Lecture Four: Cell signalling One Flashcards
What is a prerequisite for cell functionality?
Cells must exist independently from the environment
Must communicate with outside environment to obtain nutrients etc
Describe what separates the cell from the outside world; and describe its characteristics;
The plasma membrane;
- Impermeable (protect)
- Signal Transduction Mechanisms exist allowing communication
- Regulates internal environment
What triggers a signal transduction mechanisms;
- Signal molecule present at low concentrations outside the cell
i. e a hormone, that can achieve very large effects on cell function
Where do signals act in signal transduction?
Generally on EC surface transmembrane receptors, Initiating a signalling cascade, enzymes amplifying this to achieve a series of specific effects.
What does a signal transduction mechanism allow?
Such mechanisms allow organisms to use minimal amount of energy to produce highly flexible and appropriate response to their environment
What does the term hormone cover?
Cytokines
Chemokines
Growth Factors
Steroids
Describe transduction effects and time relationship
Milliseconds
- Signalling pathway ; Ion channels
- Physiological Response ; Neurotransmission
Hours
- Signalling pathway ; gene expression cascade
- Physiological response ; Growth
Must be a flexible system.
Acknowledge that system work over different time frames in the exam
What is the most common mechanism cells use to communicate with the external environment;
Specialised transmembrane proteins that have a region of hydrophobic amino acids which allow it to insert into the membrane.
How do transmembrane proteins work?
Something happens outside of the cell i.e pH change or ligand binding which leads to a conformation change in the protein and also a functional change, therefore having an effect inside the cell and transducing the signal.
What are signal transduction mechanisms responsible for in terms of acutely regulating cell functions;
- Membrane polarity
- Ion concentration
- Metabolism
- Gene expression, protein translation and degradation
- Cell growth, division death
What two categories do transmembrane transduction proteins tend to fall into?
1) Cell surface receptors, proteins that EC ligands bind to and triggers activation of the IC part of the molecule, triggering a response inside the cell. Mins to hrs
2) Ion Channels, Allows rapid (ms) regulation of cell function i.e neurotransmission and muscle function.
Regulation is mainly by;
- Ligand gated ion channels
- Voltage gated ion channels
What is a quality of cell surface receptors
Receptor binding specificity
What is the exception to extracellular receptors?
Intracellular receptors;
A limited number of hormones i.e steroid, small molecules or hydrophobic ones, can cross the membrane and have intracellular receptors
How is signal transduction tightly controlled?
Signal transduction is tightly controlled at multiple levels to ensure appropriates responses;
- Regulation of ligand levels
- Regulation of cell receptors
- Regulation of intracellular signalling
Describe the regulation of ligand levels;
The regulation of ligand levels
- Tissue specific production of ligand in a controlled manner e.g insulin
- Regulated release of ligands e.g neurotransmitters, insulin
- Circulating binding proteins for some ligands
Describe regulation of receptors in cells;
- Tissue specific expression of receptors
i. e insulin receptors are really only in muscle, liver and fat in high concentrations - Mechanisms regulating the breakdown of receptor proteins after they bind to ligands
= Ensuring receptors are expressed appropriately and at the right time.
Once the signal gets to the cell, what are some mechanisms that ensure an appropriate response is acheived?
1-3
1) Post Translational Modification of Proteins and Lipids inlcuding
- Phosphorylation of proteins and lipids by kinases
- Other transient post translational modifications of proteins including lipidation, methylation, acetylation, nitrosylation and glycosylation plus the addition of small proteins such as ubiquitin
2) Use of GTP/GDP to regulate enzyme activity
3) Small second messengers (e.g. cAMP and cGMP) which allosterically regulate protein
Once the signal gets to the cell, what are some mechanisms that ensure an appropriate response is acheived?
4-8
4) Changes in concentration of ions in specific cellular compartments
5) Cleavage of membrane lipids
6) Modification of lipids (e.g.eicosanoids)
7) Regulated formation of protein complexes at desired site of action
8) Cleavage of proteins to activate or deactivate
Describe signalling molecule composition;
Most Signalling Molecules Are Composed of Multiple Different Functional Domains
Describe signalling molecule function;
Signalling molecules often have more than one functional capability and each of these is usually performed by a specific part (or domain) of the protein.
What is a domain?
Domains are protein segments that have a distinct structure and function
Are domains similar throughout signalling molecules?
Homologues of a particular domain and will have similar functions
AA sequence of these homologous domains will be similar but not identical meaning there can be subtle differences in the way they work
Domain structures are conserved / very similar for a family of domains
Write some notes on signalling molecule evolution;
The DNA sequence coding for these domains is also very similar indicating that the domain composition of different proteins was probably achieved by gene shuffling during evolution.
The sequence, structure and function of domains have been conserved during evolution so there are many similarities in signalling in eukaryotes
Describe signalling molecule overlap;
While the ligand + receptors vary, there is a lot of overlap in the way they achieve their effects.
Signalling molecules have similarities in their functionality.
What determines the signalling molecules function?
The sequence / arrangement of domain activation that determines function
Each domain has its own function contributing to the units functionality.
What is also in the phospholipid bilayer?
Domains that allow a range of proteins to bind to it.
Specific proteins too, that allows domains to bind in a particular way.
What is a studied domain in this lecture?
Src Tyrosine Kinase
A Prototypical Domain Structure
Describe the Src tyrosine kinase structure;
Domains had aequence homologies to regions found in other proteins so the 3 domains found in Src were labelled Src Homolgy (SH) domains 1,2 and 3
SH1 is the kinase domain,
SH2 is found to bind to phosphorylated tyrosine residues - and shift Src to correct place i.e other proteins with phosphyrlated tyrosines on them
SH3 is found to bind to proline rich regions in other proteins that were associated with Src
What did studies about homologous domains in a protein show?
