Cellular Signal Trasnduction

For Professor Leibowitz's CST/Advanced Cell Biology course

Types of Cellular Signaling

• Pheromones: Between organisms

• Endocrine: Long distance signaling

• Paracrine: Local release/response

• Autocrine: Cell talks to itself

• Membrane-Attached: Cell A has signal that attaches to Cell B physically

Signal (Hormone) Categories

• Small lipophilic molecules

• Hydrophilic molecules

• Lipophilic molecules

• Gas molecules

Processing the message with…

• Specificity

• Regulation

• Flexibility

• Kinetics/Dynamics: How fast you respond to signal/amount of signal

• Amplification: Increasing signal

General Signal Pathway

Receptor → Adaptor → Second Messenger → Effector

Receptor-Ligand Interactions

• Affinity: Strength of interaction

• R + L ⇆ RL

• Kd: Dissociation constant

Molecular Switches

• Conformation

• Dimerization: Multimerization

• Phosphorylation/Dephosphorylation

• Post-Translational Modifications

• Recruitment/Subcellular Localization

Stopping/Attenuating a Signal

• Degradation of hormone, very slow

• Internal mechanisms cause an immediate halt

Points of Potential Drug Intervention

• How it works:

• Inhibit function by directly blocking target receptor

  • Receptors can have other functions/downstream effectors

• Block entire pathway (signal transduction)

  • Could work/could not, need empirical evidence

General Drug Research and Development Scheme

• Target Identification

• Target Validation

• Drug ID

• Drug Validation

• Animal Testing

• IND → Phase I-III Clinical Trials → NDA

Target Identification

• Don’t need a specific target, just need to know it works

• Starting from nothing: Comparative biology between sick and healthy

• Starting from something: Drug/modulator

Target Validation Methods

• Knockout/Knock-in mutants

• Cell-based assay with modulator

Rational Drug Design

• Understand molecular details of drugs so it binds properly

  • Structure Determination: Lots of AI used to synthesize compounds

    • NMR Spectroscopy

    • X-Ray Crystallography (very expensive/time consuming)

  • Modeling

  • Structure Activity Relationships

Random Drug Screening

• Three types:

  • Assay Development: In well plate

  • Library: Can have millions of compounds

    • Combinatorial chemistry

    • Natural product

  • Screening

Semi-Rational Drug Discovery

Know as much as you possibly can about the target so you can have a library of potential compounds (combine rational and random approaches

Validation

• Secondary Screening: Use animal models

• Move proof up a notch

Preclinical Testing

Drug design systems: How fast drug is absorbed, does drug work

Diagnostics

• Diagnosis is key

  • Importance: Arguably 50% of treatment

  • Types of Diagnostic samples: Biopsies, bodily secretions, scans

  • Diagnostic Tools: Analyte Measurement, Antibodies, genomic DNA/SNP

GPCR Activation

• GTPases linked to receptors

• Heterotrimeric G Protein Pathway: Induce guanine nucleotide exchange

  • Receptor binds ligand → Conformational change in G protein

  • Gα releases GDP to bind GTP → Gα dissociates from Gy

  • Gα and Gβy interact with different effectors

  • GTP is hydrolyzed to GDP + Pi

  • Gα associates with GDP again and Gβy

G-Proteins

• Gα: GTPase activity, Receptor and Effector Interactions

  • Gαs: Stimulates adenylyl cyclase

• Gβy: Effector interactions

  • Gβ: Interface that interacts with Gα has key residues for effector interactions

GPCR Structure

• Integral membrane protein

• Seven transmembrane α-helices

• Extracellular loops: Ligand binding site outside, G protein binding site inside

Three Classes of GPCR

• Class I: Rhodopsin-like

• Class II: Glucagon-like

• Class III: Metabotropic Glutamate-like

Molecular Switch: GPCR

• Conformational change w/ ligand binding

• Ligand-GPCR complex binds to G-protein

  • Ligand binds to receptor and changes structural conformation

GPCRs Regulate Ion Channels

• Direct regulation via G protein

• Activates a signal pathway up-regulating a kinase or phosphatase to open/close it

Second Messengers

cAMP: Activate proteins inside the cell (protein kinases, ion channels, other enzymes, transcription)

cAMP

• Formation: Adenylyl Cyclase + ATP → cAMP + Pi

• Utilization: cAMP Dependent Protein Kinase (PKA)

• Degradation: Use phosphodiesterase to hydrolyze it to AMP

GRKs

• Serine-threonine kinases recognize and phosphorylate receptors in their agonist-stimulated form

• DLEESSSSD: Intracellular sequence exposed when ligand-bound

• GRKs are alternatively regulated/expressed in different cells, different conditions

