bps 502 chapter 15 cell communication signaling through enzyme- linked cell surface receptors

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BPS 502 Chapter 15 •Cell Communication Signaling through enzyme-linked cell surface receptors

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Page 1: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

BPS 502Chapter 15

• Cell Communication

• Signaling through enzyme-linked cell surface receptors

Page 2: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Reading Assignment

MBC, Chapter 15

Levitzki A. 2003, EGF Receptor as a Therapeutic Target. Lung Cancer, 41: S9-S14.

Page 3: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Objectives

• Be able to describe and give examples of each of the six classes of enzyme-linked receptors discussed.

• Understand the importance of these signaling pathways in disease states, especially in cancer.

• Be able to discuss the various means of inhibition of these signaling pathways in clinical use and in various phases of clinical trials.

•Understand and describe the signaling pathways that depend on regulated proteolysis.

Page 4: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Enzyme-Linked Cell-Surface Receptors

• They respond to extracellular signaling proteins called growth factors that promote growth, proliferation, differentiation or cell survival

Page 5: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Enzyme-Linked Cell-Surface Receptors

• They act as local mediators at low concentrations (10- 9–10-11 M).

•The response can be slow (several hours) due to the number of intracellular steps that are required leading to changes in gene expression.

•Effects may be direct and rapid acting on the cytoskeleton to control cell movement and shape changes.

•Abnormalities in these fundamental signaling events can lead to cancer.

Page 6: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Six Classes of enzyme-linked receptors identified to date

1. Receptor tyrosine kinases – phosphorylate specific tyrosines on specific intracellular signaling proteins. (EGFR)

2. Tyrosine-kinase-associated receptors – these enzymes associate with intracellular proteins that have tyrosine kinase activity. (Cytokine/Jak-Stat)

3. Receptor-like tyrosine phosphatases – remove phosphate groups from tyrosines of specific intracellular proteins.

4. Receptor serine/threonine kinases – phosphorylate specific serines or threonines on associated gene regulatory proteins. (TGF-/Smad)

5. Receptor guanylyl cyclases – directly catalyze the production of cyclic GMP in the cytosol. (Natriuretic peptides receptor)

6. Histidine-kinase-associated receptors- activate a 2-component signaling pathway whereby the kinase phosphorylates itself on histidine (autophosphorylation) and then immediately transfers the phosphate to a second intracellular protein. Only occurs in yeasts and plants involved in chemotaxis.

Page 7: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Like G-Protein Coupled Receptors, Enzyme-linked receptors are transmembrane proteins with the ligand-binding domain on the outer surface and a cytosolic domain that either has intrinsic enzymatic activity or associates with an enzyme.

Page 8: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

1. Receptor Tyrosine Kinases

Page 9: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Activation of enzyme-linked receptors occurs by oligomerization of the receptor. This causes reorientation of the receptor chains, autophosphorylation, stimulating the kinase activity and creating docking sites for intracellular proteins.

(A) PDGF is a dimer and cross-links two receptors together.(B) FGF is a monomer and can bind in clusters to proteoglycans enabling

the ligands to cross-link the FGF receptor(C) Ephrins are membrane-bound ligands and cluster prior to binding thus

causing receptor cross-linkng.

Three ways signaling proteins can cross-link receptors:

Page 10: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Phosphorylated Tyrosines Serve as Docking Sites for Proteins with SH2 domains

Some examples:

Phospholipase C-(enzyme)Src (relay signaling protein)PI-3’-kinase

All share a highly conserved phosphotyrosine-binding domain, either SH2 or SH3 domains (Src homology region) or PTB (phosphotyrosine bindng) domainsThese proteins act as adaptor proteins to broadcast signals along multiple pathways.

Page 11: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Hypothetical Signaling Pathway Using Modular Domains

Page 12: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Binding of SH2-Containing intracellular signaling proteins to an activated PDGF receptor

Page 13: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

X-ray crystal structure of an SH2 domain that acts as a plug-in molecule recognizing phosphotyrosines and adjacent amino acid side chains.

Page 14: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Ras proteins belong to the large Ras superfamily of monomeric GTPases-Ras proteins contain a covalently linked lipid group that anchors the protein to the cytoplasmic face of the plasma membrane

-There are multiple Ras proteins, each acting in distinct cell types-Ras functions as a switch, cycling between 2 conformational states-mutations of the ras gene promote the development of cancer, 30% of human tumors have a ras activation mutation.

