06/10/2016

Cell signalling 2…specific pathways continued

• Nuclear hormone receptors

• p53

• EGF/ErbB

• Pathology – cell-cell signalling in disease

Richard Killickrichard.1.killick@kcl.ac.uk

ext 0139

Nuclear Hormone Receptors

Nuclear Hormone Receptors

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Steroid hormones

NHR ligands are all lipophilic molecules

Nuclear hormone receptors are DNA binding transcription factors!

1D

3D

Structure of NHR

Type I

The receptor resides in the cytoplasm and migrates to the nucleus upon binding with ligand

Two major types

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Type II

The receptor resides in the nucleus regardless of ligand presence

The male and female sex hormone receptors are examples of type I and type II nuclear hormone receptors

Androgen Receptor (AR) - type I

Estrogen Receptor (ER) - type II

ER and AR are two members of a large family of Nuclear Hormone Receptors.

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The Estrogen Receptor (ER) - example of a type II NHR receptor

There are 2 isoforms of the Estrogen receptor ERα and ERβ

The biological actions of estrogens are mediated by estrogen binding to one of two specific estrogen receptors(ERs) ER and ER. ER and ERare products of different genes and exhibit tissue and cell-type specificexpression.

The characterisation of mice lacking ER , or ER, or both has revealed that both receptor subtypes haveoverlapping but also unique roles in estrogen-dependent action in vivo.

DBD DNA Binding DomainLBD Ligand Binding DomainAF-1 Ligand independent transactivation domainAF-2 Ligand dependent transactivation domain

ERα is expressed in endometrium, breast cancer cells, ovarian stromal cells, and the hypothalamus. In males, ERα protein is found in the epithelium of the efferent ducts.

ERβ is expressed in ovarian granulosa cells, kidney, brain, bone, heart, lungs, intestinal mucosa, prostate and endothelial cells.

The classical (direct) pathway includes ligand activation and the direct DNA binding to estrogen response elements (ERE) before modulation of gene regulation.

The tethered pathway includes protein-protein interaction with other transcription factors after ligand activation, and thereby gene regulation is affected by indirect DNA binding.

A third mechanism, also called non-genomic with rapid effects, is not as well understood but has been observed in many tissues. The ligand activates a receptor, possibly associated with the membrane or, alternatively, a signal activates a classical ER located in the cytoplasm. Signalling cascades then activate second messengers (SM) that affect ion channels or increase nitric oxide levels in the cytoplasm, leading to a rapid response without gene regulation.

The ligand-independent pathway includes activation through other signalling pathways, like growth factor signalling. In this case, activated kinases phosphorylate ERs and thereby activate them to dimerise, bind DNA, and regulate genes.

ERs have several modes of action

ERα and ERβ are targets of several kinases ERK2, Akt/PKB, PKA etc.

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The Androgen Receptor (AR) - a type I receptor

Testosterone (T), can be converted by certain cells to 5-α-Dihydrotestosterone (DHT) by 5-α-reductase.

Both T and DHT then bind AR leading to nuclear translocation.

In the cytoplasm ARs bind chaperones (Heat shock proteins) and are inactive.Androgen binding releases them from the chaperones and they enter the nucleus, bind AREs and recruit transcriptional co-factors.

AR Mediates cell growth and differentiation in androgen responsive tissues.

Androgens play critical roles in health, regulating not only the reproductive system, but also having important effects on the muscular, skeletal, cardiovascular and central nervous systems.

The Androgen receptor can act independently of its ligandAR can be activated or modified by non-ligand factors such as EGF and IGF-1.

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p53

What is p53?

It is a DNA binding transcription factor.

Discovered in 1979, initially thought to cause cancer.

It actually protects against cancer, it is a tumour suppressor - it is the loss of p53 that causes cancer.

Is the most studied gene/protein in the whole of biomedical research.

It is mutated or lost in more than 50% of cancers (most of the remaining ~50% have problems with other genes that regulate p53, such as PKB/Akt).

It has been called the “guardian of the genome”.

The p53 protein can be divided into five domains, each with specific functions:

The amino-terminus (a.a.1-42) contains the acidic transactivation domain and the mdm2 binding site. It also containsthe Highly Conserved Domain I (HCD I)Amino acids 40-92 contains repeated proline residues that are conserved in the majority of p53. It also contains asecond transactivation domain.The central region (a.a.101-306) contains the DNA binding domain. It is the target of 90% of p53 mutations found inhuman cancers. It contains Highly Conserved Domains II to V.The oligomerisation domain (a.a.307-355, TET) consists of a β-strand, followed by an α-helix necessary fordimerisation (p53 acts as tetramer of two dimers). A nuclear export signal (NES) is located in this oligomerisation

domain.The carboxy-terminus of p53 (a.a. 356-393) contains 3 nuclear localisation signals (NLS) and a non-specific DNAbinding domain that binds to damaged DNA. This region is also involved in down-regulation of DNA binding of thecentral domain.

