Hydrophilic signal transduction

Last time, I spoke about hydrophobic signal transduction, where the premise was simple: hydrophobic molecules can pass straight through the cell membrane, and can then bind to a nuclear receptor to conduct a signal response.

Hydrophilic molecules can't do that, though, because they're unable to pass freely through the cell membrane. So instead, they need to bind to receptors, transporters, or ion channels, to let the signal pass through. This does give hydrophilic molecules a crucial advantage, though - these responses are time-sensitive, and can even span a matter of seconds; hydrophobic signalling typically occurs over a wider timescale, sometimes lasting years. Hydrophilic signalling is also responsible for a far wider variety of different responses, owing to them being able to bind to receptors which bring about signalling cascades.

G-protein coupled receptors

As you can tell from the name, these are receptors which are coupled to G-proteins. I'll call them GPCR from now on for simplicity.

Their structure is relatively simple - seven ɑ-helices spanning a cell membrane. If you don't know what an ɑ-helix is, it's just a type of protein structure where the protein is twisted into a coil. Here's an exceptionally crude diagram:

What's most important, though, is that the conformation of the GPCR changes as soon as a ligand binds to it. What I mean by that is when there's no ligand present, two of the ɑ-helices are far apart from the rest, yet once a ligand binds, these ɑ-helices are brought closer together. 

I also need to define two different molecules -  GDP, and GTP. They're both extremely similar in structure, consisting of a guanosine molecule (itself a guanine bound to a ribose sugar), bound to a series of phosphate groups. GDP has two phosphate groups, GTP has three:

You can go between the two quite easily; specifically, going from GTP to GDP involves a hydrolysis reaction that's catalysed by the GTPase enzyme, with the additional benefit that you produce a phosphate group too.

This is how they work:

• A ligand will bind to the GPCR, which induces the change in conformation. 
• The GPCR will then activate the heterotrimeric G-proteins, which, as I implied earlier, were already coupled to the GPCR. However, they will now be ready to do some signal transduction!
• There are three subunits - Gɑ, Gꞵ, and Gɣ. 
• Gɑ is a GTPase switch protein, and is where GDP is converted into GTP. When there's no ligand bound, Gɑ forms a complex with Gꞵ and Gɣ, but otherwise, Gɑ will dissociate from them and bind with GTP. We now have two states - the Gɑ, bound with GTP, and the Gꞵ and Gɣ subunits, which exist as a Gꞵɣ complex. Yet they're all still anchored to the cell membrane during all this.
• The Gɑ-GTP unit will now go and activate an effector protein, such as an enzyme, which might modify signalling pathways into and out of the cell. At the same time, Gꞵɣ might regulate an ion channel, letting ions such as Na⁺ and K⁺ in and out of the cell respectively. 
• These events can thus lead to propagating effects, such as the formation of products from enzymatic activity, or the depolarisation of the membrane if the ion channel is being operated.
• Yet none of these will last long. You see, Gɑ is a GTPase switch protein, and GTPase is an enzyme that exists specifically to hydrolyse GTP back into GDP. The whole signal transduction process therefore only lasts for a matter of seconds.
• Once we reform GDP, Gɑ will go and dissociate from the enzyme, and rejoin the Gꞵɣ complex, reforming the original heterotrimeric G-protein. 
• They'll bind to a now ligand-free receptor, and the cycle will start anew.

But for any of this to happen, we need to be able to induce the conformation change and GTPase activity necesssary for the cycle to occur:

• The guanine exchange factor, or GEF, is a protein that will induce the exchange of GDP into GTP. This process then triggers the cycle, as it means Gɑ, which is a GTPase switch protein, can dissociate and bind to effector proteins. In this cycle, the GEF will be the ligand-bound receptor itself.
• As I said earlier, Gɑ can hydrolyse GTP into GDP through GTPase. This can be stimulated by GTPase activating proteins, or GAPs. As I said before, GTPase halts the signal transduction stage, so it can also be said that GAPs are signal terminating. GEFs, on the other hand, are signal inducing. In the cycle, the effector protein will act as the GAP.

Sometimes, we might want to identify the role these GCPRs play in signal transduction. For this, we will interfere with the GCPR cycle by using some molecules. These include:

• PTX, or the pertussis toxin. This will inhibit any binding to the GCPR.
• CTX, or the cholera toxin. This will inhibit any GTPase activity, thus halting the cycle. The effector protein will continue working away in the background as a result. And yes, this is the same toxin that causes cholera.

Receptor tyrosine kinases

Enzyme-linked receptors are...receptors linked to enzymes. These enzymes can be either the receptor itself, or part of a bigger protein complex. The largest of these are receptor tyrosine kinases, or RTKs for short.