GTPase-activating proteins (GAP) inactivates Ras

Guanine nucleotide exchange factor (GEF) activates Ras

Page 15: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Activation of Ras by an activated receptor tyrosine kinase

Grb-2 is an adaptor protein linking receptor tyrosine kinase to Ras via the GEF called Sos. This is only one way of activating Ras.This is only one way of activating Ras.

Activation of ras is short-lived because it is inactivated by phosphatases and GAPS. Once activated, Ras activates other signaling proteins to relay the signal downstream along several pathways.

Page 16: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

One of the pathways activated by Ras is the MAP Kinase Pathway

MEK

MAP kinase (ERK) is phosphorylated by MEK on both a threonine and a tyrosine. ERK then travels to the nucleus phosphorylating proteins and activating immediate early genes, G1 cyclins are among them.

Raf

MAPK pathway consists of a kinase cascade of no fewer than three enzymes:

Page 17: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

The Mitogen-activated Protein Kinase (MAPK) Cascades

SIGMA-ALDRICH

At least 5 parallel MAP kinase modules can operate in a mammalian cell. These modules are activated by different kinds of cell stress such as UV irradiation, heat shock, osmotic stress and stimulation by inflammatory cytokines.

The MAP kinase pathway stimulates cell division through the action of mitogens. To prolliferate, growth factors stimulate proliferation. PI-3-kinase activation mainly stimulates cell growth. Ras can also help to activate PI-3’-kinase.

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Macaluso et al., 2002, J Cell Physiol., 192:125-130

Ras Effectors and Downstream Pathways

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EGF Receptor Signal Transduction Pathway

SIGMA-ALDRICH

Page 20: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Strategy to prevent cross-talk between MAPK Signaling Pathways:Separate pathways are organized by scaffold proteins in budding yeast and in mammalian cells

Page 21: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Phosphatidylinostitol 3-kinase (PI-3’-Kinase) Phosphorylates Inositol Phospholipids Rather than Proteins

PI-3’-Kinase catalyzes the phosphorylation of inositol phospholipids at the 3 position of the inositol ring

The 2 lipids in orange can serve as docking sites for signaling proteins with Plecstrin Homology (PH) domains

Inositol phospholipid phosphatases (PTEN) remove the 3 position phosphate. PTEN is a tumor suppressor gene, mutations result in prolonged growth signaling. These mutations are found in various cancers including endometrial, some ovarian and some breast cancers.

Page 22: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

PI 3-K stimulates cell survival and growth by activating protein kinase B (PKB or Akt)

1. A survival signal activates a RTK, with recruits and activates PI 3’Kinase.

2. PI 3’K produces the phosphorylated inositol phospholipids which serve as docking sites for 2 serine/threonine kinases PKB and PDK1

3. PKB is then further phosphorylated and activated by PDK1.

4. Activated PKB dissociated from the membrane, and phosphorylates BAD, releasing a death inhibitory protein resulting in inhibition of apoptosis.

The result is continued proliferation.

One example of transmission of the proliferation signal:

Page 23: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Five parallel intracellular signaling pathways activated by G-protein-linked receptors, receptor tyrosine

kinases, or both

Page 24: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

2. Tyrosine-Kinase-associated Receptors Depend on Cytoplasmic Tyrosine Kinases for Their Activity

•These receptors lack intrinsic tyrosine kinase activity and rely on cytoplasmic tyrosine kinases.

•The largest family of cytoplasmic TK receptors is the Src family (Src, Yes, Fgr, Fyn, Lck, Lyn, Hck, Blk).

•These proteins contain SH2 and SH3 domains and are located on the cytoplasmic side of the plasma membrane.

•Integrin receptors interact with focal adhesion kinase (FAK) that is a cytoplasmic TK.

•Cytokine receptors are stably associated with a class of cytoplasmic TKs called Jaks.

