Monday, October 22, 2012

The human machine: communication technologies

The previous post in this series can be found here.

Have you ever stopped to consider what makes you a single organism? It might sound strange, but it is a question dripping in biological significance. You may think of yourself as a neatly packaged single unit, yet you are probably also aware that this one unit is made up of tiny individual cells working together. But how do 75,000,000,000,000-odd cells cooperate with such precision? Why your cells work together is entirely separate question with answers to do with filling evolutionary niches and delegating functional roles; I'm talking about how they do it. That's the million dollar question! 

Well, to be precise, it's the $1.2 million (or 8 million Swedish Krona) question, as it was announced last week that this year's Nobel Prize in Chemistry (and the accompanying monetary reward) has been awarded to Robert Lefkowitz and Brian Kobilka for their outstanding work into the biology of G protein-coupled receptors (GPCRs). In this post I hope to give you an understanding of how GPCRs work, why they're important enough to deserve a Nobel Prize, and how they relate the question of how you stay as just one you.

GPCRs - the eyes and ears of the cell 

If you were a cell, how would you know what to do? When should you divide, where should you move, what should you make? You couldn't just do it randomly or to some pre-determined schedule because the human you're in is unpredictable and its cells must be flexible in their behaviour to match that. So, what you really need to make these decisions is information. This is exactly the same as how humans make decisions about our behaviour, we gather information about the surrounding environment through our senses and then act appropriately. A cell that receives no information from outside its own membrane is as impotent as a human with no sense of sight, smell, touch, taste or hearing. Ok, so they need information, how do they get it? As you've probably guessed given my snappy subtitle and general build-up, the answer is receptors!

Receptors are proteins expressed at the surface of cells that are capable of relaying information from the outside world into the signalling environment within the cell. If you've been following my posts for a while, you may remember that I wrote about the T cell receptor and how its mechanism of information transfer is still a contentious issue among immunologists - this is just one example of a vast array of receptors found on your cells. Within this vastness, however, is one stand-alone family of receptors that dwarfs all around it! These are the GPCRs; the undisputed King of receptor families. There are roughly 800 different GPCRs in the human body, expressed in a dizzying number of combinations across every last cell in your body. The genes encoding them make up almost 4% of the genome (an enormous amount for one family!) and they have a role in almost every active cellular processes name in all areas of human biochemistry. Without them you wouldn't be able to see, smell, think, digest, move, feel, taste, grow, heal, and pretty much anything else you can think of! This means they are a treasure trove of potential drug targets for researchers to shoot for - in fact, over 50% of licensed drugs target members of this family alone. Beta-blockers, anti-histamines, morphine, salbutamol (marketed as Ventolin for asthma-sufferers) are some of the most well known GPCR-targeting drugs in widespread use. They are, basically, a big deal. 

The genetic needle in the genomic haystack

So, how do they work? Scientists had been aware that cells could sense their environment since the late 19th century mainly due to experiments looking at the effects of adrenaline on different tissues, but it wasn't until 1970 that Robert Lefkowitz published two studies that identify the existence of an adrenaline-receptor  responsible for the cell's ability to sense external adrenaline. At this point, they only knew that there was a receptor, but had no idea what it was or how it worked. To unpick this mystery they would need to isolate the gene that acts as the receptor's blueprint and then go from there. It was in the early 1980s that Lefkowitz began his search for this elusive gene, enlisting the help of the young Brian Kobilka. Nowadays such a task has been rendered fairly trivial thanks to the efforts of the Human Genome Project, but back then is was a sizeable piece of work! Years of hard graft eventually paid off as they slowly decoded the gene responsible for what is now known as the beta 2-adrenergic receptor (B2AR), publishing the final sequence in 1986. Knowing the DNA sequence meant that they could work out the amino acid sequence of the final protein, giving some indication of its structure. What they noticed was that the receptor protein was made up of distinct regions of amino acids with different chemistries - seven areas in the protein were made up of amino acids that are highly 'hydrophobic'. As the name suggests, hydrophobic amino acids do not like to be exposed to water and so will instead try to bury themselves away either in the core of a protein or, importantly in this case, within the non-aqueous environment of the cell membrane. Voil√†! This protein must therefore consist of seven segments that cross the cell membrane and short loops connecting these 'trans-membrane' regions. Such a structure would therefore be present on both sides of the membrane and so is well placed to act an a sensor for outside stimuli.