The TP53 gene encodes a transcription factor, p53

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The p53 pathway

p53 is a DNA binding transcription factor. It sits at the crossroads of a network ofsignalling pathways that are essential for the regulation of cell growth and apoptosisinduced by genotoxic and non-genotoxic stresses.

Regardless of the type of stress, the final outcome of p53 activation is (usually) eithercell cycle arrest and DNA repair or apoptosis. However, the mechanism leading to thechoice between these fates has not yet been elucidated.

The p53 pathway can be divided into five stages.

i) Signals that activate the pathway.

ii) The upstream mediators that detect and interpret the stress signals.

iii) The core regulation of p53 through its interaction with several proteins that modulate its stability.

iv) The downstream events, mainly transcriptional activation or protein-protein interactions.

v) The outcomes; growth arrest, apoptosis or DNA repair.

p53 pathway: Upstream pathway

i) The stress signals that activate the pathwayii) The upstream mediators that detect and interpret the upstream signals.iii) The core regulation of p53 through its interaction with several proteins that modulate its protein stability

i)

ii)

iii)

p53 responds to many different forms of stress

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p53 pathways: downstream pathwayiv) The downstream events, mainly transcriptional activation or protein protein interactionsv) The final outcome(s), growth arrest, apoptosis or DNA repair.

iv)

v)

In general p53 activity is regulated by modulation of the stability of the protein - i.e. by halting its constitutive degradation.

Mdm2 is a p53 target gene. Thus p53 regulates its own destruction protein, as part of a negative feedback loop

The proteosome

In the inactive “resting” state p53 levels are kept low by constant degradation of p53 proteins, predominantly through the action of the ubiquitin ligase mdm2 (murine double minute 2).

Upstream signalling after DNA damage (γ irradiation). Phosphorylation of p53 and mdm2 disrupt the interaction between the two proteins. p53 is no longer targeted for destruction and so its levels accumulate.

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Downstream signalling. Cell cycle arrest. G1 arrest via p21 transcription. p21 prevents Rb phosphorylation via inhibition of the CDK4 and CDK2 kinases.

http://www.siliconera.com/2007/09/03/siliconera-stash-zelda-meets-biochemistry/

http://www.siliconera.com/2007/09/03/siliconera-stash-zelda-meets-biochemistry/

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That regulates protein levels.What about target selectivity?

Far from clear

Certainly involves post translational modifications (PTMs)

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of which there are many!

and which other proteins the p53 proteins interacts with

(PP-Is)

Only very recently was it discovered there are several splice variants of p53: α, β & γ. Each can also either posses (TA forms) or lack (ΔN) the transactivation domain - rendering them transcriptionally active or in active. The ΔN forms inhibit the transcriptional activity of the TA forms.

To add to that complexity, multiple splice forms of p53 have been discovered.

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And there are two other family members, p63 and p73.

Again multiple splice variants, and TA and ΔN forms, of each exist.

There are 43 different splice variants of the 3 family members.Homo- and hetero-dimers can form within and between family members.

Dimers then bind each other to form the active tetramer.The possible permutations are enormous.

Unlikely all occur in nature, but……

99

The ΔN forms of p63 and p73 repress the activity of TAp53

In epithelial cells mainly ΔNp63, in brain ΔNp73.

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ErbB or EGF receptor family

another example of receptor tyrosine kinases

ErbB or EGF receptor (EGFR) family

A family of Trk receptors (Tyrosine receptor kinases).

The founder member is the epidermal growth factor receptor, EGFR.

Strongly implicated in cancer - especially breast cancer.

Viruses “hijack” host genes.

The erythoblastoma virus hijacked several - these genes were named erb-A, erb-B, etc.

Later it became clear that erbB was the homologue of EGFR.

erbA is the the viral form of the Thyroid receptor

Epidermal growth factor (EGF) - a small peptide/growth factor/hormone but EGF is not the only ligand.

The ErbB family of receptors (4).Known ligands are listed above each receptor.ErbB2 has no known ligand.The ErbB3 kinase domain is inactive.

