Gpcr là gì
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The N termini of some peptides that regulate signal transduction, for example, GLP-1 and GLP-2, show different orientations when interacting with their respective receptors, and TIP39 adopts a unique loop conformation at its N-terminus upon binding to PTH2R. The extracellular domain (ECD) of some class B1
receptors is directly involved in signaling. For example, the N-terminus of GLP‐1R folds down towards the TMD to cover the ligand-binding pocket when bound by non-peptidic agonists. The ECD N-terminal α-helix of VIP2R inserts to the cleft between PACAP27 and ECL1 to stabilize peptide binding. The outward movement of TM6 upon receptor activation could be significantly affected by small molecules acting as allosteric modulators, including an
ago-allosteric modulator (ago-PAM) and several negative allosteric modulators (NAMs). There are two forms of signaling bias – ligand-dependent and constitutive – that are mediated by splice variants of the wild-type receptor, for example, SV1 of GHRHR. Class B1 G protein-coupled receptors (GPCRs) play important roles in human physiology and disease pathology. Using cryo-electron microscopy (cryo-EM) and X-ray crystallography, the 3D structures of all 15 members of this receptor subfamily have been determined in recent years at the near-atomic level. Although they share many structural commonalities, they show distinct features in terms of ligand recognition and receptor activation. In-depth structural analyses have yielded valuable insights into the N termini of both peptide hormones and cognate receptors, the outward movement of transmembrane helix 6 (TM6), the allosteric modulation sites located in the transmembrane domain (TMD), and the constitutive signaling bias mediated by receptor splice variants. These provide new directions for the design of better therapeutic agents, thereby making these targets more druggable. Rapid advancesG protein-coupled receptors (GPCRs, see Glossary) are the most abundant membrane proteins involved in numerous physiological functions [ 1.
How ligands illuminate GPCR molecular pharmacology.
]. One GPCR class, the B1 or secretin family, comprises 15 receptors for peptide hormones that regulate a variety of biological processes ranging from growth and development to metabolism and neuroactivities, thereby making them important therapeutic targets for many diseases [ 2.
G protein-coupled receptors: structure- and function-based drug discovery.
]. Several drugs against this class have been successfully launched to the market, and glucagon-like peptide 1 (GLP-1) and parathyroid hormone (PTH) receptor agonists have reached ‘blockbuster’ status [ 3.
Insights into incretin-based therapies for treatment of diabetic dyslipidemia.
, 4.
International Union of Basic and Clinical Pharmacology. XCIII. The parathyroid hormone receptors – family B G protein-coupled receptors.
]. In earlier years X-ray crystallography and nuclear magnetic resonance (NMR) structural studies on class B1 GPCRs mainly focused on the apo state and ligand–receptor complex structures (Table S1 in the supplemental material online), providing substantial information about the conformations of these receptor extracellular domains (ECDs) and transmembrane domains (TMDs), as well as about the structural basis of ligand recognition. To deepen our knowledge on the structure–activity relationship of these ligand–receptor pairs, the first two full-length structures were determined using single-particle cryo-electron microscopy (cryo-EM) in 2017 [ 5.
Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein.
, 6.
Phase-plate cryo-EM structure of a class B GPCR–G-protein complex.
]. Since then the cryo-EM structures for all 15 members of class B1 GPCRs have been solved (Figure 1), not only giving valuable information about hormone recognition and receptor activation from a class-wide perspective but also providing a useful template for the design and development of better peptidic and/or small-molecule drugs [ 7.
Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor–Gs complex.
, 8.
Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor.
, 9.
Structure and dynamics of the active human parathyroid hormone receptor-1.
, 10.
Molecular basis for hormone recognition and activation of corticotropin-releasing factor receptors.
, 11.
Toward a structural understanding of class B GPCR peptide binding and activation.
, 12.
Cryo-EM structure of an activated VIP1 receptor–G protein complex revealed by a NanoBiT tethering strategy.
, 13.
Structural basis for activation of the growth hormone-releasing hormone receptor.
