Cells need to protect themselves from the outside world, but they also need to interact with it and know what is going on. Bacteria have a lot of sensing mechanisms, primarily for food and toxins, but eukaryotes took this project to a whole new level, especially with the advent of multicellularity. While a few of the things cells sense come right through the cell membrane, like steroid hormones or fatty vitamins A and D, most things are blocked. This leads to the need for a large collection of proteins (receptors) that sit in the membrane and face both sides, with a ligand-binding face outside, and an effector face inside, which typically interacts with a series of other proteins that transmit signals, by phosphorylating other proteins, or modifying them with lipids, or just binding with a series of other proteins to form new complexes and activities.
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| A couple of GPCRs (red and orange) portrayed in a schematic membrane (black lines), bound by a couple of their primary intracellular targets and signaling partners, a G-protein (left, teal) and an arrestin (right, purple). |
Human DNA encodes upwards of 800 receptors of one class, the G-protein coupled receptor (GPCR), which arose early in eukaryotic evolution, and duplicated / diversified profusely due to their effectiveness as a platform for binding all sorts of different molecules on the exterior face. They dominate our sense of smell as olfactory receptors, respond to 1/3 of all drugs ever approved, such as the opioids, and also conduct our sense of vision. Rhodopsin, which detects the photon-induced conformational flip-flop of retinal, is a GPCR receptor in the photoreceptor cell membrane. The fact that photons, which could have been detected anywhere, by many sorts of mechanisms, are detected by a membrane-bound GPCR receptor illustrates just how successful and dominant this mechanism of sensing became during evolution. More GPCRs are still being found all the time, and even after receptor genes are deciphered from the genome, figuring out what they bind and respond to is another challenge. Thus over 150 of our GPCR receptors remain orphans, with unknown ligands and functions.
But how do they work? Due to their great importance in drug targeting, GPCRs have been studied intensely, with many crystal structures available. It is clear that they conduct their signal by way of a subtle shape change that is induced by the binding of their ligand to the external face/pocket, and conducted through the bundle of seven alpha helixes down to the other face. Here, the change of shape creates a binding site for the G-proteins with which the (active) receptor is coupled, so-named because they bind GTP in their active state and can cleave off one phosphate to form GDP. Binding to the activated receptor encourages an inactive, GDP-bound G-protein to alter its conformation to release GDP and bind a new GTP. The G-protein then runs off and do whatever signaling it can until its slow GTPase reaction takes place, turning it off. There are endless complexities to this story, such as the question of how cells can tell the difference between signals from the dozens of GPCRs they may be expressing on their surface at the same time, or how some ligands turn these receptors off instead of on, or the wide range of other participants such as kinases, GTP/GDP exchange factors, arrestins, etc., which have developed over the eons. But I will focus on the signaling mechanism within the GPCR receptor.
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| Rough schematic of GPCR activation. Ligands bind at the top, and a conformational shift happens that propagates a structural change to the intracellular face of the receptor, where effector signaling molecules, especially G-proteins, bind and are activated. TM refers to each trans-membrane alpha helix of the protein structure. |
A recent paper purported to have condensed a large field of work and done some mutant studies to come up with a common mechanism for the activation of the main (A) class of GPCR. This extends structural concusions that many others had already drawn about this class of receptors. As shown above, the main consequence of ligand binding is that key helices, particularly helix 6, make a substantial movement to the side, allowing the G-protein (shown in the top diagram in blue) to dock and stick a finger into the receptor. This is quite idealized, however, since GPCR receptors exist in a roiling sea of motion, being at the molecular scale, and can have subtle and partial responses to their ligands- many of which have contradictory effects. Some ligands (sometimes useful as drugs) have opposite effects from the main ligand, turning the receptors off, and others can have distinct forms of "on", or partial on effects, only fleetingly allowing the activated state to occur. Also, structures from several different GPCRs have been solved, with generally similar mechanisms, but not always informative about the dynamics of action- a structure made with an activating ligand may even show the inactive conformation, since the fraction of time spent in an active state may be much less than 100%.
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| Closeup of one switching event during receptor activation. Orange is the inactive state, where phenylalanine 6x44 (#44 on helix 6) contacts leucine 3x40 (amino acid 40 on helix 3), but it butted out of the way, upon ligand binding and activation, by tryptophan 6x48. |
These researchers analyzed 234 structures of GPCRs in various conformations to come up with an offset mechanism conducted by ~35 amino acids principally on helices 5, 6, and 7 as they conduct the tickle from the surface to the other face of the membrane. It is a classic meta-stable structure, where a small shove (by the ligand binding on the external face) causes a cascade of offsets of these amino acid side chains as they interact with each other that pushes the structure into the new, active, semi-stable conformation. A conformation that is additionally stabilized by a G-protein if one comes along, but only while in its GDP-bound state. An example of one of these individual atomic switches is shown above, where residues close to the ligand binding site undergo a dramatic shift that establishes a contact between amino acids 40 (leucine) and 48 (tryptophan), which were not close at all in the inactive state of the receptor. The larger scheme of detailed switches and shifts is shown below.
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| Detailed scheme of the authors for structural change propagation through the GPCR body. Each amino acid is referred to by a code, since this summarizes behavior of hundreds of different, though homologous, proteins. Contacts characteristic of the inactive state and broken or changed during activation are in orange, while those formed on activation are in green. For example, the "Na+ pocket", which contains a sodium ion in the inactive state, collapses in the active state. |
So this is biology descending to the level of engineering to understand an individual protein machine. We have such machines at all points, from thousands of genes, expressed in billions of copies, all cooperating and toiling in the service of us as a larger organism, blissfully unaware, certainly until the advent of molecular biology, of the wonders at work within. GPCRs have been an amazingly successful, ever-diversifying molecular machine, alerting animal and other eukaryotic cells of phenomena happening outside. A sort of instant messaging system on the cellular and organismal scale.
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