Signal Transduction II Flashcards
Exam 2
Briefly describe the importance of signal transduction receptors to the pharmaceutical industry.
Proteins targeted by approved drugs (small molecules and biologics) by human protein family class. Signaling receptors include 7TM, seven transmembrane (GPCR); LGIC ligand-gated channel; kinases.
Essentially, knowing the process of signal transduction helps us design drugs that will produce the desired therapeutic response.
What are the key parts/information of ligand-gated ion channel receptors?
Mechanism
* The first messenger binds to the channel receptor
* Then, the channel opens, which allows ions (Na+, K+, and Ca2+) to cross the membrane.
Channels have 2 domains
* Extracellular domain binds in the first messenger ligand
* Transmembrane domain is the pore through which the ions flow
Many channels are involved in neuronal signaling.
* Neurotransmitter ligands are released from excited presynaptic neuron into synaptic cleft
* Neurotransmitter binds to channel and induces ion flow, leading very quickly to a postsynaptic signal by changing the potential across the membrane.
First messenger examples:
* serotonin
* glutamate
* glycine
* GABA
* nicotinic acetylcholine
Clinical relevance: thought to be the site at which anaesthetic agents act; involved in blood pressure regulation and cardiovascular diseases
Explain how nicotinic acetylcholine receptors work.
Gating
* Acetylcholine binds to the extracellular ligand binding domain, which triggers a structural change in the transmembrane helices to widen the channel from 3 to 8 angstroms.
Channel Opening
* Sodium ions flow through the channel, down a gradient into the cells, and depolarize the post-synaptic membrane.
Characterisitics of the receptor
* Three domains: extracellular, Transmembrane, and Intracellular
* PENTAMERIC receptor (5)
What are receptor tyrosine kinases?
ex. activation of Ras
Background
* There are several families of the receptor tyrosine kinases with various extracellular domains that bind messenger.
* Many are associated with cell growth
* Dysfunction of the tyrosine kinases results in cancer.
Key Information
1. Ligands (growth factors, other proteins) bind extracellular domains and induce dimerization of the whole receptor, which activates the intracellular catalytic activity of the receptor.
2. Activated cytoplasmic domains phosphorylate tyrosine residues on its own flexible tails to form binding sites specific for a given downstream signaling proteins
3. Leads to sequential phosphorylation of other substrate proteins or downstream signaling proteins
4. Examples- insulin receptors, Growth factor receptors
5. Signal termination is mostly by receptor internalization, with ligand dissociation and degradation.
Outline the Ras/MAP signaling cascade.
- The protein growth factor on the outside of the membane binds to the RTK receptor, which induces the dimerization of the receptor and the catalytic activity.
- The P group binds to the SH2 domain, SH3 domain, and sos
- This activates the RAS by changing GDP to DTP
- An active RAS and GTP, results in other effectors by various pathways. One is the Raf, one is MEK, and the other is MAPK. They all result in other effectors. Other kinases and MAPK, go into the nucleus for gene expression.
Summary: The growth factor ligand binds to the growth factor receptor ligand to initiate the mitogen-activated protein kinase (MAP kinase) cascade.
How is the cascade to activate the G-protein Ras related to cancer? How does it work?
Works by:
* Molecular switch to activate cell proliferation, migration, transformation, and survival.
* Tyr phosphorylation of EGFR creates binding site for the GRB2 adapter protein, which also binds to the GEF protein, sos.
* Sos activates Ras
* Signaling outcome: cell division, survival, and differentiation, etc
Cancer
* Oncogenic RAS mutations- various amino acids are mutated
* Confers permanent activation of the Ras enzyme.
* 3 RAS genes are most common oncogenes in humans. Around 90% of pancreatic cancers have mutated RAS.
* Present in large majority of pancreatic ductal adenocarcinoma and high percentage of other human cancers.
* Effective diagnosis and chemotherapeutic agents are lacking.
- Binding of hormone causes dimerization and autophosphorylation of tyrosine residues
- Binding of GRB2 and Sos couples receptor to inactive Ras
- Sos promotes the dissociation of GDP from Ras; GTP binds and Sos dissociates from active Ras.
- Active Ras stimulates cell signaling
What is Ras? Why is it important?
- Ras= a membrane bound, monomeric G protein critical in cell proliferation, differentiation, adhesion, migration, and apoptosis.
- Ras is normally activated by growth factors binding to Receptor Tyrosine Kinases (RTKs) or by T cell receptors
Role in Ras/Map signaling cascade
* Switch: the conformation of two loops differs between GDP and GTP bound states; binding GTP switches the Ras structure to the “on” state. (2 switch regions)
* In activated GTP-bound state, Ras binds specific effectors and activates multiple different pathways.
