Lecture Four: Cell signalling One

Question 1

What is a prerequisite for cell functionality?

Answer 1

Cells must exist independently from the environment

Must communicate with outside environment to obtain nutrients etc

Question 2

Describe what separates the cell from the outside world; and describe its characteristics;

Answer 2

The plasma membrane;

• Impermeable (protect)
• Signal Transduction Mechanisms exist allowing communication
• Regulates internal environment

Question 3

What triggers a signal transduction mechanisms;

Answer 3

• Signal molecule present at low concentrations outside the cell
  i. e a hormone, that can achieve very large effects on cell function

Question 4

Where do signals act in signal transduction?

Answer 4

Generally on EC surface transmembrane receptors, Initiating a signalling cascade, enzymes amplifying this to achieve a series of specific effects.

Question 5

What does a signal transduction mechanism allow?

Answer 5

Such mechanisms allow organisms to use minimal amount of energy to produce highly flexible and appropriate response to their environment

Question 6

What does the term hormone cover?

Answer 6

Cytokines
Chemokines
Growth Factors
Steroids

Question 7

Describe transduction effects and time relationship

Answer 7

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

Question 8

What is the most common mechanism cells use to communicate with the external environment;

Answer 8

Specialised transmembrane proteins that have a region of hydrophobic amino acids which allow it to insert into the membrane.

Question 9

How do transmembrane proteins work?

Answer 9

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.

Question 10

What are signal transduction mechanisms responsible for in terms of acutely regulating cell functions;

Answer 10

• Membrane polarity
• Ion concentration
• Metabolism
• Gene expression, protein translation and degradation
• Cell growth, division death

Question 11

What two categories do transmembrane transduction proteins tend to fall into?

Answer 11

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

Question 12

What is a quality of cell surface receptors

Answer 12

Receptor binding specificity

Question 13

What is the exception to extracellular receptors?

Answer 13

Intracellular receptors;

A limited number of hormones i.e steroid, small molecules or hydrophobic ones, can cross the membrane and have intracellular receptors

Question 14

How is signal transduction tightly controlled?

Answer 14

Signal transduction is tightly controlled at multiple levels to ensure appropriates responses;

• Regulation of ligand levels
• Regulation of cell receptors
• Regulation of intracellular signalling

Question 15

Describe the regulation of ligand levels;

Answer 15

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

Question 16

Describe regulation of receptors in cells;

Answer 16

• 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.

Question 17

Once the signal gets to the cell, what are some mechanisms that ensure an appropriate response is acheived?

1-3

Answer 17

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

Question 18

Once the signal gets to the cell, what are some mechanisms that ensure an appropriate response is acheived?

4-8

Answer 18

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

Question 19

Describe signalling molecule composition;

Answer 19

Most Signalling Molecules Are Composed of Multiple Different Functional Domains

Question 20

Describe signalling molecule function;

Answer 20

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.

Question 21

What is a domain?

Answer 21

Domains are protein segments that have a distinct structure and function

Question 22

Are domains similar throughout signalling molecules?

Answer 22

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

Question 23

Write some notes on signalling molecule evolution;

Answer 23

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

Question 24

Describe signalling molecule overlap;

Answer 24

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.

Question 25

What determines the signalling molecules function?

Answer 25

The sequence / arrangement of domain activation that determines function

Each domain has its own function contributing to the units functionality.

Question 26

What is also in the phospholipid bilayer?

Answer 26

Domains that allow a range of proteins to bind to it.

Specific proteins too, that allows domains to bind in a particular way.

Question 27

What is a studied domain in this lecture?

Answer 27

Src Tyrosine Kinase

A Prototypical Domain Structure

Question 28

Describe the Src tyrosine kinase structure;

Answer 28

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

Question 29

What did studies about homologous domains in a protein show?

Answer 29

Studies showed that the homologous domains in other proteins had similar functionalities

Question 30

What have studies on domain structures shown?

Answer 30

Structural studies have shown that the domain structures are also conserved between different proteins.

Question 31

What was found from the Src studies in general?

Answer 31

Domain = Conserved structural shape b/w proteins

Even if AA and function is unique

Question 32

Describe the Src tyrosine kinase domain structure;

Answer 32

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

Question 33

Describe how sequence specificity of different SH2 domains dictates how they work;

Answer 33

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

Question 34

Describe domain composition in proteins;

Answer 34

Domain composition can be mixed to produce signalling molecules with specific function

Domains confer function

Question 35

Give an example of a regulatory ion channel

Answer 35

Ach receptor allows Na/K influx to trigger an AP

Question 36

Describe the importance of Ca in the cells;

Answer 36

• 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)

Question 37

Describe the regulation of Ca in the cell;

Answer 37

• 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

Question 38

What other ion is considered important in signalling?

Answer 38

H+ pumps might also be considered as players in signaling as acidification of vesicles regulates degradation of ligands and receptors

Question 39

What is the importance of maintaining the ion concentration gradient;

Answer 39

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

Question 40

How can ion channels be modulated?

Answer 40

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

Question 41

What else can modulate ion channels?

Answer 41

Temperature

TRP receptors are temperature sensing

Question 42

How do ion channels signal APs?

Answer 42

• 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.

Question 43

What is one of the most important themes in signal transduction?

Answer 43

Reversible post translational modification is one of the most important themes in signal transduction

Question 44

Why is reversible post translation modification so important?

Answer 44

• 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

Question 45

What are the most common reversible post translation modifications?

Answer 45

Phosphorylation (kinase importance)
Acetylation
N-linked glycosalyation

Question 46

What are kinases?

Answer 46

These are enzymes that add phosphate onto biological molecules via a hydroxyl residue on that molecule to form a phosphoester.

Question 47

What does phosphorylation do?

Answer 47

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

Question 48

What can kinases target?

Answer 48

Kinases can target lipids (Lipid Kinases), Sugars (sugar kinases) and proteins (Protein kinases)

Question 49

What are the three amino acids that can be targeted by kinases?

Answer 49

The three amino acids that contain hydroxyl groups (i.e. Serine, Threonine and Tyrosine) are the main target sites for protein kinases

Question 50

How many kinases are there?

Answer 50

Overall there are about 500 protein kinases in the genome, all of which share some sequence and structural homology in their kinase domains

Question 51

What are the two broad classes of protein kinases?

Answer 51

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.

Question 52

What are kinases?

Answer 52

Kinases are enzymes
so the can repeatedly add
phosphate to targets and
thus cause signal amplification

Question 53

How may reversible phosphorylation act as a regulatory mechanism?

Answer 53

• Kinases phosphorylate lipids, proteins, changing function

- Phosphotases oppose this, making it transient and reversible (regulation)

Question 54

What is often the first target of cellular phosphorylation?

Answer 54

Around 90% of all the phosphorylation in cells is on ser/thr implying tyrosine phosphorylation is at early stages in amplification cascades.