.sinauer.com/neuroscience5e/animations11.02.html

Human Growth Hormone (HGH)Human Growth Hormone (HGH)• Peptide hormone that stimulates growth, cell reproduction and regeneration. • It is a mitosis-triggering protein which is specific only to certain kinds of cells. • Single chain polypeptide consisting of 191-amino acids. • Synthesized, stored, and secreted by somatotropic cells within the lateral wings of

the anterior pituitary gland.

https://www.cgl.ucsf.edu/Outreach/midasplus/gallery/hGH.html

HGH stimulates production of Insulin-like growth factor 1 (IGF-1), which stimulates systemic body growth, and has growth-promoting effects on almost every cell in the body, especially: •skeletal muscle•cartilage•bone•liver•kidney •nerves •skin•hematopoietic cell •lungs

The growth hormone receptor: mechanism of activation and clinical implications, Brooks J & Waters MJ, Nature Reviews Endocrinology, 2010

Human Growth Hormone ReceptorHuman Growth Hormone Receptor

• Growth hormone receptor is a transmembrane receptor for growth hormone protein.

Human Growth Hormone Receptor and JAK2Human Growth Hormone Receptor and JAK2• Binding of asymmetrical growth hormone to the receptor leads to receptor dimerization and

the activation of an intra- and intercellular signal transduction pathway leading to growth. • The intracellular domain contains a proline-rich motif, termed ‘box 1’, that is required for the

binding and activation of janus kinase 2 (JAK2).• JAK2 has been identified as a tyrosine kinase associated with GHR and other receptors of the

superfamily. Binding of GH to its receptor results in dimerization of the GHR, phosphorylation of JAK2 and of the GHR.

https://www.cgl.ucsf.edu/Outreach/midasplus/gallery/hGH.html, Growth hormone receptor: structure and signal transduction, Postel-Vinay MC1, Finidori J., Eur J Endocrinol. 1995

GHR PathwayGHR Pathway

http://www.ebi.ac.uk/interpro/potm/2004_4/Page2.htm

• Phosphotyrosine residues on both GHR and JAK2 provide docking sites for a variety of signalling proteins that contain phosphotyrosine-binding motifs (such as SH2 domains).

• The GH-bound receptor induces JAK2 phosphorylation of the various target proteins, leading to different cellular responses.

Enzyme-Linked ReceptorsEnzyme-Linked Receptors

Classes1.Receptor tyrosine kinases

– Phosphorylate specific tyrosine residues on intracellular signaling proteins

2.Tyrosine kinase-associated receptors– (e.g., GHR) associate with proteins that have tyrosine kinase activity

3.Receptor protein tyrosine phosphatases– Remove phosphate groups from tyrosine residues of intracellular

signaling proteins

4.Transmembrane receptor serine/threonine kinases– Add phosphate groups to serine or threonine

5.Transmembrane guanyl cyclases– Catalyze production of cyclic GMP in cytosol

Receptor tyrosine kinases (RTKs)Receptor tyrosine kinases (RTKs)1. Single-pass transmembrane receptors.2. Intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the γ-phosphate of ATP to tyrosine residues in protein substrates. 3. Essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. 4. Part of large family that includes:

– insulin receptor– epidermal growth factor – fibroblast growth factor – vascular endothelial growth factor

5. Receptor activation occurs through ligand binding (growth factors, cytokines, and hormones), which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion.

6. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins. 7. May play critical roles in the development and progression of many types of cancers.

Structural analysis of receptor tyrosine kinases, Stevan R Hubbard, Progress in Biophysics and Molecular Biology, 1999

http://schlessinger.yalemedicine.org/picture1.htm

Insulin Receptor (IR or InsR)Insulin Receptor (IR or InsR)

1. Transmembrane (RTK) receptor activated by hormones– insulin– Insulin-like growth factor 1 (IGF-I)– Insulin-like growth factor 2 (IGF-II)

2. Regulates glucose homeostasis– Loss of function can lead to development of diabetes and

cancer.3. Transcription of alternative splice variants derived from the INSR

gene are translated to form one of two monomeric isomers; IR-A in which exon 11 is excluded, and IR-B in which exon 11 (12 additional Aas) is included.