The 7-transmembrane structure of GPCRs. The cell membrane is shown in grey; the protein consists of 7 transmembrane sections (H1-H7), 4 extracellular sections (E1-E4), and 4 intracellular loops (C1-C4).

But hang on, a seven-transmembrane receptor had already been discovered and it had absolutely nothing to do with adrenaline-reception. This protein was rhodopsin, the receptor responsible for detecting light in the retina of your eye. The only thing that these proteins were known to have in common was that they associated with a class of signalling proteins in the cell known as 'G proteins', but so what? About 30 other proteins were known to signal via G proteins, and they had very little in common in terms of their biological roles. So the fact that B2AR and rhodopsin both associate with G proteins doesn't mean much, unless....all of these proteins have the same structure?  This was the realisation that Robert Lefkowitz would later describe as his "real Eureka moment" and that would eventually earn him the Nobel Prize: all of these proteins were members of the same, vast family that all share the same architecture and signalling mechanism! 

An emerging family - deciphering how they work

As the field of molecular biology and its associated technologies advanced during the 1990s, Lefkowitz and Kobilka were proved correct. Receptor after receptor was found that not only associated with G proteins but also shared this seven-TM structure. It wasn't until the publication of the human genome that the full scale of the family and its 800 members was fully realised, but it had been clear for some time that this was going to be an important group of proteins and so much work had been done on their biochemistry. What had been found was that GPCRs all signal in much the same way. Each GPCR is capable of binding its various ligands on the external side of the cell membrane - which ligands it binds depend on the chemistry of the amino acids that make up the binding-site, and so it is unsurprising that this is where the greatest differences are seen between GPCRs given that the ligands range from full proteins (in the case of chemokine receptors, for example), to small molecules (such as adrenaline or histamine), all the way down to fundamental particles (the rhodopsin families bind to incoming photons via an attached retinal molecule). No matter what the ligand might be, the binding event had the same effect of altering the arrangement of the receptor's seven transmembrane domains.

This rearrangement affects the regions of the GPCR on the inside of the cell membrane, causing them to change their shape too. This new shape is able to bind to G-proteins on the inside of the cell. One G-protein is actually made up of three distinct proteins associated with one another: alpha, beta, and gamma. When the activated GPCR binds to a G-protein, it causes the G-protein to let go of a bound molecule called GDP, and replace it with another called GTP. This GTP-bound form of the G-protein is now active and is released from the GPCR whereupon it splits into two halves, the alpha section goes off on its own whilst the beta and gamma domains remain together. These two halves are able to bind to secondary signalling proteins and so start a whole complex cascade of signalling that determines how the cell behaves, all started off by the humble GPCR. There are a number of different types of G protein available within the cell and different GPCRs associated with different types, thereby allowing for subtlety and complexity to arise from this one, simple mechanism.  

The GPCR signalling cycle. The GPCR is shown in blue, the ligand in brown, and the G-protein in pink.

Slowing down the film

Simply knowing that this is what's happening is, however, not enough! Molecular biologists still wanted to know what these things actually look like, and what exactly are the shape changes that take place when the receptor is activated so that the movie of GPCR activation can be pieced back together from the individual frames. This, as you may have guessed, was not easy. In fact, saying it 'was not easy' makes me look like a delusionally blind optimist; it was actually one of the most daunting challenges ever faced by modern molecular biology. The problem was not what to do - the structures of hundreds of proteins had been solved to atomic resolution using the technique of X-ray crystallography - but rather how to do it. X-ray crystallography involves purifying your protein of interest and then treating it in such a way that it forms an ordered crystal through which you can fire an X-ray beam to give a diffraction pattern that tells you your protein's structure right down to the last atom. However, proteins that enter membranes are notoriously difficult to crystallise for a number of reasons: firstly, it's difficult to get enough protein out of a membrane to make a crystal; secondly, it's difficult to ensure all of the protein is in the same state of activity; and thirdly, because the hydrophobic regions must be yanked out of their cosy membrane home and exposed to the water in the crystallisation solution, which they do not like at all! The degree of difficulty is proportional to how much of the protein is embedded in the membrane, and for GPCRs there is very little that is not in the membrane! It is unsurprising, therefore, that it was nearly fifty years between the determination of the first protein structure by crystallography and the publication of the first GPCR structure obtained in this way. It was worth the wait, however, as we were able to see for the first time how the transmembrane regions of GPCRs pack together to form a tight bundle spanning the cell membrane. 

