Yanting Yin

Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation.

An intrinsic agonist mechanism for activation of glucagon-like peptide-1 receptor by its extracellular domain.

The glucagon-like peptide-1 receptor is a class B G protein coupled receptor (GPCR) that plays key roles in glucose metabolism and is a major therapeutic target for diabetes. The classic two-domain model for class B GPCR activation proposes that the apo-state receptor is auto-inhibited by its extracellular domain, which physically interacts with the transmembrane domain. The binding of the C-terminus of the peptide hormone to the extracellular domain allows the N-terminus of the hormone to insert into the transmembrane domain to induce receptor activation. In contrast to this model, here we demonstrate that glucagon-like peptide-1 receptor can be activated by N-terminally truncated glucagon-like peptide-1 or exendin-4 when fused to the receptor, raising the question regarding the role of N-terminal residues of peptide hormone in glucagon-like peptide-1 receptor activation. Mutations of cysteine 347 to lysine or arginine in intracellular loop 3 transform the receptor into a G protein-biased receptor and allow it to be activated by a nonspecific five-residue linker that is completely devoid of exendin-4 or glucagon-like peptide-1 sequence but still requires the presence of an intact extracellular domain. Moreover, the extracellular domain can activate the receptor in trans in the presence of an intact peptide hormone, and specific mutations in three extracellular loops abolished this extracellular domain trans-activation. Together, our data reveal a dominant role of the extracellular domain in glucagon-like peptide-1 receptor activation and support an intrinsic agonist model of the extracellular domain, in which peptide binding switches the receptor from the auto-inhibited state to the auto-activated state by releasing the intrinsic agonist activity of the extracellular domain.

Molecular assembly of rhodopsin with G protein-coupled receptor kinases

G protein-coupled receptor kinases (GRKs) play pivotal roles in desensitizing GPCR signaling but little is known about how GRKs recognize and phosphorylate GPCRs due to the technical difficulties in detecting the highly dynamic GPCR/GRK interaction. By combining a genetic approach with multiple biochemical assays, we identified the key determinants for the assembly of the prototypical GPCR rhodopsin with its kinase GRK1. Our work reveals that the regulatory G-protein signaling homology (RH) domain of GRKs is the primary binding site to GPCRs and an active conformation of the GRK1 kinase domain is required for efficient interaction with rhodopsin. In addition, we provide a mechanistic solution for the longstanding puzzle about the gain-of-function Q41L mutation in GRK5. This mutation is in the RH domain and increases the capacity of the GRK mutant to interact with and to desensitize GPCRs. Finally we present the principal architecture of a rhodopsin/GRK complex through negative stain electron microscopy reconstruction. Together, these data define the key components for the rhodopsin/GRK1 interaction and provide a framework for understanding GRK-mediated desensitization of GPCRs.

Cryo-EM structure of human rhodopsin bound to an inhibitory G protein

G-protein-coupled receptors comprise the largest family of mammalian transmembrane receptors. They mediate numerous cellular pathways by coupling with downstream signalling transducers, including the hetrotrimeric G proteins Gs (stimulatory) and Gi (inhibitory) and several arrestin proteins. The structural mechanisms that define how G-protein-coupled receptors selectively couple to a specific type of G protein or arrestin remain unknown. Here, using cryo-electron microscopy, we show that the major interactions between activated rhodopsin and Gi are mediated by the C-terminal helix of the Gi α-subunit, which is wedged into the cytoplasmic cavity of the transmembrane helix bundle and directly contacts the amino terminus of helix 8 of rhodopsin. Structural comparisons of inactive, Gi-bound and arrestin-bound forms of rhodopsin with inactive and Gs-bound forms of the β2-adrenergic receptor provide a foundation to understand the unique structural signatures that are associated with the recognition of Gs, Gi and arrestin by activated G-protein-coupled receptors.The cryo-electron microscopy structure of human rhodopsin bound to the inhibitory Gi protein-coupled receptor provides insights into ligand–receptor–G-protein interactions.

Crystal structure of the human 5-HT 1B serotonin receptor bound to an inverse agonist

Abstract5-hydroxytryptamine (5-HT, also known as serotonin) regulates many physiological processes through the 5-HT receptor family. Here we report the crystal structure of 5-HT1B subtype receptor (5-HT1BR) bound to the psychotropic serotonin receptor inverse agonist methiothepin (MT). Crystallization was facilitated by replacing ICL3 with a novel optimized variant of BRIL (OB1) that enhances the formation of intermolecular polar interactions, making OB1 a potential useful tool for structural studies of membrane proteins. Unlike the agonist ergotamine (ERG), MT occupies only the conserved orthosteric binding pocket, explaining the wide spectrum effect of MT on serotonin receptors. Compared with ERG, MT shifts toward TM6 and sterically pushes residues W3276.48, F3306.50 and F3316.51 from inside the orthosteric binding pocket, leading to an outward movement of the extracellular end and a corresponding inward shift of the intracellular end of TM6, a feature shared by other reported inactive G protein-coupled receptor (GPCR) structures. Together with the previous agonist-bound serotonin receptor structures, the inverse agonist-bound 5-HT1BR structure identifies a basis for the ligand-mediated switch of 5-HT1BR activity and provides a structural understanding of the inactivation mechanism of 5-HT1BR and some other class A GPCRs, characterized by ligand-induced outward movement of the extracellular end of TM6 that is coupled with inward movement of the cytoplasmic end of this helix.

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.

The structural basis of the dominant negative phenotype of the Gα i1 β 1 γ 2 G203A/A326S heterotrimer

Aim:Dominant negative mutant G proteins have provided critical insight into the mechanisms of G protein-coupled receptor (GPCR) signaling, but the mechanisms underlying the dominant negative characteristics are not completely understood. The aim of this study was to determine the structure of the dominant negative Gαi1β1γ2 G203A/A326S complex (Gi-DN) and to reveal the structural basis of the mutation-induced phenotype of Gαi1β1γ2.Methods:The three subunits of the Gi-DN complex were co-expressed with a baculovirus expression system. The Gi-DN heterotrimer was purified, and the structure of its complex with GDP was determined through X-ray crystallography.Results:The Gi-DN heterotrimer structure revealed a dual mechanism underlying the dominant negative characteristics. The mutations weakened the hydrogen bonding network between GDP/GTP and the binding pocket residues, and increased the interactions in the Gα-Gβγ interface. Concomitantly, the Gi-DN heterotrimer adopted a conformation, in which the C-terminus of Gαi and the N-termini of both the Gβ and Gγ subunits were more similar to the GPCR-bound state compared with the wild type complex. From these structural observations, two additional mutations (T48F and D272F) were designed that completely abolish the GDP binding of the Gi-DN heterotrimer.Conclusion:Overall, the results suggest that the mutations impede guanine nucleotide binding and Gα-Gβγ protein dissociation and favor the formation of the G protein/GPCR complex, thus blocking signal propagation. In addition, the structure provides a rationale for the design of other mutations that cause dominant negative effects in the G protein, as exemplified by the T48F and D272F mutations.

Publisher Correction: Cryo-EM structure of human rhodopsin bound to an inhibitory G protein

In the PDF version of this Article, owing to a typesetting error, an incorrect figure was used for Extended Data Fig. 5; the correct figure was used in the HTML version. This has been corrected online.