Lecture Five: Cell signalling Two Flashcards
Whats an example of an intracellular receptor mediated signalling pathway?
Steroid Hormone receptors
What do steroid hormones typically do?
Generally bind transcription regulators directly and activate gene expression
No mechanism for amplifying or shutting off enzyme cascades so tend to work at low level over a longer time frame.
What other hormones act in a similar manner to steroid hormones?
Non-steroidal hormones such as retinoic acid, VitD3 and thyroid hormone also work via similar mechanisms
Describe steroid hormone similarities;
Subtle differences (all have cholesterol back bone) but huge functional differences because of very specific receptors
Whats a property that is essential to steroid hormones?
Typically hormones must get through the lipophilic membrane on their own accord (without carrier molecules)
Tend to be lipophilic i.e cholesterol backbone
What do steroid hormones act on?
Steroid hormone receptors bind directly to intracellular receptors that act to regulate gene expression.
One hormone can bind to a promotor- turns on gene expression = multiple proteins if stays bound
Whats an intracellular receptor mediated signalling pathway?
Nitric oxide
- Cell permeable gas
- Acts only over short range
- Signals via production of cGMP
- Plays a major role in endothelial cells regulating smooth muscle tone and thus blood flow
How is cGMP produced?
- cGMP produced by transmembrane receptor guanylate cyclases or by soluble guanylate cyclases whose expression is inducible and activity is stimulated by NO
What is cGMP?
A intracellular signalling mechanism
How does cGMP work?
- One effector is allosteric activation of cGMP dependent protein kinase
How is cGMP regulated?
In transducin signaling cGMP breakdown increased when Gi subunits of heterotrimeric G-proteins interact with cGMP PDE
Describe an intracellular cascade using the previous examples of signalling molecules;
NO can diffuse into the cell and activate Soluble Guanylate Cyclase.
This converts GMP into cGMP which can activate (allosterically modulate) Protein kinase G
What are some examples of cell surface receptor mediated signalling pathways?
GPCR (most common)
Receptor Tyrosine Kinases
Describe the structure of RTK and GPCR;
GPCR: 7 transmembrane domains
RTK; One transmembrane domain, but always in a dimer
How do GPCR signal to the cell?
The signal to the cell using a hetrodimeric G-protein
Describe the heterotrimeric g protein;
The consist of three subunits (GaGbGg).
There are 22 different genes for Ga subunits, 12 different genes for Gb subunits and 7 different genes for Gg subunits
Each different combination has a unique signaling capability but there are some broad themes.
What is typically the function of the Ga subunit?
Activity of subunit is controlled by guanylate nucleotides, being inactive when bound to GDP and active when bound to GTP
GTP bound G separates from the other two subunits and binds to target proteins and modifies their activity
What is the function of Gb and Gg subunits?
When released from the trimer the subunit dimer also binds to target proteins and regulate their activity
The subunit is a GTPase so as soon as the GTP is bound it starts to remove the -phosphate to return it to GDP (and hence inactive form)
How many GPCR genes are there?
There are ~800 different genes for GPCRs and they are broken down into five different structural classes
Describe the importance of GPCR structure;;
- Hydrophobic intermembrane domain
- Ligands fit perfectly into the binding pocket(very tight binding)
Describe step 1/6 for GPCR signal transduction;
Once the ligand binds to the binding pocket, it triggers a 3D conformational change that signals dissociation of Ga GDP from the hetrotrimeric g protein, and it binds the receptor.
Describe step 2/6 for GPCR signal transduction;
Ga binds GTP and its function is now completely changes and it has become activated. (still bound to the receptor)
Describe step 3/6 for GPCR signal transduction;
Ga has several sub classes and the signals it produces now that it is activated depends on the subclass Ga protein.
Different receptors couple to different g proteins.q
What are the subclasses of Ga proteins?
Ga (q)
Ga (s)
Ga (i)
What is the consequence of Ga (q) activation?
Stimulates phospholipase C
What is the consequence of Ga (s) activation?
Stimulates adenylate cyclase (increases cAMP)
What is the consequence of Ga (i) activation?
Inhibits Adenylate cyclase (decreases cAMP)
What may Ga + GTP do?
Bind downstream proteins and allosterically modulate these
Describe step 4/6 for GPCR signal transduction;
Gb ands Gg (dimer)
Localise certain enzymes to the plasma membrane
i.e GRK and BTK
Describe step 5/6 for GPCR signal transduction;
G proteins are GTPases that remove phosphate from GTP and so automatically shut off the reaction
(time dependant switch)
- Heterodimeric G protein reforms
- Ligand disappears, degrades, this shuts off the signal
Describe step 6/6 for GPCR signal transduction;
Many GPCRs are also down regulated when the activated GPCR binds to a specialised protein ; Beta arrestin
B arrestin can diffuse into and out of a cell to turn them off.
Creates the right amount of signal for the right amount of time.
Are all signalling components in all cells;
Not every signalling molecule is present in every cell.
Cells are defined by their genes expression
Ratio / mix of receptors + Heterotrimers g protein functionality
How does the Gby and Gaby protein structure differ?
When Ga is bound it blocks the key binding interface preventing function
Protein structure is fluid and extremely important
What does Ga (q) do and how?