GPCR Regulation: GTP

• For G-proteins/GTP: Built in self-timer (shut-off mechanism)

• RGS Proteins: Accelerate GTP hydrolysis by binding Gα (higher catalytic rate)

GPCR Regulation: Receptor Phosphorylation

• PKA: Doesn’t matter if ligand is still bound, receptor is turned off due to dissociation from G Protein

• GRK (G Protein-Coupled Receptor Kinase): Targets ligand directly, desensitization

GPCR Regulation: cAMP

• Bind to regulatory subunit in a cooperative fashion, first ligand makes second ligand bind better

  • Regulatory domain has pseudosubstrate that binds active site, inhibit catalytic domain

  • cAMP binding causes regulatory and catalytic domains to separate, allow phosphorylation of substrate

GPCR Specificity

• Link specific GPCR to specific G-Protein via

  • Effector specificity

  • Differential cell expression

  • Toxins

Coupling Promiscuity

• Many GPCRs can couple to more than one G protein subtype

• Isoforms of cellular components can promote signal integration by having partially overlapping connectivity

Adrenergic Receptors

• Three Types:

  • Gs-coupled: More camp, stimulate adenylyl cyclase

  • Gq-coupled: α1

  • Gi-coupled: α2 for smooth muscular lining blood cells, inhibit Adenylyl Cyclase

Gs vs. Gi

• Gs stimulates adenylyl cyclase

  • Cholera: ADP ribosylates Gαs to lock it in a GTP-bound form → No GTP hydroysis → Overproduce cAMP

• Gi inhibits adenylyl cyclase

  • Pertussis: Stuck in GDP-bound/inactive form → Inhibit adenylyl cyclase → Overproduce cAMP

Crosstalk | GPCR and RTK Pathway

• Gα+GTP dissociates and activates Phospholipase C

• Phosphoinositol pathway activated by RTKs produce PIP2

• Phospholipase C hydrolyzes PIP2 → IP3 + DAG

• IP3 associates with voltage-gated Calcium ion channels to release CA2+

• DAG + CA2+ → Release Protein Kinase C which can modulate RTK pathway proteins

FULL GPCR PATHWAY

• Ligand binds GPCR, triggering a conformational change in the G-protein subunits

• Gα loses GDP for GTP binding, dissociates from Gβy

• Gαs + GTP bind to adenylyl cyclase, cause conversion of ATP to cAMP

• cAMP binds to inactive PKA complex

• Two catalytic domains dissociate from two regulatory domains, so PKA is now active

• PKA travels to the nucleus and phosphorylates CREB TF, binds to DNA

Receptor Tyrosine Kinases

• Signal Transduction receptors that can form homodimers or heterodimers w/ many combinations depending on the cell and varying numbers based on expression

• Structure:

  • Extracellular Ligand Binding Domain

  • Single Transmembrane Helix

  • Cytosolic Domain including a protein Tyrosine Kinase

Molecular Switch: RTK

• Dimerization: Activates all RTKs

• Ligand binding locks dimers in active conformation when they were once inactive

Tyrosine Phosphorylation

• Ligand forces kinase domains to interact → Chains phosphorylate each other making open kinase domains for even more phosphorylation

  • Structure on its own inhibits range of interaction/action, need ligand to alter conformation permanently

RTK Amplification: Adaptor Proteins

• Has binding site for phosphotyrosine, binds to exposed phosphate on tyrosine chain

• Can branch off and link tyrosine to multiple pathways

  • Membrane interactions

  • Enzymatic activity domain

RTK Regulation | Desensitization

Limited number of cell surface receptors, so regardless of the concentration of ligand, prevent prolonged receptor activity

Specificity | Phosphotyrosine Interacting Domains

• Recognize phosphotyrosine directly on chains to recruit adaptor proteins

• SH2: Src Homology 2 domains

  • Looks for 1-6 AAs on C-terminal side of chain, if there’s recognition, binding happen

• P-tyr Binding Domain: Recognizes phosphotyrosine and 3-5 AAs in N-Terminal

Specificity | Protein-Protein Interactions

• SH3: Ser Homology 3 domains

  • Bind to Proline Rich Domains (PXXP); bind in multiple places for stronger overall interaction

• WW domains: PXPX

• PD2: Bind to hydrophobic AA at C-Terminus of protein

Specificity | Membrane-Interacting Domains

• Stick to surface of membrane, interact with head groups of phospholipids

  • Pleckstrin Homology (PH) domain: Bind PIP3

  • FYVE domain: Bind PI-3-P

  • Docking proteins

RTK Effectors

• Ras-GEF: MAP Kinase

• PLCy

• PI-3 Kinase: Kinase phosphorylates PI

How to Activate Effector Proteins | Membrane Translocation

• Protein in cytosol

  • Ligand binds receptor → Translocation from membrane to cytosol to find substrate

