Chapter 28: Intracellular Receptors and Regulated Proteolysis

Question 1

Regulated roteolysis of Latent Gene Regulatory Proteins

Answer 1

• Many signaling systems rely on the activation of latent gene regulatory proteins to transmit the signal to the DNA that gene transcription must be altere.
  
  • Stats (cytokine receptors) and Smads (receptror ser/thr kinases) are activated by phoshorylation
  • β-catenin, NF-κB, Notch, and Hedgehog/sonic hedgehog, are activated by regulated degradatioin of a component in the signaling pathway
• This ‘regulated proteolysis’ involves polyubiquitination (Lys 48 – Gly 76) followed by proteasomal degradation. It differs from the proteolysis triggered by misfolded proteins in that the targets of the E3 ubiquitin ligases are very specific – not just an exposed hydrophobic region.

Question 2

Regulated Proteasomal Degradation can be Controlled by a Variety of Mechanisms

Answer 2

• Wnt signaling>dephosphorylation of the protein to be degraded
• NF-κB signaling > phophorylation of the protein to be degraded

Question 3

Regulated Proteolysis: The Wnt Signaling Pathway

Answer 3

• ‘Wnt’ was coined by combining the names for two orthologs into a single name:
  
  • Wingless: a segment polarity gene in Drosophila. Discovered because mutations made flies wingless
  • Int: a gene that promotes mammary tumors in mice when a virus (MMTV) integrates next to it and activates it.
• Wnts are a family of secreted ligands (19 in humans) that act as paracrine mediators to control many aspects of development. They also contribute to cancer formation.

Question 4

Signaling Through ‘Canonical’ Wnt Pathway

Answer 4

• There are 3 Wnt signaling pathways, we are focusing on most common one
• In all 3 pathways, the Wnt signal molecule binds to the Frizzled family of 7 cell surface receptors.
• Frizzled receptors transverse the membrane 7 times and thus resemble GPCRs. However, no G protein has been identified that is activated by Frizzled.
• Activation of Frizzled involves a co-receptor known as LRP (LDL-receptor-related protein).
• The Wnt pathway acts by regulating the proteolysis of β-catenin, which functions both in cell adhesion (adherens junctions) and in gene regulation.

Question 5

Basal State of Wnt Pathway

Answer 5

• In its inactive state, the b-catenin (Armadillo in flies) that is not bound to cell-cell adherens junctions is sequestered in the cytoplasm in a “degradation” complex with 4 other proteins: Axin, APC, GSK3, CK1
• Phosphorylation of b-catenin by CK1 and then by GSK3 marks it for ubiquitination and rapid degradation.
• Wnt responsive genes are kept inactive by the Groucho co-repressor bound to the LEF1/TCF activators.

Question 6

Axin

Answer 6

Scaffold protein

Question 7

APC

Answer 7

(Adenomatous polyposis coli), scaffold protein

Question 8

GSK3

Answer 8

(glycogen synthase kinase 3)

ser/thr protein kinase

Question 9

CK1

Answer 9

(Casein kinase 1)

shr/thr kinase

Question 10

Activation of Wnt Signaling

Answer 10

• Wnt ligands cause the Frizzled and LRP receptors to cluster together, which in a poorly understood process, activates the Dishevelled (Dsh) scaffold protein.
• Somehow, Dsh recruits GSK1 and CK1 to the receptor complex, and LRP gets phosphorylated.
• Axin is also brought to the receptor.
• All of this disrupts the degradation complex, so b-catenin is no longer phosphorylated and recognized by a ligase for ubiquitination.
• Unphosphorylated b-catenin accumulates and goes to the nucleus, where it kicks off Groucho and binds to LEF1/TCF This recruits the degradation complex to the receptors, inhibiting GSK3 activity and thus the degradation of b-catenin.
• Target genes for b-catenin include Myc, which is a transcription factor that promotes cell growth and proliferation.
• 1, 2, 4, 6, most important

Question 11

(Familial) Adenomatous Polyposis Coli

Answer 11

• First hereditary colorectal cancer syndrom analyzed
• 100’s to 1000’s of polyps appear early in adult life
• If the colon is not removed, one or more will progress to cancer within about 12 years. About 80% of the time this is the result of mutation/inactivation of both copies of the Apc gene.
• Apc is a tumor suppressor gene**

Question 12

beta-Catenin, APC, and the Biology of Colonic Crypts

Answer 12

• In normal intestine, the stromal cells send out Wnt ligand, which causes the cells at the bottom to replicate, and it also prevents their differentiation.
• As the epithelial cells migrate away from the stromal cells, the Wnt signal decreases, the cells stop replicating and they differentiate. After 3 – 4 days, the cells at the lumen go through apoptosis.
• In cells lacking APC, β-catenin remains high even without Wnt because β-catenin is not sequestered in the degradation complex by APC.

