Interaction with heterotrimeric G proteins is a hallmark of G-protein-coupled receptor (GPCR) family members, and it is the key step for a diverse range of cell-signaling cascades. A recent cryo-EM structure of the human calcitonin receptor (CTR) in complex with a G-protein heterotrimer reveals novel insights into receptor–G-protein coupling.
The large family of GPCRs encompasses more than 800 members and is characterized by its agonist-stimulated interaction with heterotrimeric G proteins, comprised of α, β and γ subunits. Coupling and activation of G proteins results in the generation of a range of second messengers, such as cAMP, inositol triphosphate and Ca2+, which in turn initiate a diverse array of signaling cascades1. Although crystal structures of nearly 30 different GPCRs bound to different ligands have been determined2,3, high-resolution structures of GPCR signaling complexes have been limited to the crystal structures of the β2 adrenergic receptor (β2AR)–G-protein complex4 and a rhodopsin–arrestin fusion protein5. Owing to the small size of GPCR signaling complexes (150–200 kDa), direct application of cryo-EM to obtain high-resolution structures has been challenging, although negative-stain-based single-particle EM has provided an overall architecture of the β2AR–G-protein complex6 and a β2AR–β-arrestin1 (βarr1) complex7 at lower resolutions (20–40Å) (Fig. 1). Liang et al. successfully break this resolution barrier and obtain a cryo-EM structure of the human CTR in complex with heterotrimeric G proteins (Gαs, Gβ1 and Gγ2) at <5-Å resolution8 (Fig. 1). This represents a major advance in the area of GPCR structural biology that is likely to open new doors for structural analysis of GPCR signaling complexes.
CTR, a member of the class B GPCR (or secretin-receptor) family, is critical for maintaining calcium homeostasis, and its agonist calcitonin is clinically approved for the treatment of bone disorders, such as osteoporosis9. In order to assemble the CTR–G-protein complex, the authors co-expressed the receptor and the G-protein subunits and then performed agonist stimulation and addition of a nanobody (Nb35) directed against the Gαs–Gβ1 interface to stabilize the complex for subsequent purification8. This approach is very similar to that used recently for isolating an antibody-fragment stabilized β2AR–βarr1 complex7. Purified complex preparations were analyzed by means of cryo-EM using a Volta Phase plate for data collection, a recent development that has dramatically improved the image contrast. This has emerged as a promising tool for analyzing relatively small protein complexes by cryo-EM10,11. Overall, the structure of the CTR–G-protein complex displays an arrangement of the receptor and G-protein subunits similar to that observed in the β2AR–G-protein complex (Fig. 2), although some key differences are evident. The major interaction interface in the CTR–G-protein complex is constituted by the Ras-like domain of Gαs, although Gβ1 also contributes significantly. Similar to that of the β2AR–G-protein complex structure, the alpha-helical (AH) domain of Gαs is highly dynamic in the CTR–G-protein complex and thus cannot be resolved well. On the intracellular side, the cytoplasmic end of transmembrane domain 6 in CTR reveals a significant outward movement compared to the structural model of the inactive state of CTR. This feature is consistent with the active state of the receptor in this complex. Significant structural rearrangements are also observed in the ligand-binding pocket of CTR, which harbors calcitonin, a 32-amino-acid long peptide agonist; however, the extracellular end of transmembrane helices and the loops display structural flexibility.
An interesting feature observed in the CTR–G-protein complex is the extended helix 8 in the receptor, which constitutes an interface for the interaction with the Gβ1 subunit (Fig. 3). Interestingly, CTR mutants containing a truncated helix 8 display significantly reduced levels of agonist-induced cAMP generation, which also indicates a potential role of helix 8 in G-protein coupling. In contrast, helix 8 of β2AR in the β2AR–G-protein crystal structure is shorter and pointed away from the Gβ1 subunit (Fig. 3). Thus, in the β2AR–G-protein crystal structure, the Gβ subunit is not directly involved in the interaction interface with the receptor4. Whether this interaction of helix 8 with the Gβ subunit is conserved for other class B GPCRs and whether it represents a key difference between the G-protein coupling of class A and B receptors remains to be experimentally evaluated. It is worth noting that simultaneous binding of G proteins (or a Gαs mimetic nanobody) and βarrs, a regulator of GPCR signaling, to selected class A GPCRs has been documented recently12,13. Such complexes are referred to as super complexes, and they are proposed to be responsible for sustained cAMP generation from endosomal compartments. In such super complexes, the Gβγ subunits remain bound to the receptor–βarr complex even after dissociation of the Gαs subunit12. It would be interesting to explore whether class B GPCRs can also form such super complexes, and if they do, whether this interaction of helix8–Gβ1 might help tether the complex. Interestingly, the parathyroid hormone receptor, another class B GPCR, not only exhibits sustained cAMP response, even after internalization, but also forms a receptor–Gβγ–βarr complex14.
Since the first crystal structure of the β2AR was determined in 2007, close to 100 crystal structures of nearly 30 different GPCRs have been solved. Consistent with the conserved architecture, signaling and regulatory mechanisms of GPCRs, many of the methodological breakthroughs pioneered with β2AR (such as fusion-protein and nanobody approaches) have been by and large directly transferrable to other GPCRs2,15. Although stable assembly and structural characterization of GPCR signaling complexes present very different challenges compared to the characterization of isolated receptors, the current cryo-EM breakthrough with the CTR–G-protein complex promises exciting prospects over the next few years. A major challenge that still remains is to capture distinct conformations of receptor–G-protein complexes during various steps of nucleotide exchange. It is also interesting that some GPCRs have strict preferences for specific Gα subtypes, whereas others can couple to two or even more subtypes of Gα. Investigating the structural basis for such preference and promiscuity of G-protein coupling depending on the context (for example, cell or ligand dependent) is likely to be a challenging but rewarding avenue to explore in order to better understand the activation and signaling of GPCRs.
What are the next structural revelations in the field of GPCRs? The structure of a GPCR–GRK (GPCR kinase) complex? The structure of a receptor–transducer–effector complex? Or the structure of a dimeric class C GPCR? Whichever of these it may be, it is likely to take us a step closer to a better understanding of GPCR signaling and regulatory paradigms with direct implications for novel drug discovery. So, stay tuned!
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The research program in A.K.S.'s laboratory is supported by the Wellcome Trust DBT India Alliance (IA/I/14/1/501285), the Department of Biotechnology (DBT), the Department of Science and Technology (DST), the Council for Scientific and Industrial Research (CSIR) and the Institute of Technology, Kanpur.
The authors declare no competing financial interests.
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Baidya, M., Dwivedi, H. & Shukla, A. Frozen in action: cryo-EM structure of a GPCR–G-protein complex. Nat Struct Mol Biol 24, 500–502 (2017). https://doi.org/10.1038/nsmb.3418
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