Studies showed that the homologous domains in other proteins had similar functionalities
What have studies on domain structures shown?
Structural studies have shown that the domain structures are also conserved between different proteins.
What was found from the Src studies in general?
Domain = Conserved structural shape b/w proteins
Even if AA and function is unique
Describe the Src tyrosine kinase domain structure;
Each domain has a distinct pattern of folding i.e SH3 may vary b/w protein in terms of AA sequence and function, but same beta barrel shape
Domains are linked by linker regions that have no particular shape
Conserved functional domains that can be mixed and matched, but together changes function
Describe how sequence specificity of different SH2 domains dictates how they work;
Subtle variations, subtly change function despite conserved shape
i.e in protein families, there are 6 examples given to us. Each SH2 domain has similar shape, but every SH2 has unique function and slightly varied AA sequence
Describe domain composition in proteins;
Domain composition can be mixed to produce signalling molecules with specific function
Domains confer function
Give an example of a regulatory ion channel
Ach receptor allows Na/K influx to trigger an AP
Describe the importance of Ca in the cells;
- Changes in level of Ca++ (up to 1000 fold) are important for signal transduction.
- A major effector of Ca++ is the Ca++ binding proteins such as calmodulin which change their activity state when Ca++ is bound
- Calmodulin activates myosin light chain kinase (hence promotes contraction) and calmodulin dependent protein kinase (regulates metabolism, transcription etc)
Describe the regulation of Ca in the cell;
- Cytoplasmic levels of Ca++ are regulated via voltage sensitive PM receptors and receptors on the ER sensitive to Ca++ (ryanodine receptors) and IP3.
- ATP dependent pumps transport Ca++ back to where it came from, allowing rapid signal on/off - very important in situation like muscle contraction
What other ion is considered important in signalling?
H+ pumps might also be considered as players in signaling as acidification of vesicles regulates degradation of ligands and receptors
What is the importance of maintaining the ion concentration gradient;
Many cellular functions are regulated by the concentration gradient.
i.e calcium activates many intracellular processes and cytosolic Ca2+ concentrations in the resting state are very low (<0.1µM). Stimuli can increase cytosolic Ca2+ levels (upto 100 µM) thus activating Ca2+ dependent processes.
However, Ca2+ levels rapidly return to basal levels so shutting off these processes
25% energy dedicated to maintaining these gradients
How can ion channels be modulated?
Ion channels can be modulated by events that occur inside the cell to ensure correct activity.
Fine tunes receptor response
Molecules include;
- Src
- CDK5
- CAMKII
What else can modulate ion channels?
Temperature
TRP receptors are temperature sensing
How do ion channels signal APs?
- Initial signal to allow influx of +ve ions (e.g. Na+ or K +)
- Membrane potential rises to -60 to -40 mV.
- This activates voltage gated Na+ channels which further depolarises membranes right up to +40 mV
This has a two fold effect
- the next voltage activated Na+ channel becomes activated so propagating the signal
- voltage activated K + channels become activated which pump K + out of the cell and restore the action potential to -70 mV
In nerves this just propagates down the nerve to the next synapse where the change in voltage releases neurotransmitters.
In muscle the depolarization induced by the wave of action potential activates voltage sensitive Ca ++ channels which allows the influx of Ca ++ . This binds allows contraction to proceed.
What is one of the most important themes in signal transduction?
Reversible post translational modification is one of the most important themes in signal transduction
Why is reversible post translation modification so important?
- Addition of a molecule onto a protein can induce different functional states for that protein
- One protein can have multiple post-translational modifications so have a wide range of different functional states
- Because there are enzymes to add and also remove these molecules it means the function of proteins can be changed very quickly
What are the most common reversible post translation modifications?
Phosphorylation (kinase importance)
Acetylation
N-linked glycosalyation
What are kinases?
These are enzymes that add phosphate onto biological molecules via a hydroxyl residue on that molecule to form a phosphoester.
What does phosphorylation do?
This drastically changes the properties of the target molecule as it :
- adds 2 negative charges so can drastically change overall charge
- it allows more hydrogen bond formation
Together this can both affect the intramolecular connections (i.e. the 3-dimensional structure) of the target molecule and also alter the inter-molecular connections that the molecule can make
What can kinases target?
Kinases can target lipids (Lipid Kinases), Sugars (sugar kinases) and proteins (Protein kinases)
What are the three amino acids that can be targeted by kinases?
The three amino acids that contain hydroxyl groups (i.e. Serine, Threonine and Tyrosine) are the main target sites for protein kinases
How many kinases are there?
Overall there are about 500 protein kinases in the genome, all of which share some sequence and structural homology in their kinase domains
What are the two broad classes of protein kinases?
There are two broad classes of protein kinases – those that phosphorylate ser/thr and those that phosphorylate tyrosine. This is because serine and threonine are small and tyrosine is big so they need different shapes in their catalytic sites.
What are kinases?
Kinases are enzymes
so the can repeatedly add
phosphate to targets and
thus cause signal amplification
How may reversible phosphorylation act as a regulatory mechanism?
- Kinases phosphorylate lipids, proteins, changing function
- Phosphotases oppose this, making it transient and reversible (regulation)
What is often the first target of cellular phosphorylation?
Around 90% of all the phosphorylation in cells is on ser/thr implying tyrosine phosphorylation is at early stages in amplification cascades.