Question 13

NF-kB proteins

Answer 13

• NF-kB proteins are latent transcription factors that are present in most animal cells and are central to stressful, inflammatory, and innate immune responses.
  
  • There are 5 NF-kB proteins in mammals (RelA, RelB, c-Rel, NF-kB1, and NF-kB2 ).
  • These proteins form a variety of homodimers and heterodimers, each of which activates its own characteristic set of genes.
  • However, the signaling pathway that activates these various transcription factors is the same

Question 14

Regulated Proteolysis: Activation of NF-kB

Answer 14

• NF-kB activates the transcription of >150 genes involved in cell responses to inflammation and the immune response
• NF-kB activity is regulated by inflammation, infection, and other stressful situations such as ionizing radiation
  
  • Two inflammatory cytokines that activate NF-kB are TNF-a and interleukin-1 (IL-1)

Question 15

NF-kB

Answer 15

• In its inactive state, NF-kB is stored in the cytoplasm complexed with an inhibitory protein, I-kB, which hides its NLS (nuclear localization signal)
• Binding of an inflammatory cytokine to its receptor triggers a protein kinase cascade that leads to the phosphorylation of I-kB by I-kB kinase.
• The phosphorylated I-kB is recognized by a specific E3 ligase, which ubiquitinates I-kB
• Polyubiquitinated I-kB is recognized and degraded by the proteasome
• Removal of I-kB exposes NLS on NF-kB so that it can enter the nucleus

Question 16

Intracellular Receptors=

Answer 16

Nuclear Receptors=Steroid Receptors

Question 17

NRs Play Profound Roles in Normal and Abnormal Physiology

Answer 17

• NRs generally have long-term effects on the body
  
  • The endogenous ligands are synthesized on demand (not stored).
  • The ligands have relatively long half-lives (hours to days).
  • The effects of NRs are mostly at the level of gene transcription.

Question 18

Normal Physiological Effects of Estrogen

Answer 18

• Cardiovascular and neuronal activity; protects against dementia
• Liver, fat, and bone cell metabolism
• Reproduction – development and function
• Immune responses
• Estrogen receptor targets genes involved in cellular proliferation, cellular differentiation, cell survival

Question 19

The Nuclear Receptor Superfamily

Answer 19

• The receptors for many lipophilic hormones, metabolites, and xenobiotic endocrine disruptors are intracellular proteins that function as ligand-activated transcription factors.
• ~13% of the FDA approved drugs target these receptors.
• Nuclear receptors are only found in metazoans (animals).
• There are 48 nuclear receptors in humans, 49 in mice, and 250 in C. elegans

Question 20

NRs Have Many Functional Domains

Answer 20

A/B region: 100 – 500 aa long with essentially no aa identity among family members

C = DNA binding domain = DBD: ~68 aa long and 42 – 94% aa identity

D = hinge region. Involved in receptor folding

E = ligand binding domain = LBD = 15 – 57% aa identity. Also involved in receptor dimerization, interaction with other proteins, and nuclear localization

AF-1: transcriptional activation function 1. Thought to be activated primarily by phosphorylation.

AF-2: transcriptional activation function 2. Activated by ligand binding

Question 21

NR Type I

Answer 21

Bind to inverted repeats as homodimers. May be cytoplasmic and/or nuclear (the steroid receptors)

Question 22

NR Type II

Answer 22

Bind to direct repeats as heterodimers with RXR. Are nuclear. Usually called the orphan receptors

Question 23

NR Type III

Answer 23

Bind to direct repeats as homodimers

Question 24

NR Type IV

Answer 24

Bind as monomers or heterodimers but only one receptor binds to DNA

Question 25

Nuclear Receptor Ligands

Answer 25

• All steroid hormones are derived from cholesterol.
  
  • They are lipophilic and enter cells by passive diffusion.
  • Vitamin D is also a steroid hormone, although it binds to a Type II receptor.
• Ligands for the Type II receptors are varied in structure but are also lipophilic.
  