  • Convert PIP2 → PIP3

How to Activate Effector Proteins | Conformational Change

• Two molecular switches

• Membrane translocation then conformational change

How to Activate Effector Proteins | Tyrosine Phosphorylation

• Inactive Phospholipase C recruited to membrane, kinase of receptor phosphorylates Phospholipase C to activate it

Getting Your RAS in Gear

• RAS: Guanine Nucleotide Binding Protein (GTPase)

• Nucleotide exchange needs GEF (Guanine Exchange Factor: Interacts with small G protein with only one subunit)

  • Inactive: GDP + GEF → Active: GTP until GTP hydrolyzes

  • Hydrolysis needs GAPs: Change conformation of RAS to increase hydrolysis capabilities

• RAS lives near membrane, undergoes palmitoylation (palmitoyl group is covalently attached, stuck in membrane)

Identifying RAS as an RTK Downstream Inhibitor w/ VEGF

• Remove RAS with anti-RAS Abs

  • No cell activation because VEGF is a downstream growth factor

• Have mutant RAS that is constitutively active

  • Out of control cell growth, regardless of presence of VEGF

Linking RAS and RTK: Bug Eyes

• Sevenless: RTK (named after mutated eye cell in drosophila)

• Bride of Sevenloss (BOS): RTK ligand/agonist that causes dimerization after binding

• Grb2: Adaptor protein (SH2/SH3 Homolog) that grabs receptor w/ SH2 domain and recruits SOS with SH3 domains in cytosol

• Son of Sevenless (SOS): GEF that interacts with and activates RAS via conformational change

  • Ras+GDP + SOS → RAS+GTP

Amplification | MAP-Kinase Pathway

• RAF: A MAP-KKK activated by RAS+GTP, further activated by RAS+GDP

• MEK: MAP-KK phosphorylated by RAF

• ERK: MAPK phosphorylated by MEK

• ELK-1: TF in nucleus is phosphorylated by ERK, leads to cell cycle progression

  • Other MAPKs: Jun, Myc, Fos, ATF-2

Crosstalk | Phosphoinositol Pathways I

• Phospholipase Cγ: Inositol or PI can be further phosphorylated to get PIP, PIP2, or PIP3

  • PIP2 cleaved by Phospholipase C → DAG + IP3

  • GPCRs activate Phospholipase Cβ!

Phosphoinositol Pathways II

• PI3 Kinase: Activates PKB

• AKT/PTEN: pH domain interacts with PIP3; phosphorylation by PDK1,2

  • PTEN: Phosphatase and Tensin homolog, promotes apoptosis

    • BAD: Induces apoptosis, inhibited when phosphorylated by PKB

    • p27: Regulator, inhibitor of inhibitor of cell cycle

RTK Regulation | Attentuation/Termination

• Antagonistic Ligands

• Heteroligomerization with receptor mutants

• Inhibiting the RTK:

  • Ser/Thr phosphorylation: Turn on receptors

  • Protein Tyrosine Phosphatase: Remove phosphate, turn receptors off

• Receptor Endocytosis/Degradation

RTKs in Pathophysiology | Cancer Treatments

• Herceptin: Erbitux mAb - binds to receptor outside of cell

  • Improved by releasing toxin when degraded and killing cancer cells

• Tarceva: Small molecule tyrosine kinase inhibitor

RTK | Role of EGFR in Cancer

• Stimulating motility factor of cells (metastasis) and angiogenic factors

• Binding of specific ligands, leads to different outcomes

• Regulates tumor cell division, repair, survival

• Block pathway → Block phenotypes

FULL RTK PATHWAY

• RTK: Sevenless

• Ligand, Bride of Sevenless (BOS) binds to RTK

• Tyrosine Kinase domains phosphorylate one another, bind Grb2 adaptor protein

• Grb2 recruits SOS via SH3 domain, which activates RAS

• RAS exchanges GDP for GTP, and activates RAF

• RAF activates MEK with ATP exchange

• MEK activates ERK with ATP exchange

• ERK translocates to nucleus and activates TF, ELK-1 to allow cell cycle progression

  • Other TFs: jun, myc, fos

How to Control Transcription?

