Cellular Signalling Flashcards
What does the Ca2+/CaM switch do?
Non-covalent binding of the Ca2+/CaM to a kinase opens catalytic sites on the kinase allowing it to phosphorylate its substrate -> increase in ca2+ acts as “on” switch
What is the purpose of the GTPase switch?
GTPase is an enzyme that hydrolyses GTP -> GDP
What is Guanine Nucleotide Exchange Factor?
GEF – speeds up the replacement of GDP with GTP (Confers switch turning ON)
What is GTPase Activating Protein?
GAP – Increases GTPase activity (confers switch turning OFF)
What are the effects of acetylation on chromatin?
Unacetylated -> Chromatin highly condensed (heterochromatin) -> no expression
Acetylated -> chromatin less condensed (Euchromatin) -> expressed
What is a disease associated with nucleus?
Laminopathies – mutations that impact lamins
- Muscular dystrophies & cardiomyopathies
- Partial lipodystrophy
- Peripheral and sensory neuropathies
- Premature aging
What is Progeria?
Premature aging caused by the overexpression of Progerin gene
- Mutations cause accumulation of unprocessed lamins or misprocessed lamins
- There is a little progerin in all individuals -> increases with age
- HGPS individuals have high levels of progerin at birth
What signal tends to act on membrane receptors?
Hydrophilic signals tend to act on membrane receptors (as they cannot pass through membrane)
What signal tends to act on cytosolic receptors?
Hydrophobic signals tend to act on cytosolic receptors (as can pass through membrane)
What are the four key steps in signal transduction?
- Conformational Change,
- Signal relay and amplification,
- Effector protein mediates cellular response,
- Signal shutdown.
What happens in the first stage of signal transduction?
What happens in the first stage of signal transduction?
What can signal transduction do to cells?
Signal transduction can cause the cell to mature and differentiate, move, acquire/lose specific functions and die (apoptosis)
How does Endocrine signaling work?
Hormone secreted into blood by endocrine gland -> hormone binds to receptors of the target cells (long distance signaling)
How does paracrine signaling work?
Ligands released by secretory cells which act on receptors of nearby target cells
How does autocrine signaling work?
Ligands released by cell act on its own receptors
How does juxtracrine signaling work?
Signaling cell has plasma-membrane-attached proteins which make contact with receptors on adjacent cells (i.e. cells must be in contact)
What does the cell surface receptor allow?
Allows water soluble ligand binding (e.g. growth factors, hormones)
What do cytoplasmic / nuclear receptors allow?
Binding of lipophilic molecules that can pass through membrane (e.g. steroids)
What are some examples of second messengers?
- cAMP
- cGMP
- DAG
- IP3
- Ca2+
What are effector proteins?
Effector proteins are proteins that can bring about a cellular response (e.g. kinases, phosphatases, metabolic enzymes)
How do second messengers and signaling proteins allow for signal amplification?
Production of lots of second messengers in response to a signal -> each second messenger molecule might activate a signaling protein -> might activate number of downs tread further second messengers -> massive amplification (like a pyramid scheme)
How are signals recognized by effector proteins?
Signaling proteins and messengers move signal around the cell to convert it to a format understood by effector protein
What are the three primary means by which conformational changes are triggered?
- Allosteric modification
- Covalent modification
- Cleavage (proteolysis)
What are allosteric modifications?
Molecule binds non-covalently to protein, alters protein conformation
What are covalent modifications?
Modification of chemical structure of target protein – this process can be reversible (e.g. Phoshporylation, ubiquitination, Lipidation, SUMOylatio)
What is Lipidation?
Transfer of fatty acid group to protein, which enhances protein hydrophobicity
What is SUMOylation?
The addition of small SUMO peptides which can alter protein function/localisation
What is Proteolysis?
Protein cleaved by protease protein – this is not reversible and activate or inactivate protein
• Insulin produced by cleavage of precursor protein called pro-insulin
• Some proteases activated by cleavage
What are some effects protein modification can have?
- Activate enzyme activity
- Unmask active sites
- Alter protein localisation
- Facilitate protein-protein interactions
- Alter protein stability
What are GPCRs components?
- 7 Trans-membrane Alpha-Helix Domains
- 4 Cytosolic Domains
- 4 Extracellular Domains
How are GPCR’s activated?
- Hormones
- Metabolites
- Light
What are the sub-units of Trimeric G-Protein Complexes?
- Alpha (GDP/GTP bind here)
- Beta
- Gamma
GPCR Effector Pathways
- Adrenylate Cyclase
- cAMP
- Protein Kinase A
&
- Phospholipase C
- Protein Kinase C
- Calcium
- Calmodulin
What does the ligation of GPCR cause?
- Triggers a structural change that enables the following receptor functions:
o Converts the inactive receptor to a Guanine-Nucleotide Exchange Factor (GEF)
o Allows the receptor to interact with the trimeric G-Protein Complex to initiate signalling
How many different subunits do humans have?
- 21 different G-Alpha subunits (gas, gai, gaq)
- 6 different G-Beta subunits
- 12 different G-Omega (y) subunits
Which GPCR subunits activate effectors?
- Usually activated by G-Alpha – but this is not always the case as some ion channels are activated by the G-Beta-Omega (G-B-Y) Complex after it dissociates
What is Adenylate Cyclase’s structure?