  • Thyroid hormone (T3), derived from tyrosine
  • Retinoids, derived from Vitamin A
  • Bile acids (FXR)
  • Oxysterols (LXR)
  • Fatty acids and derivatives (PPAR)
  • Xenobiotics

Question 26

Ligands for Type I NR

Answer 26

1. All are derived from cholesterol
2. Are present in serum in pM to nM amounts
3. Bind NR with high specificity
4. Bind NR with high affinity (K_D=10^-9 to 10^-12 M)

• Progestins (C21)
• Androgens (C19)
• Mineralocorticoids (C21)
• Glucocorticoids (C21)
• Estrogens (C18)

Question 27

Some Type II Nuclear Receptors Bind Metabolic Ligands

Answer 27

PPARalpha, PPARgamma, LXR, FXR

These receptors are promiscuous and bind many compounds with relatively low affinity and specificity (10^-6 M)

Question 28

PPARα

Answer 28

The fibrate receptor, Lowers serum triglycerides

Question 29

PPARγ

Answer 29

Glitazone receptor, lowers serum glucose

Question 30

LXR

Answer 30

The oxysterol receptor, lowers serum cholesterol

Question 31

FXR

Answer 31

the bile acid receptor, raises serum cholesterol

Question 32

Selective Receptor Modulators (SRMs)

Answer 32

Ligands for specific nuclear receptors. The goal is to develp tissue-specific regualtors

Question 33

SERMs

Answer 33

Selective ER modulators (birth control-agonists, chemotherapy-tissue dependent)

Question 34

SARMs

Answer 34

Selective AR modulators (steroid abuse-agonists, chemotherapy-antagonists)

Question 35

Agonist

Answer 35

Binds to receptor and activates it

Question 36

Antagonist

Answer 36

Binds to R and prevents agonist from binding

Question 37

Determination of Agonist or Antagonist Activity Is Determined by Tissue-Specific CoRegulatory Proteins

Answer 37

• The conformation of ER changes depending on which SERM binds.
• The proteins that recognize the different ER forms sometimes vary with different cell/tissue types.
• The differences in the intercellular proteins provides the basis for tissue-selective agonism/antagonism.

Question 38

NRs bind to DNA via…

Answer 38

Zinc Fingers

Question 39

NRs Typically Bind to Short Sequences of DNA That Function as Enhancers

Answer 39

• Enhancers are short segments of DNA (~6 – 15 bp) that bind transcription factors that activate genes
• Enhancers may be thousands of bp 5’ or 3’ of the protein coding region or within introns
• Enhancers may be oriented in either direction with regard to transcription (they may be flipped)
• Enhancers can be moved to another gene and will regulate that gene

Question 40

Response Elements for Steroid Receptors Are Inverted Repeats (Palindromes)

Answer 40

•Response elements are called HREs (hormone response elements), SREs (steroid response elements), or by the receptor name such as GRE for glucocorticoid response element. These HREs are usually enhancers.

Question 41

Summary of the Mechanism of Action of Type I NRs

Answer 41

• The NR resides in the cytoplasm and/or the nucleus in a heat shock protein inhibitory complex.
• The binding of steroid hormone to the NR releases the chaperone complex, and two receptors dimerize.
• The dimerized NRs travel to the correct site on chromosomes and binding to their HREs. In the process, chromatin structure is often opened up.
• Coactivators associate with the NRs and gene transcription is increased.

Question 42

Type II Receptors Behave Somewhat differently from Classic Steroid Receptors

Answer 42

• Nuclear receptors for non-steroid hormones (thyroid hormone, retinoic acid, vitamin D3, PPARS, LXR, etc.) typically bind DNA as heterodimers with a common heterodimerization partner, RXR (retinoid X receptor)
• These Type II nuclear receptors are typically bound to DNA in a co-repressor complex in the absence of ligand (rather than in a hsp complex), silencing the gene
• The DNA binding sites (response elements) for these nuclear receptors are composed of two direct repeats of the hexamer AGGTCA in which the distance between the repeat imparts specificity

Question 43

Summary of the Mechanism of Action of Type II NRs

Answer 43

• Type II NRs bind to DNA as heterodimers with a common heterodimerization partner, RXR (retinoid X receptor)
• Type II nuclear receptors are typically bound to DNA in a corepressor complex in the absence of ligand (rather than in a hsp complex), which silences transcription.
• Binding of ligand to the non-RXR receptor partner releases the corepressor molecules, which are replaced by coactivators.
• Coactivators associate with the NRs and gene transcription is increased.

Question 44

Comparison of the Mechanism of Action of Type I and Type II NRs

Answer 44

Question 45

NRs Can Directly and Indirectly Regulate Gene Transcription

Answer 45

Although NRs have major effects on the transcription of genes, each receptor type binds directly to relatively few genes. However, those genes may encode transcription factors, which then go on to bind to other genes. Thus, NRs can indirectly regulate a large number of genes through transcriptional cascades. Bottom line: they have many indirect effects and their effects can be quite delayed because of this.