• Get region of DNA to extend and not be condensed AND recruit DNA Pol

• Use transcription factors

  • Steroid Hormone Receptors

  • TFs recruited by sequence-specific TFs, and general TFs with RNA Pol II to start transcription

Molecular Switch: Intracellular Receptors

Subcellular localization with AhR

Intracellular/Nuclear Receptors

• Ligands: Small lipophilic molecules, thyroid hormones

• Receptor Activity:

  • Ligand binding

  • DNA binding

  • Protein-Protein Interactions

Nuclear Receptor Superfamily

• Steroid Receptors: Binds palindromic sequences

• RxR Heterodimers

• Dimeric Orphan Receptors: Binding a direct repeat

• Monomeric Orphan Receptors

DNA Binding Domain

• Zn² Fingers: Coordinated by cysteine residues

• P Box: DNA recognition

• D Box: Dimerization

• Recognition Helix: Binds major grroove because it needs to bind to specific sequences

• C-Terminal Extension: T/A Boxes

Hormone Translocation

• Hormone enters cell and binds to complex

  • If in cytosol, translocates to nucleus

  • Bind to promoter region of gene in DNA, start transcription to allow genes to be expressed (produce mRNA)

  • DNA Binding Domain binds DNA at major groove

Receptor Structure

Domain Structure of Nuclear Hormone Receptors

• NLS: Nuclear Localization Sequence in between C and D

• Absence of ligand: NLS is hidden

Hormone Response Elements (HRE)

• Hormone response element is where TF binds

  • Consensus Motif: 6 bp core element

  • Steroid Class II: AGAACA

  • The rest: AGG/TTCA (/ represents G or T)

Dimeric HRE Recognition

• Receptors may recognize singular motif but as dimers, there are two consensus motifs that are recognized

  • Direct repeats, palindromes

  • Inverted Palindrome: ACTT/GGA or ACAAGA

DNA Binding Sites of Receptors

• DR5: Consensus motifs, in between are spacers

• DBD: Only binds when it recognizes a core sequence, but it still looks at nucleotides nearby

Dimerization

• Monomers: When binding as monomer, binds to DNA elements with 5’ A/T-rich sequence before the consensus sequence

• Homodimers: Competitive binding, first one binds making second bind easier

  • Binding palindromes

Heterodimers

• RXR as promiscuous partner (Pal., DR, Inverted Pal.)

• Non-Permissive: Ligand binds RAR first activating transcription ALWAYS

• Permissive: Ligand binds either PPAR or RXR activating transcription

  • 2nd ligand binding increases activity

  • Low level transcription at first, then high level for the second

  • Two signal pathways, crosstalking

Intracellular Nuclear Receptors | Regulation

• A/B Domain: Size/length varies between TFs

• AF-1: Autonomous transcriptional activation factor, upregulate/downregulate activity of TFs via phosphorylation with protein kinase

• AF-2: Ligand dependent; can only phosphorylate region if ligand is bound

Crosstalk | Intracellular Receptor and RTK

• Phosphorylation of Steroid Nuclear Receptors

  • Use MAPK and AKTin A/B region of AR Basal

• Direct phosphorylation of TFs

  • Phosphorylation of ELK-1 leads to conformational change → Stronger DNA binding → Transcriptional repression of ELK-1

Controlling Chromatin Structure (KNOW FOR EXAM)

• Histone Acetylation/Deacetylation

• HDAC: Removes acetyl groups from histones to condense DNA (deacetylation)

• HAT: Add acetyl groups

• TRAP/DRIP Complex: Holoenzyme recruitment

  • Recruit RNA Pol II and initiate RNA transcription

Recruitment Patterns

• Sequential: One after the other is recruited

• Combinatorial: Work together at same time

• Parallel: Sort of combinatorial but two promoters near each other are bound by RNA Pol II, one recruits HAT, the other TRAP/DRIP

Crosstalk | Intracellular Receptors and GPCRs

• GPCR/cAMP: CREB (TF)

• Phosphorylation of CREB by Protein Kinase A due to increase in cAMP → Binds DNA with DBD

• Binding of CREB to CRE → GPCRs can directly control transcription

  • Coactivator CBP/p300

Intracellular Receptors | Estrogen Receptor

• In cytosol, released from HSPs, goes into nucleus; recruits co-activates to open DNA, recruit RNA Pol.

• Estrogen..