, 14.
A unique hormonal recognition feature of the human glucagon-like peptide-2 receptor.
, 15.
Structural insights into hormone recognition by the human glucose-dependent insulinotropic polypeptide receptor.
, 16.
Molecular insights into differentiated ligand recognition of the human parathyroid hormone receptor 2.
]. Figure 1A family portrait of the 3D structures of class B1 G protein-coupled receptors (GPCRs). Show full caption The structures of all 15 members, divided into five subfamilies, have been determined at the near-atomic level by cryo-electron microscopy (cryo-EM) and X-ray crystallography. Representative structures of each receptor are shown as cartoons. A complete list of class B1 GPCR structures is presented in Table S1. The location of the orthosteric binding pocket of class B1 GPCRs is indicated by a red broken oval box, as exemplified by GLP-1R. Abbreviations: CTR, calcitonin receptor; CGRPR, calcitonin gene-related peptide receptor; CRF1R and CRF2R, corticotropin-releasing factor 1 and 2 receptors; GCGR, glucagon receptor; GHRHR, growth hormone-releasing hormone receptor; GIPR, glucose-dependent insulinotropic polypeptide receptor; GLP‐1R and GLP‐2R, glucagon-like peptide 1 and 2 receptors; PAC1R, pituitary adenylate cyclase-activating polypeptide (PACAP) 1 receptor; PTH1R and PTH2R, parathyroid hormone receptors 1 and 2; SCTR, secretin receptor; VIP1R and VIP2R, vasoactive intestinal peptide (VIP) 1 and 2 receptors.
Overall structureGPCRs can be divided into five families: class A (rhodopsin), class B1 (secretin), class B2 (adhesion), class C (glutamate), and class F (frizzled/smoothened) based on sequence homologies [ 17.
The concise guide to pharmacology 2019/20: G protein-coupled receptors.
]. They differ in their ECD architecture and follow a different activation mechanism. Orthosteric ligands of class A GPCRs primarily bind within the extracellular half of the TMD, whereas class B1 receptors have a large ECD of 120–160 residues that is crucial for peptide binding and receptor activation [ ]. It is generally recognized that peptide ligands operate through a two-domain binding mechanism in which the peptide C terminus initially interacts with the ECD and the N terminus then binds to the orthosteric pocket within the TMD to activate the receptor [ 19.
Structure and mechanism for recognition of peptide hormones by class B G-protein-coupled receptors.
]. Class B1 receptors couple to a variety of G proteins, including Gi/o and G11/q, but predominantly signal via Gs to modulate intracellular cAMP levels. All the 15 class B1 receptor structures determined by cryo-EM to date are agonist-bound, Gs-coupled, and are stabilized by Nb35, dominant negative Gs protein, or NanoBiT tethering technology, except for the glucagon receptor (GCGR)–Gi complex [ 12.
Cryo-EM structure of an activated VIP1 receptor–G protein complex revealed by a NanoBiT tethering strategy.
, 20.
Structural basis of Gs and Gi recognition by the human glucagon receptor.
, 21.
Dominant negative G proteins enhance formation and purification of agonist–GPCR–G protein complexes for structure determination.
]. Compared to the inactive full-length GLP-1 receptor (GLP‐1R) structure [ 22.
Full-length human GLP-1 receptor structure without orthosteric ligands.