Page 25: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

The Jak-STAT signaling pathway activated by -interferon

Cytokines are secreted by cells in response to a viral infection and bind to neighboring cells to induce proteins that increase resistance to virus. All cytokine receptors are associated with one or more Jaks (4 types)

Janus kinases (Jaks)Signal transducers and activators of transcription (STATs). 7 known STATs

Page 26: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors
Page 27: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

3. Some Protein Tyrosine Phosphatases Act as Cell-Surface Receptors

Protein tyrosine phosphatases (PTPs) remove selected phosphotyrosines on a subset of tyrosine-phosphorylated proteins. Exhibit high degree of substrate selectivity.

These enzyme ensure that the tyrosine phosphorylations are short-lived and are responsible for regulating the intensity of the signal.

There are about 30 known PTPs and occur as both transmembrane and cytoplasmic forms.

Page 28: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Protein Tyrosine Phosphatases

The role of receptor-like tyrosine phosphatases is not yet clearly understood.

They are thought to act as receptors, but their ligands have not been identified.

Some have been shown to display features of cell-adhesion.

Some can activate receptors on neighboring cells.

Page 29: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

4. Receptor Serine/Threonine Protein KinaseSmad-dependent signaling pathway activated by TGF-

Transforming growth factor (TGF) consists of a large number of structurally related, secreted, dimeric proteins. They mediate a wide range of biological functions in animals: proliferation, differentiation, ECM production, cell death, tissue repair and immune regulation.

They act through receptor serine/threonine kinases type I and type II

Smad family members are directly phosphorylated by the type I receptor, and moves to the nucleus to direct gene transcription

Page 30: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

The size and location of protein kinases

Page 31: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

5. Receptor Guanylyl Cyclases•Single pass transmembrane proteins with an extracellular binding site for a signal molecule and intracellular guanylyl cyclase catalytic domain.

•Binding of ligand activates the cyclase domain to produce cyclic GMP, which in turn binds to and activates a cyclic GMP-dependent protein kinase (PKG).

•PKG phosphorylates specific proteins on serine or threonine.

•Some signaling molecules that use these receptor types include natriuretic peptides (NPs) (regulate salt and water balance and dilate blood vessels).

Page 32: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

6. Histadine-Kinase Associated ReceptorsInvolved in Bacterial Chemotaxis

•Dimeric transmembrane proteins that bind specific attractants and repellants

•The cytoplasmic tails of the receptors are stably associated with an adapter protein CheW and a histidine kinase, CheA – couples the receptor to the flagellar motor.

•Binding of a repellent activate CheA causing autophosphorylation on a histidine, and immediate transfer of the phosphate to an aspartic acid on a messenger protein, CheY.

•CheY dissociates from the receptor, diffuses through the cytosol, and causes the flagellar motor to rotate clockwise and the bacterium tumbles.

•This type of signaling is used in yeast and plants, but not found in animals.

Page 33: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

What are the opportunities for therapeutic applications?

Page 34: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

A. Levitzki. 2003. Lung Cancer, 41: S9-S14

Modes of EGFR Inhibition

Page 35: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Iressa/Gefitinib (EGFR Inhibitor)

• Used to treat non-small-cell lung cancer (NSCLC) – 85% of all lung cancers

• Works in 25% of cases in Japan, but only effective in 10% of U.S. patients.

• Suspected a genetic factor in efficacy

• Has variable effects based on a single gene

Page 36: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors
Page 37: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Science. 2004 Jun 4;304(5676):1458-61

Page 38: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors
Page 39: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

EGFR Mutations in Lung Cancer:

Correlation with ClinicalResponse to Gefitinib

TherapyJ.Guillermo Paez,1,2 *Pasi A.Ja ¨nne,1,2 *Jeffrey C.Lee,1,3 *

Sean Tracy,1 Heidi Greulich,1,2 Stacey Gabriel,4 Paula Herman,1

Frederic J.Kaye,5 Neal Lindeman,6 Titus J.Boggon,1,3Katsuhiko Naoki,1 Hidefumi Sasaki,7 Yoshitaka Fujii,7

Michael J.Eck,1,3 William R.Sellers,1,2,4 †Bruce E.Johnson,1,2 † Matthew Meyerson 1,3,4 †

SCIENCE VOL 304 4 JUNE 2004

Page 40: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Convergence of Results With Different Approaches

Lynch et al.:

• Sequenced the entire coding region of the EGFR from 9 patients treated with gefitinib with clinical response.

