G protein-coupled receptors (GPCRs) are the largest family of membrane proteins and are involved in many signalling pathways related to neurotransmission, development, and the senses [1-3]. Their relevance to human physiology and medicine is demonstrated by the fact that GPCR genes make up 4% of the human genome and that 34% of FDA-approved drugs target GPCRs [1-3]. Despite the significance of GPCRs to human physiology and the effort spent researching them, many GPCR structures and binding sites are still unknown. This is due to a combination of factors related to the dynamism of GPCRs [2-3]. This article will explain the function of GPCRs, their general structure, and why it is difficult to visualise and determine the molecular structure of specific GPCRs.

GPCRs are membrane proteins, meaning they exist at the cell’s surface and typically interact with G proteins [1-5]. GPCRs have seven transmembrane domains which pass through the membrane in a serpentine fashion [1-5]. At one end, there is an extracellular N-terminus, and at the other, there is an intracellular C-terminus [1-5]. This means that the N-terminus interacts with the ligand while the C-terminus interacts with the G protein to which the GPCR is coupled [1-5].

There are six families of GPCRs, classified from A to F, with each family having unique structures and functions [1-5]. All the classes have the same general structure as mentioned above, with minute differences. For example, class A GPCRs have a short extracellular N-terminal chain, while class B GPCRs have a longer one [2]. Class C GPCRs also have large extracellular domains, with their chains resembling a “C” or venus fly trap [2]. Despite research efforts, the class D-F GPCR structures are still largely unknown and have little to no clinical significance [2]. The functions of proteins are usually dependent on their structure – and GPCRs are no different. Although all GPCRs have the same broad function of binding to a ligand and promoting a downstream signalling process, the specifics of this process varies for each individual GPCR [2]. For example, research on the visualisation and binding of class A GPCRs has shown that in their transmembrane domain are a conserved proline, a NPxxY motif, and a DRY motif [2]. When a ligand binds to the transmembrane seven domain, it causes a conformational change. The transmembrane domain seven then positions itself closer to transmembrane domain three, locking the ligand in the active site [2]. For class B GPCRs, the ligand first binds to the extracellular N-terminus. Then, the N-terminal domain swings towards the transmembrane domains, causing the peptide to bind to the transmembrane domain, activating the complex [2]. Lastly, for class C GPCRs, the ligand binds to their large extracellular domain which causes the GPCR to dimerise so it can be fully activated [2].

Once a GPCR is activated, it associates with a specific G protein [2-5]. G proteins are heterotrimeric, meaning they have three different subunits: the alpha, beta, and gamma subunits, and it is the alpha subunit that binds to the GPCR [2-5]. This initiates the G protein cycle, where the alpha subunit dissociates from the beta and gamma subunits of the G protein to associate with a downstream effector protein, initiating a change within or outside the cell [2-5].

Furthermore, other features of GPCRs promote their function and specificity. For example, GPCRs have specific ligands and specificities for each ligand, called biased agonism. This bias can affect the GPCR protein itself, as in receptor bias, or it can affect the peptide that the GPCR binds, as in ligand bias [3-5]. Receptor bias occurs when the receptor itself is biased towards a specific signalling pathway as it only activates a specific downstream effector [3-5]. Ligand bias is when the ligand itself causes the GPCR to adopt a specific conformation, which only allows a specific G protein to bind, causing that signalling pathway to become activated [3- 5].

Being such an important aspect of the signalling pathway, GPCRs must have processes in place to keep them in check so they do not produce excessive signalling within the cell. Thus, there are some key processes that can reduce or halt the function of GPCRs: phosphorylation and desensitisation, internalisation, recycling, and degradation [5]. Phosphorylation reduces the GPCR’s affinity for its G protein, resulting in decreased signal transduction [5]. This phosphorylation is typically done by kinases, which can be facilitated by arrestin proteins, while the reverse process, dephosphorylation, is carried out by phosphatases [5]. This process inevitably leads to the GPCR becoming desensitised [5]. Internalisation is when the GPCR is internalised into an endosome due to ubiquitination, causing it to be absent from the cell’s membrane [5]. This can then lead to recycling and degradation, where the GPCR is either recycled to the surface again at a later time or is sent to a lysosome to be degraded [5].

The relevance of GPCRs to the normal functioning of the human body is exemplified by the sheer number of medications on the market that target GPCRs. This is due to the fact that they exist on the plasma membrane at the cell’s target and are responsible for many downstream effects, making them ideal candidates for drug targets [6]. The malfunction of GPCRs are implicated in many disease states, ranging from mild, such as migraines, to lethal cases, such as myocardial infarctions [6].

There have been mounting research efforts to determine the molecular structure of different GPCRs so that their mechanism of action can be elucidated and better drug targets can be developed [2-3]. The advancement of technologies like cryo-electron microscopy has accelerated the visualisation of many GPCRs, but despite this, around a third of all GPCRs still have their structures to be revealed [2-3]. The reason why it is difficult to visualise GPCRs is because many visualisation techniques require the GPCR to be static or crystallised, and thus require specific conditions to induce that state [3]. However, these conditions are typically contradictory to the conditions needed for the GPCR to be functional, and thus the active sites, conformational changes, and mechanisms are difficult to determine [3]. For example, to make the GPCR soluble and crystallised for X-ray crystallography, shortchain detergents need to be applied to the protein, while for normal function, the GPCR requires long-chain detergents [3].

However, the future is bright; there have been major advances in microscopy and visualisation techniques, as seen in the case of cryo-electron microscopy. This method does not require protein crystallisation to resolve the minute molecular structures of GPCRs [3]. With the aid of such tools, we look forward to new information on GPCR structures to contribute to more effective drug development in the near future.