Signalling

Signalling is vital for respsone to hormones, growth factors, infection etc. These signals range from very short term such as pain or vision to very long term such as cell differentation or sex hormones. Most signals are short term and only occur when a ligand is bound Many signals need turning off e.g. growth factors, but some need to remain on such as sex hormones. 

The basic problem is how an external signalling molecule can affect something inside the cell. All signalling molecule can do is bind to a receptor it cannot pass through membrane so receptor has to change inside once signal binds. The cell membrane is very thickk in molecular terms and proteins are quite 'floppy' so have a hard job accurately transporting signals. 

• Hydrophobic molecule travels through into cell and directly binds to an intracellular receptor e.g Steriods, NO
• Ion channels - ligand gated ion channels 
• G-protein coupled receptor - Undergoes conformational change which activates something inside cell (most commonly G-proteins) 
• Enzyme coupled receptors - binding causes enzyme activity inside cell (most commonly kinases) 

Ligand-gated channels, need to be very responsive to ligand concentration. A sharp transition need allostery (multiple subunits). Therefore ion channels often have multiple subunits (commonly 5), to make them respond more dramatically to change in ligand concentration. 

An advantage to ion channels is that they can turn on a signal rapidly (1ms) but in order to reverse the signal all the moved ions need to pumped out again which can use a lot of energy. Speed is sometimes critical such as in vision or muscle cells so ion channels are used here. Ion channels aren't very specific so more specific signals tend to use another type of signalling. 

It is a ligand-gated sodium/potassium channel and is typically found on postsynaptic membranes. 

There are 5 possible binding sites as it has 5 subunits but only binds at two of them. This is enough to produce a sharp transition however it is not a cooperative as it could be. 

Ligand binds a long way from the membrane so the conformational change is transmitted a long way from where the ligand binds. 

The conformational change works like the lens a camera opens. Simulatneous rotation of each subunit open up the channel. 

A molecular on/off switch used in receptor kinase and GPCR pathways. G-proteins are switched on when bound to GTP and off when bound to GDP. Strurctural changes occur on hydrolysis of GTP to GDP. Residues near the end of the protein loops of G proteins recognise and hydrogen bond to the terminal phosphate of GTP. When hydrolysis occurs the terminal phosphate is released and loses the hydrogen bonds and causes a conformational change in the sturcture. This is amplified if additional proteins/domains are attachted to the protein loops. 

Switch is turned on by dissociation of GDP and binding of GTP and turned off by GTP hydrolysis. Cell concentration of GTP is 10x higher than GDP so GTP is favourable to bind you just need to encourage GDP to dissociate. This is done using guanine exchange factors (GEFs) which aid turning the signal on. GTPase- activating proteins (GAPs) aid hydrolysis and turning the signal off. 

A classic example is Ras. 

To get a good on/off signal they use dimerisation. Kinase domains on the inside of these receptors phosphorylate other proteins. This is a switch because when the ligand is bound phosphorylation occurs and when the ligand isn't bound there is no phosphorylation, all cell need to do is recognise the phosphate. 

When a ligand binds (typically a small protein like a hormone) dimerization occurs and brings two kinase domains together and autophosphorylation occurs. This is where they phosphorylate each other. 

Autophosphorylation fixes the position of the activation loop which in the inactive kinase state (before phosphorylation) is floppy and disordered. This means a substrate cannot bind correctly and ATP, bound to the other domain isn't in the correct position to catalyse. A phosphorylated activation loop allows the substrate to bind in between the two domains and ATP is in the correct place for catalysation. (Note the activation loop can fit in the active site in when the activation loop is disordered as this has to happen in one kinase for autophosphorylation) 

Once the receptor is phosphorylated it is classed as on and acts as a binding site for modular adaptor proteins e.g. Grb2 and Sos. These proteins are a way of connecting different parts of the system. 

Grb2 has 3 modular domains which lots of signalling proteins have. It has 2 SH3 domains which recognises polyproline helices (different types of this domain but share similarities). It also has an SH2 domain which recognises phosphotyrosines and residues either side (specific SH2 for eac receptor). Sos proteins plug together to bring the GEF (which is Sos) to the cell surface to activate Ras. 