• 8/9 mutations found in responders; 0/7 in nonresponders

Paez et al.:• Sequenced the exons of 47

TK activation loop domain 58 lung cancers from Japanese patients and 61 from U.S. patients.

• Narrowed search to EGFR in 119 cases of NSCLC

• Mutation found in 5/5 responders; 0/4 non-responders

Page 41: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Both Studies Reveal Mutations in the EGRF Tyrosine Kinase Domain

-either small in frame deletions or amino acied substitutions -Cluster around the ATP binding pocket-activating mutations

Page 42: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors
Page 43: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Table 1. Protein kinase inhibitors in clinical trials

                                                                                            

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Genentech.com

Page 45: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors
Page 46: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Genentech.com

Page 47: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Genentech.com

Data from Phase III clinical trials of Herceptin

Page 48: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Table 2. Antibody protein kinase inhibitors in clinical trials

                                                                                             

Page 49: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Overview of the PI3K/Akt Pathway

Page 50: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Compounds that Directly Inhibit the PI3K/Akt Pathway

Page 51: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Signaling Pathways that Depend on Regulated Proteolysis:

-Notch-Wnt-NF-B

Page 52: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

The Notch Receptor Protein is Widely Used in Animal Development

Notch genes encode heterodimeric transmembrane receptors that regulate differentiation, proliferation and apoptosis.

Mammals have 4 known Notch genes and 2 families of Notch ligands: Delta and Jagged.

Both Notch and Delta are single-pass transmembrane proteins that require proteolytic processing to function.

Well known for its role in nerve cell production in Drosophila

Deregulated expression of Notch receptors, ligands and downstream targets is described in many cancers

Page 53: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Activation of Notch by Proteolytic Cleavage1. Full-length Notch is cleaved in the trans-golgi by a furin and is transported to the plasma membrane. 2. Notch binds delta that is displayed on an adjacent cell triggers the next 2 proteolytic cleavages. 3. extracellular cleavage mediated by TACE leaving 12 amino acids. 4. Cleavage mediated by presenilin that releases the Notch tail to migrate to the nucleus.

Notch binds to the transcriptional repressor CSL and activates gene transcription.

Page 54: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Wnt Proteins Bind to Frizzled Receptors and Inhibits -Catenin Degradation Wnt proteins are secreted

glycoproteins that act as mediators to control many aspects of development.

Wnt binds to the Frizzled receptor (family of 7 transmembrane proteins resembling G-proteins.

Signaling is mediated by the cytoplasmic protein dishevelled.

Dishevelled acts by regulating the proteolysis of a multifunctional protein, -catenin, which functions in cell-cell adhesion and as a latent gene regulatory protein.

In the absence of a wnt signal, catenin is phosphorylated in a degradation complex and is degraded in the proteosome.

Page 55: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

In the presence of Wnt, in association with a co-receptor protein LRP and leads to inhibition of -catenin phosphorylation and degradation.

As a result, the unphosphorylated -catenin accumulates in the nucleus and displaces the repressor protein Groucho.

Wnt target genes are expressed including c-myc , a powerful stimulator of cell growth and proliferation

APC (adenamatous polyposis coli) gene mutations occur in 80% of benign colon polyps that lead to cancer.

Wnt ligand overexpression is documented in many tumors including breast.

Page 56: BPS 502 Chapter 15 Cell Communication Signaling through enzyme- linked cell surface receptors

Multiple Stress and Proinflammatory Stimuli Act Through an NF-B-Dependent Signaling Pathway

Two cytokines that are important in inducing the inflammatory response are TNF- and interleukin-1 (IL-1). These cytokines bind to cell-surface receptors and activate NF-B.

There are 5 NF-B proteins in mammals (RelA, RelB, c-Rel, NF-B1, NF-B2). These form a variety of homo- and heterodimers each activating a specific set of genes.

Inhibitory proteins IB, bind tightly to the dimers and hold them in an inactive state.

TNF- signals lead to recruitment of several intracellular signaling proteins (RIP, TRADD, TRAF2) that in turn activate a specific serine/threonine kinase, IB kinase. This releases the IB exposing the nuclear localization signal of NF-B and it translocates to the nucleus.