], these active-state structures revealed common conformational changes upon receptor activation. The key features include outward movement at the top of transmembrane helix 6 (TM6)/extracellular loop 3 (ECL3)/TM7 and inward movement of TM1, forming a ‘V-shaped’ pocket to accommodate the peptide ligand at the extracellular side. At the intracellular side, an outward shift of TM6 is facilitated by a sharp kink in the middle at the P6.47xxG6.50 motif, thus opening the base of the receptor to allow G protein coupling, where the α5 helix of Gs forms a highly conserved interaction with TM2, TM3, and intracellular loop 2 (ICL2), indicative of a common mechanism in Gs coupling (Figure 2A ). Figure 2The overall conformational states of class B1 G protein-coupled receptors (GPCRs). Show full caption (A) Conformational transition from inactive to active states. Superimposition of the inactive GLP‐1R structure (6LN2) in grey with the GLP‐1R in complex with Gs bound to GLP-1 (6X18) in blue. (Left) Side view of the TMD). (Middle) The TMD viewed from the extracellular face. (Right) The TMD viewed from the intracellular face. (B) Comparison of ECD conformations in the active GLP‐1R structures bound by peptidic, non-peptidic, and allosteric ligands. (C) Class B1 GPCRs display similar active state conformations. Superimposition of one receptor from each of the subfamily: GLP‐1R (6X18) in green, CGRPR (6E3Y) in blue, PAC1R (6M1I) in grey, PTH1R (6NBF) in pink, and CRF2R (6PB1) in yellow. Peptide and G protein were omitted for clarity. ECD, ECL1, and ECL3 give close-ups of the ECD showing its position relative to the TMD, ECL1 viewed from the side, and ECL3 viewed from the top, respectively. Abbreviations: ECD, extracellular domain; ECL, extracellular loop; TM, transmembrane helix; TMD, transmembrane domain.
However, the ECD region in all the cryo-EM maps is poorly resolved, reflecting greater flexibility in this region. The 3D structures of the calcitonin (CT) receptor (CTR), parathyroid hormone receptor 1 (PTH1R), and secretin receptor (SCTR) revealed multiple ECD conformations, and molecular dynamics (MD) simulations on these structures showed a high degree of ECD mobility which may be required for initial binding or partial disengagement of the peptide from the receptor [ 6.
Phase-plate cryo-EM structure of a class B GPCR–G-protein complex.
, 9.
Structure and dynamics of the active human parathyroid hormone receptor-1.
, 23.
Structure and dynamics of the active Gs-coupled human secretin receptor.
]. At a global level, the ECDs of GLP-1R, GCGR, gastric inhibitory polypeptide or glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR), SCTR, PTH1R, and parathyroid hormone receptor 2 (PTH2R) adopt similar vertical conformations with subtle differences in the positions of the N terminus α-helix relative to the TMD [ 9.
Structure and dynamics of the active human parathyroid hormone receptor-1.
, 15.
Structural insights into hormone recognition by the human glucose-dependent insulinotropic polypeptide receptor.
, 16.
Molecular insights into differentiated ligand recognition of the human parathyroid hormone receptor 2.
, 20.
Structural basis of Gs and Gi recognition by the human glucagon receptor.
, 23.
Structure and dynamics of the active Gs-coupled human secretin receptor.
, 24.
Differential GLP-1R binding and activation by peptide and non-peptide agonists.
] (Figures 2B,C). By contrast, the ECDs of the CTR family are tilted because of the unstructured C terminus of the peptide ligand, resulting in a distinct orientation [ 6.
Phase-plate cryo-EM structure of a class B GPCR–G-protein complex.
, 8.
Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor.
], whereas all the other class B1 peptides adopt a continuous α-helix. This may be a feature of an individual receptor that is not of direct relevance to druggability. The conformation of receptor ECD is agonist-dependent, as exhibited by different ECD orientations observed in the active GLP‐1R structures bound by peptidic, non-peptidic, and allosteric ligands [ 24.
Differential GLP-1R binding and activation by peptide and non-peptide agonists.
, 25.
Activation of the GLP-1 receptor by a non-peptidic agonist.
, 26.
Molecular insights into ago-allosteric modulation of the human glucagon-like peptide-1 receptor.
]. The peptide-bound GLP‐1R adopts a vertical ECD conformation that allows the ECD to move further downwards to interact with the TMD upon activation by non-peptidic agonists bound to the TMD, whereas the ECD N terminus penetrates into the TMD when activated by an ago-allosteric modulator (ago-PAM) interacting with a site far away from the orthosteric pocket (Figure 2B). As a comparison, the ECD of apo-GLP‐1R adopted a closed TMD-interacting conformation, which highlights an essential role of ECD movement during receptor activation [ 22.