Phosphorylated kinases are recognised by SH2 domain on Grb2. Sos binds to Grb2 via SH3 domains as it has a proline rich tail. Sos is now at the membrane surface positioned next to Ras as Ras is linked to the membrane surface. Sos activates Ras which then becomes an active G-protein whose main function is to activate a kinase called Raf. 

Raf sits at the top of a kinase cascade ( a sequence of kinases which phosphorylate each other and amlify the signal at each step). The final kinase (ERK in example) moves into nucleus and phosphorylates several transcription factors. 

Lots of variation on basic theme and it is a complicated way of getting a signal to nucleus but you can modify the signal in lots of ways. Slower than ion channels but more powerful. 

Basic principal is that a ligand binds to the outer fac a receptor and this leads to conformational change to switch on the inner face, which is detected and carried on by a G-protein that binds to the inner face. 

Confromational switch occurs by rotation of helices and as helices are rigid if the top moves the bottom end moves too (especiallt large if kinked helices). G protein binds to the inner face and is activated by the receptor which acts like a GEF. 

GPCRs all have same basic 7 transmembrane helices structure, starting outside the cell and ending inside. Third intracellular loop is longer and is where the G protein binds. 

We have about 800 GPCRs, 400 are olfactory (smell) and of the others only just over half have known ligands. GPCRs are also a common drug target and half of current drugs interfere with them. 

Ligand binding occurs well within the membrane of GPCRs. A tryptophan ring is crutial in creating the on/off behaviour as the ring changes orientation when the ligand binds. This creates a switch as tryptophan is a large amino acid so causes a large change.

The G protein that interacts with the receptor forms a heterotrimeric complex with two other proteins. These are G alpha (G-protein), G beta and G gamma (the last two go around together). Sometimes G beta and G gamma have their own signalling roles but its often only G alpha that takes part in signalling. Lipid anchors keep the proteins attached to the membrane. 

G alpha when bound to GDP (off) has a bump on top, which is the part that interacts with the GPCR. When the GPCR is bound to ligand the intracellular loop 3 changes shape and the bump fits into a corresponding groove in the GPCR. This opens up the space where GDP binds and allows GDP to dissociate and GTP to bind. 

Signalling pathway 

• Signalling molecule (first messenger) binds to GPCR
• GCPR activates G-protein which activates adenylyl cyclase 
• Adenylyl cyclase synthesises cAMP (second messenger) 
• cAMP activates protein kinase A
• Activates protein kinase A subunits enter nucleus
• PKA phosphorylates CREB
• activated CREB recruits CREB binding protein (CBP) 
• Genes activates by CREB-CBP

Some indirectly open or close ion channels eg smell and vision. Binding of odorants to specific receptors activates a G alpha which actuvates adenylyl cyclase. cAMP opens sodium channels, which initiates neuron depolarisation. Light leads to alteration in conc of cGMP which works in similar way. 

An important class of GPCRs work by the G alpha activating phospholipase C (PLC) usually in the isoform PLC beta. Phospholipases separates head group from fatty acid tails. 

Examples 

• Tissue - signal molecule - response
• Liver - vasopressin - glycogen breakdown 
• Pancreas - acetylcholine - amylase secretion 
• Smooth muscle - acetylcholine - muscle contraction 
• Platelets - thrombin - platelet aggregation

Inositol is a sugar, which can be phosphorylates at several positions eg IP₃ is a 1,4,5-triphosphate. Inositol can also be attached to a diacylglycerol lipid via a phosphate becoming phosphotidyl inositol 4,5-bisphosphate (PIP₂). Different inositol phosphate are recognised by different proteins. They are mainly on the inner leaflet of the cell membrane. PIP₂ binds to proteins involved in actin cytoskeleton, and in endocytosis. 

Activation of PLC leads to cleavage of PIP₂ into IP₃ and DAG, which go on to act as second messengers. IP₃ leads to release of Ca²⁺ from ER, which activates cells in various ways. High conc of Ca²⁺ in the ER flows through specific channels in ER. Ca²⁺ and DAG activate protein kinase C.