- A multi-pass trans-membrane protein with two cytosolic and two catalytic domains
What happens with Adenylate Cyclase interacts with GaS?
- Interaction with GaS induces a conformational change allowing ACs two cytosolic catalytic domains to interact forming an active site
What happens when Adenylate Cyclase interacts with G-aI?
- Interaction with GaI induces a conformational change forcing the catalytic domains away from each other
Which intracellular loop and terminal tail are phosphorylated, and by what kinase?
Activated GPCRs are phosphorylated on the 3rd intracellular loop and C-terminal tail by GPCR-Kinases (GRKs)
Describe three mechanisms by which GPCR signalling is turned off?
- Arrestin proteins – direct GPCR for internalisation, leading to either recycling of the dephosphorylated inactive GPCR to the plasms membrane, or degradation
- A interaction between G-alpha and its effector protein increases GTPAse activity (GTP->P+GDP) – this returns the trimeric G-Protein to its “off” state
- By turning off secondary messengers (cAMP and IP3)
What is the process for Hydrophobic Signalling?
• Steroids, Retinoids, Thyroxines -> Cytosolic Receptor -> Modification of gene expression development in the nucleus
What is the process of Hydrophilic Signalling?
• Small molecules, peptides, proteins -> attach to cell-surface receptor -> Signal transduction proteins and second messengers -> effector protein -> ONE OF: modification of cellular metabolism, function, movement -> OR -> Modification of gene expression and development
Receptor tyrosine kinase (RTK)
- inactive RTK
- Ligan binds->dimerization, kinase activation
- Active RTK-> autophosphorylation of tyrosine residues (cross phosph)
- Binding/activation of signaling proteins->initiation of cascade
MAP kinase cascade
Mitogen-activated protein kinase cascade
- adaptor protein
- Ras activation protein
- active Ras
- activated MAPKKK (serine/threonine kinase)
- activate to MAPKK, threonine/tyrosine)
- activate to MAPK - effector protein (serine/threonine kinase)
- Phosphorphylates cystolic membrane proteins of nuclear gene regulatory protein
- Change in cystolic/membrane proteins or change in gene expression
Cytokine receptor mechanism
- Inactive
- Cytokine binds–> dimerization of JAK
- JAK cross phosph, subunit phosphorylation
- STATs bind to subunit
- Phosphorylation of STATs, activated
- STATS dissociate and dimerize
- Translocation to nucleus–> altered gene expression
Cytokine receptor termination
phosphatases remove tyrosine phosphates from receptor/STATS
SOCS (suppressor of cytokine signaling) protein inhibit STAT phsophorylation by binding/inhibiting JAKs or competing with STATs for phosphotyrosine binding sites on receptor
-multimeric formation of receptor after ligan binding triggers endocytosis of ligand receptor complex
G protein coupled receptor mechanism
- ligand binds
- Conformational change, recognition site exposure for G protein binding
- GDP/GTP exchange, G protein alpha dissociates from beta-gamma
- Subunit alpha binding to enzyme (release second messengers)
- Intrinsic GTPase activation, hydrolysis of GTP to GDP, release enzyme
- G-protein reformation with GDP, returns to receptor
G protein coupled receptor termination
extracellular enzymes metabolize or inactivate many of the small ligands
- Receptor mediated endocytosis accounts for some desensitization
- receptor phosphorylation by protein kinases is the major mechanism of sensitization
- protein kinase A–> receptor +/- ligand
- GPCR specific protein kinases (GRKs)->receptor +ligand
cAMP action
ligand binds to G protein and sends alpha subunit w/ bound GTP binds to adenylate cyclase, which w/ ATP produces cAMP, which binds to receptors
Regulation: Depending on which G protein is activated can gave +/- effect
cAMP termination
cAMP phosphodiesterase w/ h20 makes 5’-AMP
What is the function of IP3?
Interacts with IP3 receptors on the ER to release intracellular store of Ca2+. Ca2+ activates PKC.
PKC translocates to the plasma membrane where it becomes activated by DAG.
What are some of the functional roles of Ca2+
Can bind to the calcium binding protein calmodulin to activate calcium-dependent protein kinases e.g CaM Kinases
Ca2+ can cause muscle contraction
Ca2+ can influence gene expression
what is the diff b/w a RTK and GPCR?
GPCRs have 7 transmembrane segments
RTKs have a single transmembrane segment, but each RTK functions as a dimer so it has a total of 2
RTKs are involved in the regulation of what processes?
Growth (growth factor receptors), trigger mitosis
Cell division (defects lead to cancer)
Cell survival and death (programmed)
Cell attachments, migrations
what are the 2 ways RTKs dimerize?
1 ligand binding to both monomers (ligand causes dimerization)
each monomer binds their own ligand & the 2 monomers come together
what does dimerization trigger in RTKs?
auto-phosphorylation
the 2 monomers phosphorylate each other
EF Hand Domain
Characteristic: Binds Calcium
Cellular Process: Calcium Dependent
SH2 / PTB Domain
Characteristic: Binds Phospho-tyrosine
Cellular Process: Tyrosine Kinase Pathways
PH Domain
Characteristic: Binds Phospho-inositides
Cellular Process: Recruitment to membranes and motility
C1 Domain
Characteristic: Binds Diacylglyercrol
Cellular Process: Recruitment to membranes