Full-length human GLP-1 receptor structure without orthosteric ligands.
] (Figure 2B). Clearly, the TMD is a key region that attracts much attention: in addition to the orthosteric binding pocket often used for ligand design, TMs (TM6 in particular) could be targeted by allosteric modulators including positive allosteric modulators (PAMs), negative allosteric modulators (NAMs), and ago-PAMs. The ECL1 in most active-state structures adopts an α-helical conformation, but with several exceptions. It partially unwinds and demonstrates an elevated conformation in the glucagon-like peptide 2 receptor (GLP-2R) active structure [ 14.
A unique hormonal recognition feature of the human glucagon-like peptide-2 receptor.
]. A similar feature was also observed for vasoactive intestinal polypeptide (VIP) 2 receptor (VIP2R) structure bound to pituitary adenylate cyclase activating polypeptide 27 (PACAP27) [ 27.
A distinctive ligand recognition mechanism by the human vasoactive intestinal polypeptide receptor 2.
], whereas the ECL1 in growth hormone releasing hormone (GHRH) receptor (GHRHR) stretches towards the peptide to make broad contacts with the middle region of GHRH [ 13.
Structural basis for activation of the growth hormone-releasing hormone receptor.
] (Figure 2C). Among the class B1 receptors, conformational changes in TM6/ECL3/TM7 are most variable upon activation. The CT and corticotropin releasing factor (CRF) families have a common fold in the N terminus, where CT has a conserved cysteine-mediated disulphide bridge and CRFs have a similar loop. The ECL3 conformations of active-state CRF and CT receptors are most similar owing to a comparable peptide binding position within the TM core, and TM6/ECL3/TM7 therefore adopts a conformation further away to form a larger peptide-binding pocket [ 6.
Phase-plate cryo-EM structure of a class B GPCR–G-protein complex.
, 10.
Molecular basis for hormone recognition and activation of corticotropin-releasing factor receptors.
, 11.
Toward a structural understanding of class B GPCR peptide binding and activation.
, 21.
Dominant negative G proteins enhance formation and purification of agonist–GPCR–G protein complexes for structure determination.
] (Figure 2C). This probably contributes to the difference observed between PTH1R and PTH2R bound to long acting-PTH (LA-PTH) and tuberoinfundibular peptide 39 (TIP39), respectively. TIP39 has a similar N-terminal loop, which is not present in LA-PTH, thereby TM7 in PTH2R adopts a more outward conformation to accommodate the bulky N-terminal peptide [ 9.
Structure and dynamics of the active human parathyroid hormone receptor-1.
, 16.
Molecular insights into differentiated ligand recognition of the human parathyroid hormone receptor 2.
]. From a ligand perspective, peptide residue composition and length could be manipulated to change the pharmacological property of a peptide from agonist to antagonist or vice versa. Collectively, the ECD and ECLs of class B1 receptors demonstrate the ability and diversity to regulate the binding of different peptides. Subfamily featuresClass B1 GPCRs are divided into five subfamilies: glucagon-like, PACAP/VIP-like, PTH-like, CRF-like, and CT-like (Table S1 and Figure S1). Glucagon-like subfamilyThe structural features of this subfamily include: (i) ligands are tightly surrounded by the N-terminal domain and TMD core; (ii) ECLs are involved in stabilizing ligand binding; and (iii) similar conformational changes take place in the TMD upon activation. PTH-like subfamilyPTH, PTH-related protein (PTHrP), and TIP39 are natural ligands for PTH receptors. Similarly to many class B1 GPCR ligands, most parts of LA-PTH (a PTH/PTHrP chimera) are helical [ 9.
Structure and dynamics of the active human parathyroid hormone receptor-1.
], with its N terminus inserting deeply into the TMD core, while the ECD of PTH1R adopts three distinct conformations and an ordered annular lipid belt surrounding the TMD [ 9.
Structure and dynamics of the active human parathyroid hormone receptor-1.