It activates Ca metabolism via phospholipase C
1) Ga(q) binds Phospholipase C -beta
2) Ph/C - beta cleaves PIP2 (lipid bilayer bound)
3) Inositol tri-phosphate signals - Activating Ca channels in ER, increasing cytosolic Ca
4) Diacyl-glycerol (in membrane still) recruits and activates C1 domain containing proteins i.e PKC
What are enzymes proteins typically bound to?
Regulatory units that inhibit their function
What are the effectors of cAMP?
- Protein Kinase A . It is activated when cAMP binds regulatory subunits freeing active catalytic subunits. PKA phosphorylates a wide range of targets including key enzymes in metabolism plus transcription factors
- EPAC, a GEF for small G-protein Rap1
- In olifactory system cAMP directly binds and activates Na+ channels
Where is cAMP found?
In a regulatory complex with two regulatory units.
What do Ga(s) and Ga(i) do?
Regulate adenylyl cyclases
How do Ga(s) and Ga(i) Regulate adenylyl cyclases?
Space between the 2 intracellular domains is where catalytic activity occurs
One of the intracellular domains has Gαs binding site and this promote AC enzyme activity while the other intracellular domain has a Gαi binding site which acts to inhibit AC activity
What does PKA control (activated by cAMP)?
Lipases Kinases Ca metabolsim Glycolysis CREB cycle
Dose dependant effect;
- Low = PKA
- High = EPAC + RAP1
What do RTK do?
These phosphorylate tyrosine residues on target proteins
Give an example of an RTK;
A number of important cell surface receptors are tyrosine kinases e.g. insulin and PDGF receptors
There are also a number of important intracellular tyrosine kinase enzymes
What do a number of cellular proteins contain?
A number of cellular proteins (including many cell surface receptors) contain peptide consensus sequences containing tyrosines that allow them to be phosphorylated by the many receptor and non-receptor tyrosine kinases
What do tyrosine residues insight?
In general sites phosphorylated by tyrosine kinases act as specific binding sites for SH2 domains which are contained in a number of important signaling molecules.
This results in the initiation of specific signalling cascades
What are RTK important in?
Growth factors (biggest class of RTKs)
I.e the insulin receptor
Give some examples of RTK growth factor receptors
Includes receptors for Insulin, Insulin like Growth Factor (IGF), Epidermal Growth Factor (EGF), Fibroblast Growth Factor(FGF), Platelet Derived Growth Factor (PDGF), Vascular Endothelial Derive Growth Factor (VEGF), Nerve Growth Factor (NGF).
Describe the characteristics of RTKs;
- Ligand binding site on extracellular surface and a tyrosine kinase domain inside the cell.
- Receptors must dimerise to bind hormone
- Most receptor tyrosine kinases exist as monomers in the absence of ligand (Inactive)
- Hormone binding induces receptor dimerisation which in turn induces conformational changes which allow the receptor to autophosphorylate.
Describe what happens when ligand binds to RTK;
Rather than phosphorylating itself each chain phosphorylates the other in a process called transphosphorylation (fail safe mechanism if inadvertently activated)
The phosphorylated tyrsosine residues on the receptors act as docking sites to recruit and activate downstream signalling pathways
Whats the exceptions to RTK existing as monomers in the absence of ligand;
Insulin and IGF receptors preformed heterotetramer
Still uses transphosphorylation mechanisms
How does insulin then not produce a continuous signal?
Tyrosine phosphorylation of the receptor is not the main signalling event for insulin, instead phosphorylation of intracellular proteins by the tyrosine kinase is the more important event
Describe what happens when insulin binds the insulin receptor;
There is a conformational change in the protein receptor structure and the TK domains become open allowing each chain to phosphorylate the other
What does the activated insulin receptor do?
Active kinase phosphorylates downstream targets
Whats they key to how receptors work?
It is the subtle differences in the system
i. e signalling
i. e enzymes
i. e AA sequences of the families of these.
Describe key features for different receptor tyrosine kinases achieving specific outcomes;
- Different kinases have different optimal target sequences in substrates
- Sequence specificity of different SH2 domains dictates how they work
- The differences in tyrosine kinase target sequence results in differences in optimal signalling
What makes a good target for insulin receptors? (RTK)
IRS proteins
- they have many consensus sites for tyrosine phosphorylation
i. e the structure makes them a great signalling molecule
How does signalling molecule AA sequence influence effects?
Each domain that for example can be phosphorylated on a signalling molecule will have a different down stream effect
i.e some molecule can have different effects
How does binding different SH2 domains influence the outcome?
Subtle difference sin binding sequences and receptors = different outcomes
Binding of Different SH2 Domains to Different Phospho-Tyr Motifs Contributes to Differences in Signalling
Whats a variation of the RTK specificity rules?
Both Ligand and Receptor Heterodimerisation in PDGF System Allows Specificity And Flexibility
How is the PDGF system flexible?
PDGF functions as a dimer
There are two different forms of PDGF
These can heterodmerise or homodimerise so 3 possibilities
There are also two different forms of receptors so 3 combinations
What does the differing PGDF receptor combination means?
Not all forms of PDGF bind to all forms of receptor
This allows cells to both choose which ligand they will respond to and which reponse they will generate
What are the possible PGDF combinations?
AA
AB
BB
but ligand AA can only bind AA while BB can bind them all and AB can bind AA and AB
What does heterodimerisation mean for the system?
Receptor heterodimerisation can regulate signalling
