About one-third of all drugs, including opioid painkillers, antihistamines and many antipsychotics, target members of a family of proteins called G-protein-coupled receptors (GPCRs)1. This reflects the fact that GPCRs are important in almost all aspects of human physiology, and suggests that many more of them will be promising drug targets for numerous diseases. GPCRs span the cell membrane and convert myriad extracellular signals, including neurotransmitter molecules, hormones, and even light, into a cellular response by activating cellular G proteins and other transducer proteins. Four papers2–5 in this issue help to unravel the mystery of how GPCRs selectively activate a particular group of G proteins known as Gi/o, and provide clues that might aid the design of improved GPCR-targeting drugs.
Although more than 800 GPCRs are encoded in the human genome, they couple to only a small number of intracellular signal transducers, including 16 Gα proteins6. The latter proteins assemble with Gβ and Gγ proteins to form heterotrimeric G proteins. The G-protein complex disassembles on activation by GPCRs, whereupon the various subunits activate different signalling pathways. For instance, stimulatory Gα proteins (known as Gs) increase cellular levels of cyclic AMP molecules, which regulate various cellular processes. Structures of Gs-bound GPCRs have been reported7,8 that have begun to elucidate the general activation mechanism of Gα proteins, and of Gs in particular. But much less is known about how GPCRs selectively activate inhibitory Gα proteins, which include Gi1, Gi2, Gi3 and Go, and are collectively known as Gi/o.
The four papers in this issue report structures of Gi/o-bound GPCRs obtained using cryoelectron microscopy: Koehl et al.2 report the structure of the µ-opioid receptor bound to Gi1; Draper-Joyce et al.3 describe the adenosine A1 receptor in complex with Gi2; García-Nafría et al.4 report the 5HT1B receptor bound to Go; and Kang et al.5 reveal the structure of the light receptor rhodopsin in complex with Gi1. The G-protein activation cycle involves the binding and release of nucleotides to and from the G proteins, and all of the reported structures capture the receptors bound to the nucleotide-free state of their respective G proteins.
In some respects, the four structures are similar to those of the previously published GPCR–Gs complexes7,8, probably because Gs- and Gi/o-containing complexes have the same overall conformation at the stage of the G-protein activation cycle captured by the structures. Nevertheless, the Gi/o-containing structures reveal striking differences at the receptor–G-protein interface when compared with the Gs-containing structures. For example, there are no interactions between the receptors and the Gβ subunits in the Gi/o-containing structures.
The four structures uncover several key interactions at the GPCR–Gi/o interface mediated by the α5 helix — an α-helix structure in the carboxy terminus of Gα subunits. It is known that the binding of this helix to the receptor’s cytoplasmic site triggers conformational rearrangements in Gα that cause the release of a nucleotide (GDP) bound to Gα, initiating G-protein activation9. The positioning of the Gi/o α5 helices in the new structures is different from that of the analogous helices in the GPCR–Gs complexes. Specifically, the Gi/o α5 helices are rotated and translated slightly towards transmembrane helix (TM) 7 in the GPCR and away from TM6. Moreover, TM6 is displaced outwards from the receptor core by a smaller amount than occurs in the Gs-bound GPCRs (Fig. 1). The authors of all four papers therefore suggest that the smaller displacement of TM6 might preclude binding of Gs and help to explain how GPCRs can bind selectively to Gi/o proteins.
The difference in the positioning of the α5 helices seems to be due to the Gs α5 helices containing bulkier amino-acid residues than those of the Gi/o α5 helices. Moreover, Kang et al. analysed and compared the amino-acid sequences for TM6 in the Gs- and Gi/o-coupled receptors, and suggest that the different patterns of hydrophobic and hydrophilic residues observed in the two systems might affect the amount of displacement of TM6, and thus contribute to Gi/o specificity.
Comparison of the four GPCR–Gi/o structures reveals considerable structural plasticity at the interface. This is not surprising, given that Gi/o proteins are engaged by hundreds of GPCRs that have diverse structures and sequences. Draper-Joyce et al. thus suggest that G-protein specificity is not necessarily encoded by evolutionarily conserved interactions between specific amino-acid residues, but might be based on “pocket complementarity”, in which conformational rearrangements produce regions on the GPCR cytoplasmic site that are conducive to the binding of specific G proteins. Further evidence for this comes from the fact that all the structures of the GPCR–Gi/o complexes display markedly smaller GPCR–G protein interfaces than do the structures of GPCR–Gs complexes. This is particularly pronounced for the 5-HT1B receptor–Go interface surface, which García-Nafría et al. report has an area of 822 square ångströms; this compares with 1,260 Å2 and 1,135 Å2 for the interfaces in the Gs-bound β2-adrenergic7 and adenosine A2A receptors8, respectively.
Finally, Koehl et al. report subtle, yet potentially crucial, differences in the conformations of Gi1 and Gs that occur during the transition between the GDP-bound and the nucleotide-free states of the proteins. Given that GPCRs catalyse specific structural transitions in specific G-protein subtypes, it is tempting to speculate that the observed conformational differences might also contribute to the G-protein specificity of GPCRs.
This body of work provides a key step towards delineating the molecular mechanisms by which GPCR conformations drive the activation of one signalling pathway in preference to another. Many more such structures are sure to follow, and will probably reveal structural hallmarks that drive GPCR coupling to other G proteins and signal transducers, such as arrestin proteins. However, as the authors of all four papers point out, these studies provide only snapshots of the G-protein activation pathway, and are thus incomplete. The coupling specificity of GPCRs depends on several factors not addressed by the new structures, including the pre-coupling of G proteins10 (a preliminary step in which GPCRs and G proteins associate with each other, before actually coupling), and the binding of the GDP-bound form of G proteins11. The lifetimes of distinct receptor conformations can also determine the specificity of GPCRs for transducers12,13, adding a kinetic dimension to GPCR signalling that needs to be considered.
A comprehensive molecular model of GPCR specificity for G proteins and transducers would not only improve our understanding of how GPCRs elicit complicated signals involving multiple, occasionally intersecting, pathways, but also facilitate the design of better drugs that target GPCRs. In particular, it could allow the structure-based design of drugs that selectively activate or inhibit particular signalling pathways, thereby making them safer and more effective than currently available therapeutics.
For example, the painkilling properties of opioid medications such as morphine are thought to arise from the activation of a Gi protein by the μ-opioid receptor, whereas coupling of the receptor to arrestin probably causes the drugs’ addictive properties and the — often fatal — depression of respiratory functions. Much effort has thus been dedicated to designing opioid compounds that provide pain relief, but that reduce the risk of addiction or overdose. A flurry of structures of isolated GPCRs has already greatly facilitated the discovery of compounds that bind to the receptors, and that are useful tools for laboratory studies14. But it is the structures of GPCR signalling complexes that will allow the rational design of pathway-selective drugs. After all, GPCR signalling is, literally, a complex story.
Nature 558, 529-530 (2018)