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The receptors act as homodimers or heterodimers.

work best as heterodimers containing one ErbB2

A major ligand is neuregulin-1 NRG1. a)The six types of neuregulin 1 (NRG1) isoforms are classified according to their distinct amino-terminal sequences.b) Most NRG1 isoforms are synthesised as transmembrane precursor polypeptides (pro-NRG1s) with the EGF domain

located in the extracellular region, but in Type III NRG1 both the N- and the C-terminal regions are located inside thecell. Cleavage by tumour necrosis factor-α converting enzyme, β-site of amyloid precursor protein cleaving enzyme ormeltrin β (indicated by the lightning arrow) generates mature NRG1s that are soluble, except in the case of Type IIINRG1, which is thought to function in a manner that requires cell contact.

Canonical NRG1–ErbB signalling pathways.In response to neuregulin 1 (NRG1) stimulation, ErbB proteins become dimerised to form homo- and heterodimers. ErbB2 does not bind to NRG1 (indicated by the black crosses) but has an active kinase domain; ErbB3 binds to NRG1 but has an impaired tyrosine kinase domain (indicated by the purple cross). Therefore, ErbB2 and ErbB3 need to form heterodimers with each other or with ErbB4 to be functional, whereas ErbB4 homodimers can bind to NRG1 and become activated. Activation of the tyrosine kinase domains leads to auto- and trans-phosphorylation of the intracellular domains, generating docking sites for the adaptor proteins Grb2 and Shc, which activate the Raf–MEK–ERK pathway, and for the p85 subunit of PI3K, which activates the PI3K pathway and subsequently mTOR-dependent protein synthesis.

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Non-canonical NRG1–ErbB4 forward signalling (bottom cell).In non-canonical forward signalling (bottom cell, right-hand pathway), the carboxy-terminal fragment (ErbB4-CTF) iscleaved by γ-secretase to produce ErbB4-intracellular domain (ErbB4-ICD), which can translocate to the nucleus toregulate gene expression. When it is overexpressed in transfected cells, ErbB4-ICD interacts with several transcriptionalregulators, including Eto2, STAT5, Mdm2 and YAP, to mediate the transcriptional activation or repression ofheterologous promoters. This interaction might require the phosphorylation of either ErbB4-ICD or the transcriptionalregulator and/or the kinase activity of the ICD domain.

Back signalling (top cell).by pro-NRG1 can proceed by two mechanisms. First, the C-terminal fragment of pro-NRG1 (NRG1-CTF), which is generated

by extracellular cleavage, can be cleaved again by γ-secretase to generate NRG1-intracellular domain (NRG1-ICD), whichcan relocate into the nucleus to regulate gene transcription (left-hand pathway).

Cells have evolved many different (sometimes apparently peculiar) ways

to communicate with each other.

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Cell signalling and disease(when cell-cell signalling goes array).

Components of the Wnt pathway can be classed as oncogenes (i.e., promote tumour formation) or as tumour suppressors (i.e., repress tumour formation).

Adenomatous polyposis coli (APC) also known as deleted in polyposis 2.5 (DP2.5) is a protein that in humans is encoded by the APC gene. Mutations in the APC gene are the

major cause of colon cancer.

Several Wnt antagonists are being developed and trailed to treat cancer.

Schizophrenia.Nrg-1 is (was) the most highly replicated Schizophrenia (SZ) risk gene - its receptorErbB4 is also a SZ risk gene.

ErbB4 and Nrg-1 both involved in synapse formation.

Which of Nrg-1/ErbB4 activities is the most relevant for Schizophrenia has not been determined.

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16 - 18 months

1.5 - 2 months

Loss of one copy of p73 leads to tau phosphorylation and the formation of tangle like structures

Crossing p73+/- mouse with TgCRND8(double APP mutant) dramatically

exacerbates tau pathology

β-amyloid activates the expression of Dickkopf1 (Dkk1)

It is claimed Dkk1 inhibits canonical Wnt and thereby “activates” GSK3 - the major tau kinase

Alzheimer’s disease

The sFRPs (soluble frizzleds) - mop up Wnts before they can bind receptor complex

Ca2+ and PCP pathways

Dkk1 permits non-canonical pathway activation

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Neuronally expressed Wnt-PCP target genes are needed for tau phosphorylation and neuronal cell death and memory impairment in vivo

whilst the RhoA/ROCK arm regulates synapse loss.

β-amyloid activation of Dkk1 switches off canonical Wnt and turns on the Wnt-PCP pathway in neurons (in a p53-dependent manner).