https://www.science.org/content/blog-post/good-old-bewildering-gpcrs
Ah, G-protein coupled receptors. They’re an absolute mainstay of drug discovery and have been for many decades, and one of the first projects I worked in when I joined the industry was an effort to produce selective muscarinic M2 antagonists. A few years back I had the chance to meet Bob Lefkowitz (Nobel for GPCRs, along with Brian Kobilka), and I mentioned to him that back in the early 1990s when I was working on them I thought I understood them reasonably well. He grinned when I told him that as time went on I decided that I actually didn’t know jack #$! about them, and that this became more apparent every passing year. This new paper emphasizes that situation!
To recap for folks who don’t do this for a living, GPCRs are ubiquitous proteins found on the cell surface, which extend all the way to the inside cytoplasm. There are whole families of these things (with the classic opioid, dopamine, and serotonin ones being famous examples), and they are a way for cells to receive chemical signals from outside that get turned into intracellular changes as these receptors get activated or inactivated.
Those “G proteins” in the name are associated with the inner poking-into-the-cell region of the GPCR. The receptor itself changes shape as small molecule ligands (like dopamine!) bind to it or leave on the outside surface, and the activities of the G proteins on the inside change in response to those movements in turn. And those activities include formation or cleavage of a number of prominent “second messenger” molecules like cyclic AMP and others, which set off all kinds of activity as their concentrations move up and down. It’s really a sort of physical toggle switch to get signals through the cell membrane, and on close inspection they truly resemble an old Rube Goldberg machine where the cat jumps for the toy mouse, moving a lever that makes the ball go down and hit the bell that wakes up the bird, which flies over to the. . .
Complications ensue. As mentioned, there are often a whole list of GPCRs that respond to the same ligand but are hooked up to different second messengers and are found on different types of cells. That’s one level. Another is that the ligands that activate these receptors (“agonists” in the nomenclature) don’t always activate them to the same degree - there are “partial agonists” that can have different effects than the “full agonists”. Then you have molecules that bind to the receptor but don’t activate it (and in fact can keep agonists from binding while they’re around) - those are “antagonists”, and there are several ways that they can bind to make this happen. Yet another variation is the way that some receptors are set in the “always on” position, activating their second messenger proteins all the time, until a ligand comes along and binds to them to change their shape that then actually shuts them off instead. These are “inverse agonists”. Then you have the way that some particular GPCR subtypes seem to cluster together in defined groups on the cell surface and affect each other’s signaling and behavior as opposed to how they act when they’re studied in isolation. I’m not even going to start on the subtleties on the inner loops of these proteins; suffice it to say that there are whole other signaling families that can also bind down there in addition to the various G-proteins. Oh, it’s a mess.
This latest paper is actually trying to bring some of that mess into better focus, and they’re using my old friend and sparring partner the M2 muscarinic receptor as an example. It’s a good choice because it’s been shown to have some large structural changes on ligand binding. Through genetic manipulation and fluorescence microscopy, they’re able to set up a number of structural reporters for the physical state of the receptor protein (the fluorescent groups respond to changes in their environment, such as being shifted into a more or less polar region). And what they find is that this garden-variety GPCR has a whole range of behaviors when exposed to various agonist molecules, shifting around on millisecond time scales between several conformational states with equilibrium constants between each of them. Different agonist molecules hit in different ways when examined at this level of resolution - there are several distinct active states for this GPCR and they interact with the G-proteins in different ways.
The authors dryly note that “Overall, our study reveals that the activation of a GPCR in intact cells may be far more complex than previous biophysical studies with isolated receptors have suggested”. And as I mentioned above, it’s not like the previous studies have all led to a simple or orderly model themselves! But this is probably a first look at what’s really happening with these receptors, and we’re going to have to get more details as we try to figure out whether we can use this knowledge to our advantage with new ligands. One way or another, we all now officially know more about GPCRs than we did, and we all now officially understand them less. Welcome to the club!
https://www.science.org/content/blog-post/good-old-bewildering-gpcrs