The structure of GPCRs within the membrane. The outside of the cell is at the top, the inside at the bottom.

This was a major leap forward - for the first time researchers could analyse in detail how new drugs might bind to the receptors, or how different G-proteins might be selected by different GPCRs. Nonetheless, the additional goal of working out what shape changes occur upon ligand-binding took quite a bit longer to come. Brian Kobilka was working on this for nearly two decades before finally, in 2011, managing to capture an image of a GPCR at the very moment of information transfer to a G-protein. Combining this structure with a number of others of GPCRs in different states of activation has given us an atomic-level understanding of what happens when a GPCR is activated.

The Holy Grail! B2AR (blue) caught in the act of activating its cognate G-protein (red) having bound its ligand (gold). 

Holding you together

This relatively simple mechanism is responsible for all of the outcomes that I mentioned above. Cells rely on GPCRs and other receptors for cell-cell communication, i.e. one cell will release a molecule, which is then received and understood by another. I referred to this above as the 'eyes and ears' of the cell, but in fact it is far more akin to the taste or smell of the cell. When you smell or taste something, the taste you register is the combination of a multitude of different tastes all coming together to give a unique outcome. This is how cells see the world, they constantly 'taste' a huge number of different molecules through their GPCRs and other receptors and the results depend on the combination of flavours rather than any one in particular. This is how your cells can be induced to take on such different shapes and functions, and so how you can be formed as one organism made up of trillions of cells working in unison. 

An elegant example of this comes in the unlikely shape of the humble slime mould, Dictyostelium, which exists for much of its life as single celled organisms that live independently of one another. When it come to spreading their spores, however, the individual organisms come together to form a single organism that takes on a number of different shapes to allow maximum spore dispersal, before the cells part ways to become individuals again. The communication needed to allow such cooperation is achieved through cell surface receptors, including GPCRs. Indeed, a GPCR was identified recently that is responsible for sensing cell density in multicellular Dictyostelium, a process not dissimilar to those taking place in the developing organs of a human being. 

Dictyostelium cells cooperating thanks to GPCRs and other receptors. Each structure is made up of millions of individual cells. 

Where next?

Once the champagne's been drunk and party poppers popped, the GPCR field will be quietly getting back into the work that is currently pushing back the boundaries of our understanding of this extraordinary family of proteins. GPCR-targetted drug development is a multi-billion dollar industry, easily the biggest single field in all of pharmaceuticals. In a more academic sense, however, there is still a huge amount to learn about GPCRs. A lot of work is now focussed on how the G-protein signalling described above is linked in to signalling by another group of proteins called 'arrestins', which have also been shown to associate with GPCRs. This holds the promise of developing so-called 'biased' drugs that only activate the G-protein- or arrestin-associated pathways of GPCRs, thereby broadening the scope of numerous treatments. A morphine-based sedative, for example, has been shown to have similar effects but be far less addictive than morphine when it targets only the arrestin-associated signalling of the morphine receptor GPCR. 

Similarly, there is still a great deal of controversy over the organisation of GPCRs at the cell surface and how this relates to their signalling crosstalk. Many researchers believe that GPCRs are able to come together in the membrane to form complex structures that signal differently to individual GPCRs, whereas others (myself included) do not believe this to be the case. Solving this issue is likely to be one of the next great advancements in our understanding of GPCR biology. 

What's more, our structural understanding of GPCRs is far from complete. We have a good understanding of their conformational changes, but this is based on extrapolation from a small number of receptors to the family as a whole. Who's to say that there aren't differences based on the relationships between GPCRs? This is another fruitful area of research that is still being headed by the great Brian Kobilka. 

The GPCR family tree, including those receptors for which we have at least one structure. Taken from Katritch et al, Trends Pharma. Sci. 33, 2012.

All in all, GPCRs are pretty damn cool and are likely to keep being cutting edge for a long time yet to come. The exciting thing for those of us in the field is that we are working on something that pervades all of molecular biology and not just our own little bit of it. These days it is rare in biology to work on something that is significant across so many fields, and I for one relish the opportunity! I congratulate the Nobel Committee on their selection - they made a good choice!

The next post in this series can be found: here.

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