* Hydrolysis of GTP to GDP switches the Ras structure to the off state. GTPase-activating proteins, GAPs, increase the hydrolysis rate.
* Guanine nucleotide exchange factors, GEFs, increase the rate of GDP dissociation (by 100 fold). Slow hydrolysis, and prolonged lifetime of activated Ras, promotes cancer.
What effects can you expect from KRAS?
KRAS is a single-base missense mutations
Effect
* Decreased GTPase activity (less hydrolysis of GTP)
* Increased affinity for GTP
* KRAS always active–> increased cell proliferation and survival.
* KRAS is very difficult to inhibit because it has a very high affinity for GTP (increased affinity for binding) and GDP.
How may irreversible inhibitors target G12C KRAS mutants?
- Structural studies discovered an allosteric pocket formed with switch II in a certain conformation.
- Screening identified to lead compound that convalently links to C12.
- Covalently bound inhibitor prevents GTP binding and lowers KRAS function in cells.
- This lead compound is being pursued to develop effective treatments against G12C mutant cancers.
- Contains Cys 12.
Describe G-protein coupled receptors.
- ~1000 different GPCRs in human genome
- GPCRS are the most common target for pharmaceuticals.
- Many hormone a neutotransmitter receptors
- High degree of structural similarity (& alpha-helical transmembrane regions, conformational change upon receiving signal)
- No enzyme activity; no scaffolding (compare RTKs)
Examples of hormones and neurotransmitters: Ca2+, Odorants, Pheremones, Small molecules: amino acids, amines, nucleotides, nucleosides, prostaglandins, PAF, peptides, TSH, LH, FSH, interleukins, chemokines
Descibe the G-protein cycle for the trimeric G protein.
General Information:
* The release of GDP is intrinsically slow.
* Transient association with an agonist-bound GPCR increwases GDP release from Galpha.
GEF activity:
* One agonist bound GPCR can activate 10-100 G proteins
Big amplification
* Intrinsic GTPase activity converts the active/on form (GTP-bound) to the inactive/off form (GDP-bound)
* Sometimes facilitated by regulators of G protein signaling, RGS proteins (same as GAPs in previous slides).
The Process:
1. Basal state- alpha/beta gamma subunits with GDP and pi
2. Nucleotide exchange- the GTP is exchanged for GDP (bye GDP)- alpha/beta gamma moves to center- Ag binds
3. Beta gamma and alpha subunit separate and Ag dissociates slightly- this is the activated state. When effector reacts with Beta gamma, beta gamma joins with alpha. When alpha binds with effector and GTP–> some effectors and RGS proteins result in alpha and GDP + Pi——> Overall back to the basal state.
The phosphorylation of GPCR residues in this region (S316) by kinase leads to desensitization. We have the alpha, beta, and gamma subunits.
G proteins are: timeric
GPCR-> have membrane helices
Describe the different types of GPCRs and proteins.
Heterotrimeric G-proteins that couple to GPCRs
* Three subunits: Galpha, Gbeta, and Ggamma
* Galpha has GTPase activity (20 distinct subtypes–> (as, ai, aq)
* Association with an agonist-bound GPCR induces dissociation of GDP, and GTP binding.
* The activated Galpha dissociates from the Beta gamma complex and stimulates or represses a particular effector protein.
* Many types of GPCRs and G proteins that couple to different downstream effector proteins.
Which protein families and signaling proteins are the most targeted by the pharamaceutical industry?
7TM1, Nuclear Receptor, Kinase, Proteases, VGIC, Reductase, LGIC, and others.
Most commonly targeted receptor by drugs: GPCRs.
What are the structure/function characteristics of ligand-gated ion channels?
- Oligomeric with extracellular domain (ligand binding) and transmembrane helices (channel)
- Involved in neuronal signaling, where ligands are neutotransmitters. Gating leads to change in potential of neuronal membranes.
What are the structure/function characteristics of receptor tyrosine kinases and pathways?
- Numerous extracellular domains that recognize a variety of ligands
- RTKs autophosphorylate on intracellular domains for docking in initials steps of signal pathways
- Function: growth, metabolic, important anticancer treatment
- Pathway cascade of regulated protein-protein interactions (ex example Ras/MAP kinase cascade
How is KRAS regulated? Why is KRAS difficult to inhibit?
* Very high affinity for GTP & GDP (KD>nM)
* Binds GTP with increased affinity
* Constitutively active
Why does a covalent inhibitor offer some promise as an anticancer drug?
* G12C KRAS mutant has irreversible inhibitor
* Lead compound covalently links to C12
* Covalently bound inhibitor prevents GTP binding and lowers KRAS function in cells
* Lead compound is pursued to develop effective treatments against G12C mutant cancers