  • Affects heart, brain, bone, breast, uterus, and liver due to estrogen receptors

  • Produces proteins to decrease LDL (low density lipids) to protect good cholesterol

  • Stimulates uterine wall/breast cells to proliferate → Increase cancer risk

Antiestrogens

Block estrogen’s growth stiulation → Bind ER, no coactivator interaction, no gene activation

SERMs

• Selective Estrogen Receptor Modulator

  • Stops breast cell proliferation, not uteruine cell proliferation

Tamoxifen

• Blocks binding at receptor

• First: Surgery

• Second: Check if ER+, use Tamoxifen

  • Even without recurrence, risk of uterine cancer increases

  • Need better SERMs

Raloxifene

• Prevents osteoporosis; at menopause, lose estrogen → Decrease bone strength, blood clots

• Less breast/uterine cancers compared to tamoxifen

Progesterone

• Birth control

• HRT

• Abortion

HDAC Inhibitors

• Zolinza: HDAC inhibitor for cutaneous T-Cell lymphoma

• Allow continued expression of proteins that decrease cell growth

FULL INTRACELLULAR (ESTROGEN) RECEPTOR PATHWAY

Receptor Ser/Thre Kinases | Ligands

• TGF-β: Dimer found by ligands

• BMP: Bone morphogenetic protein

• GDF: Growth and differentiation factor

  • Proteolytic cleavage → Activation

  • All homodimers

Receptor Ser/Thre Kinases | Regulation of TGF-β

• TGF-β Bioavailability

  • Pre-pro-TGF-β: Made signal protein and have it secreted via secretory pathway

  • Synthesize part of protein, ribosome binds to ER to continue synthesis, then go to Golgi Apparatus and release a secretory vesicle

  • Signal peptide cleaved in ER, (that’s the “Pre”): Left with pro-TGF-β

  • TGF-β binds to surface of receiving cell, controlling when and how signal is made

Receptor Ser/Thre Kinases | Type I Receptor

• Intrinsic Ser/Thr kinase, GS-domain, single transmembrane helix

  • Self inhibitory w/ GS domain

Receptor Ser/Thre Kinases | Type II Receptor

• Same features as Type I but..

• Phosphorylation/activation of Type I receptor: Ser in GS domain, removing inhibitory effect

• Brought together by ligand

Receptor Ser/Thre Kinases | Type III Receptor

Regulatory proteoglycan for receptor activity

Receptor Activation

• Ligand binding increases receptor hetero-tetramerization

• Type II phosphorylates Type I → Activation (completely inactive unless interacting)

Receptor Ser/Thr Kinases | Flexibility

• How does heterotetramerization increase flexibility?

  • Conformation can vary, lead to multiple downstream pathways being affected

Receptor Ser/Thr Kinases | Amplification

Direct link to nucleus: TF that is phosphorylated via signaling cascade, goes to nucleus

Receptor Ser/Thr Kinases | How to Find a Protein Downstream of the Receptor?

• Type I phosphorylates something

• Take a cell with TGF-β receptor and add to a well, solubilize, stain with antibody serine, add lots fo protein + 1 phosphorylated

• Use gel to find protein

SMADs

• TFs, three types to know:

  • R-SMADs: Receptor regulated, phosphorylated

  • Co-SMADs: Work with phosphorylated R-SMADs

  • Antagonistic SMADs: Regulate R-SMADs with non-productive interactions

Crosstalk: R-SMADs and MAPK (Receptor Ser/Thr Kinases and RTK)

• MH1 + MH2 Domains

  • MH1: Recognizes DNA sequence CAGAC

  • MH2: Transcritpional activation via regulation of interactions with..

    • Co-SMADs (work as trimer)

    • Other TFs

    • Co-activators

  • Crosstalk with MAPK pathway to recruit new co-repressors (ERK-1 blocks SMAD complexes)

Receptor Ser/Thr Kinases | Specificity

R-SMADs: Differential interactions with receptors

Receptor Ser/Thr Kinases | Regulation

• SARA: SMAD anchor for receptor activation

  • SARA presents R-SMAD to receptor → R-SMAD can only interact with Co-SMAD after dropping SARA, becoming a trimer → Enter nucleus

  • NES keeps Co-SMAD out of the nucleus

    • R-SMAD has a hidden NLS, revealed by SARA

  • SMAD4 links different downstream effectors and expressions

TGF-β and Cancer

• Acts as a tumor suppressor

• Mutations to pathway → Cancer (mainly pancreatic and colon)

TGF-β Signaling

• Epithelial cells:

  • Leads to adhesion, death, cell cycle arrest

  • Cancer-inhibiting

• Endothelial cells:

  • Motility, morphogenesis, growth

  • More migration and invasiveness

  • Cancer-promoting

Crosstalk | Receptor Ser/Thr Kinase and Intracellular Receptor

• SMAD complex (2 R-SMADs + Co-SMAD) associate with CBP/p300 intracellular nuclear co-activators

  • R-SMADs: SMAD2/3

• Increase transcription

FULL RECEPTOR SER/THR KINASE PATHWAY