Worth explaining what a kinase is first, though - a kinase is an enzyme which catalyses a phosphorylation reaction, which is basically "adding a phosphate group to a substrate". The typical reaction for this process is this:

ATP + substrate → ADP + phosphorylated substrate 

ATP, or adenosine triphosphate, is very similar in structure to GTP - this time, however, its nucleobase is adenosine, not guanosine. Ditto for ADP/GDP. 

RTKs actually work very similarly to GPCRs:

• A ligand will bind to the RTK, inducing a conformational change in the RTK.
• Specifically, we need dimers - molecules with two of the same functional group. This will be either a dimeric ligand binding to a monomeric receptor or a pre-exisiting, inactive dimeric receptor accepting a monomeric ligand.
• This binding will trigger the kinase to do kinase things, such as autophosphorylation of the receptor. This creates docking sites for intracellular signalling molecules.
• These molecules can be monomeric G-proteins, effector enzymes, and adaptor proteins.
• Adaptor proteins in particular can then be phosphorylated themselves. An example of this is the STAT protein. A specific part of STAT can be phosphorylated by the Janus kinase (JAK), inducing dimerisation, and subsequent phosphorylisation of the dimer, within the STAT protein. 
• The adaptor can then be released, before being translocated into the cell's nucleus.

If you want some chemical intervention tools against kinases, here's a list:

• FSBA is an irreversible inhibitor. Acts against all kinases, as well as ATPases and any other ATP binding proteins.
• Staurosporine is a very potent ATP competitive inhibitor. Acts against all kinases.
• Hypericin is a very potent ATP competitive inhibitor. Acts against all RTKs, but can also inhibit some serine kinases.

Glucose transport

A useful example of a transporter can be found in the urine, where they transport excess glucose into the kidneys. This is the SGLT2 transporter, which is coupled to a sodium gradient in the kidneys. On the other hand, this glucose will then be transported into the blood from the kidneys using the GLUT2 transporter. To remove any excess sodium ions, we'd then need an additional pump which facilitates the GLUT2 transporter. 

SGLT2 is a symport, where the Na⁺ ions and glucose are both transported in the same direction - out of the urine, into the kidneys. Symports need two things to bind - here, glucose can only bind if sodium is bound first. That's quite good, in fact, as otherwise you could end up with a far higher blood sugar concentration than you'd otherwise want. However, SGLT2 actually works in both directions, meaning it will act according to how much sodium there is in the kidneys. Quite clever.

GLUT2, on the other hand, is a uniport. All that needs to happen is the glucose binds, and the transporter is activated. Bear in mind, though, that this is driven by the sodium pump I mentioned earlier.

You might now be curious over how we can target these transporters:

• SGLT2 can be targeted using phlorizin, which competes with glucose for binding to the transporter. If no glucose binds, then no transport.
• The only problem with phlorizin is that it's hydrolysed, once consumed, by enzymes in the small intestine. You can get around this through using canagliflozin, which is similar in structure, but contains a hydrolysis-resistant bond that gets around the enzyme issue.
• This is used as a drug target for type 2 diabetes, in fact!

Ion channels

Comparatively simpler, ion channels are regulated by a membrane receptor. A ligand binds to the ion channel, the channel opens or closes, ions can pass through or are blocked by the channel, done. A common ion channel is used for Ca²⁺ ions, which will often go on to then bind with calcium binding proteins, before the protein is bound to an intracellular receptor and the signal is transmitted. 

The rate of transport through an ion channel is dependent on the concentration difference across the membrane. We can exploit this fact by using a voltage-gated ion channel, which will be regulated by the polarity across the membrane. 

If we wanted to measure the rate of transportation across an ion channel, we'd use a patch clamp to detect changes in permeability of the ion channel. Permeability happens to be directly proportional to the net transport rate, which is why this works. 

We can also block ion channels using channel blockers. In Ca²⁺ voltage-gated ion channels, we can use nifedipine to stick itself into the ion channel, and quite literally stop the ions from getting inside. Nifedipine is used as a drug target against cardiovascular conditions, and it can bind in the ion channel since it has an amine group that can have a positive charge on the nitrogen. This is really important - it means that it can insert itself into the channel since it has a positive charge, like a calcium ion.

We can do similar with receptor-operated channels, such as the NMDA receptor. This receptor is typically activated by neurotransmitters like glutamate and glycine, and it typically has an Mg²⁺ ion inside. This ion is often removed anyways due to depolarisation, but at this site, we can bind channel blockers here by inducing depolarisation across the receptor. 

A major reason to do this is because if we have high levels of calcium in the cell, that can lead to cell death - by limiting the levels of calcium, we can limit these effects. Examples of drug targets used are memantine and ketamine, the former selectively binding to the NMDA channel. Both memantine and ketamine have amino groups that can be ionised, and mimic the positive charge on the Mg²⁺ ion, enabling them to bind there. Memantine is used to treat Alzheimer's, whereas ketamine is an anaesthetic (and popular party drug), if you're wondering why we do this in the first place.