4. Downstream post-translational modification to IR-A or IR-B result in the formation of a proteolytically cleaved α and β subunits, which can be combined to form homo or hetero-dimers leading to production of the disulfide-linked transmembrane insulin receptor.

Ward CW, Lawrence MC (April 2009). "Ligand-induced activation of the insulin receptor: a multi-step process involving structural changes in both the ligand and the receptor". BioEssays; Belfiore A, Frasca F (Oct 2009). "Insulin receptor isoforms and insulin receptor/insulin-like growth factor receptor hybrids in physiology and disease.". Endocr Rev

InsulinInsulin• Peptide hormone produced by beta cells in the pancreas. • Regulates the metabolism of carbohydrates and fats by

promoting the absorption of glucose from the blood to skeletal muscles and fat tissue and by causing fat to be stored rather than used for energy.

• Inhibits production of glucose by the liver.• Except in certain metabolic disease states (e.g., diabetes

mellitus and metabolic syndrome), insulin is provided within the body in a constant proportion to remove excess glucose from the blood.

• Active insulin is a monomer.• Inactive insulin binds zinc to form a hexamer.

http://en.wikipedia.org/wiki/Insulin; Sonksen P, Sonksen J (2000), "Insulin: understanding its action in health and disease". Br J Anaesth

Structural biology of insulin and IGF1 receptors: implications for drug design, Pierre De Meyts & Jonathan Whittaker, Nature Reviews Drug Discovery 1, (October 2002)

Each isometric monomer is organized into 8 distinct domains: •leu-rich repeat domain (L1), •cys-rich region (CR), •leu-rich repeat domain (L2), •Three fibronectin type III domains; Fn0, Fn1 and Fn2.•insert domain (Ins) is found in Fn1, containing the α/β furin cleavage site, from which proteolysis results in both IDα and IDβ domains. •Within the β-chain, lies the Fn2 domain, transmembrane helix (TM) and intracellular juxtamembrane (JM) region, just upstream of the intracellular tyrosine kinase (TK) catalytic domain, responsible for subsequent intracellular signaling pathways.•Structure stabilized by disulfide bonds

Insulin Receptor Domain StructureInsulin Receptor Domain Structure

http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-14/CB14.html

Receptor protein tyrosine phosphatases - RPTPsReceptor protein tyrosine phosphatases - RPTPs

• RPTPs are enzymes that regulate a variety of cellular processes including:– cell growth– differentiation– mitotic cycle– oncogenic transformation

• Specific ligands that bind RPTPs remain to be identified. • Once activated, the receptor phosphatases remove phosphate groups from

phosphorylated tyrosine residues on proteins.

http://www.ncbi.nlm.nih.gov/gene/5786 ; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753797/

Protein tyrosine phosphatases: from genes, to function, to disease, Nicholas K. Tonks, Nature Reviews Molecular Cell Biology 7, 833-846 (November 2006)

RPTPRPTPαα

• RPTPα contains an extracellular domain, a single transmembrane segment and two tandem intracytoplasmic catalytic domains (D1 and D2).

• RPTPα dephosphorylates and activates Src family non-receptor tyrosine kinases, and is implicated in the regulation of integrin signaling, cell adhesion and proliferation.

http://www.ncbi.nlm.nih.gov/gene/5786 ; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753797/; Protein tyrosine phosphatases: from genes, to function, to disease, Nicholas K. Tonks, Nature Reviews Molecular Cell Biology 7, 833-846 (November 2006)

ProximalPTP domain D1

DistalPTP domain D2

Receptor sub-type R4

1yfo

Protein tyrosine phosphatases: from genes, to function, to disease, Nicholas K. Tonks, Nature Reviews Molecular Cell Biology 7, 833-846 (November 2006)

Phosphorylation of PTPα by PKCδ causes detachment of Grb2 followed by attachment of pp60src, effectively through SH2 domain exchange.

Binding of the SH2 domain of pp60src to PTPα exposes phosphotyrosine 527 in pp60src (yellow star), which is then dephosphorylated by PTPα.

RPTPRPTPαα