]. In the TIP39-bound PTH2R–Gs structure (7F16), TIP39 shows a unique loop conformation at the N terminus indispensable for PTH2R activation [
16.
Molecular insights into differentiated ligand recognition of the human parathyroid hormone receptor 2.
]. This demonstrates that the N terminus of a peptide could be modified to drive pharmacological action to a desired direction. PACAP/VIP subfamilyPACAP and VIP are shared ligands of pituitary adenylate cyclase activating polypeptide 1 receptor (PAC1R), VIP1R, and VIP2R [ 28.
Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery.
]. PACAP (PACAP38 and PACAP27) and VIP have comparable affinity for VIP1R and VIP2R, but PACAP displays a higher selectivity for PAC1R than for VIP [ ]. PACAP27 adopts an α-helical conformation, engages a V-shaped binding pocket, and interacts with each of the TM helices except TM4. In the PACPA38-bound PAC1R–Gs structure (6M1I) the ECL1 protrudes to anchor the ligand bound in the orthosteric site. A unique conformation was found in the PACAP27–VIP2R–Gs complex where the N-terminal α-helix deeply inserts into a cleft between PACAP27 and the ECL1, thereby stabilizing the peptide‐receptor interface [ 27.
A distinctive ligand recognition mechanism by the human vasoactive intestinal polypeptide receptor 2.
] and possibly offering a new site for therapeutic development. CRF-like subfamilyCRF1R and CRF2R are activated by CRF and urocortins 1–3 (UCN1–3). Whereas UCN1 binds to both receptors, CRF is specific for CRF1R, and UCN2 and UCN3 are selective for CRF2R. The ECD of CRF1R lacks the N-terminal helix seen in most other class B1 structures [ 11.
Toward a structural understanding of class B GPCR peptide binding and activation.
]. Seven residues in the N terminus of UCN1 are folded to form a ring, followed by a 33‐amino acid vertical insertion into the TMD as a long α-helix. Many ordered lipid and cholesterol molecules were found around the TM regions of the UCN1–CRF1R/CRF2R–Gs complexes. Also observed in Gαs and Gβγ subunits, lipid modifications at different sites have been reported to affect receptor signaling, subcellular localization, and trafficking [ 30.
Post-translational modifications of G protein-coupled receptors control cellular signaling dynamics in space and time.
] that may have potential implications for drug discovery. CT-like subfamilyCTR and calcitonin receptor-like receptor (CLR) interact with single-span TM proteins (receptor activity-modifying proteins, RAMP1–3) to regulate their ligand-binding specificity [ 31.
Molecular mechanisms of class B GPCR activation: insights from adrenomedullin receptors.
]. The N terminus of CTR (5UZ7) is very flexible and the bound CT has a secondary N-terminal structure [ 6.
Phase-plate cryo-EM structure of a class B GPCR–G-protein complex.
]. The calcitonin gene-related peptide (CGRP) receptor (CGRPR) is a heterodimer of CLR and RAMP1, and its full-length structure shows that the RAMP TMD sits at the interface between TM3, TM4, and TM5 of CLR, and stabilizes ECL2 of CLR. RAMP1 makes only limited direct interaction with CGRP, consistent with its key function of allosteric modulation at CLR [ 8.
Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor.
]. The extent to which RAMPs modify the actions of some B1 receptors was comprehensively revealed by a recent study [ 32.
Modulating effects of RAMPs on signaling profiles of the glucagon receptor family.
], thus offering a new dimension for rational drug design. Ligand recognitionTo date, 27 peptidic and 10 non-peptidic ligands in complex with at least one class B1 receptor were structurally determined, and GLP‐1R is the most extensively studied (Figure 3).  Figure 3Ligand recognition in class B1 G protein-coupled receptors (GPCRs). Show full caption (A) Schematic of ligand-binding sites shown as ovals, colored according to the location. (B) Family-wide comparison of the peptide-binding conformations in class B1 GPCRs: glucagon subfamily (top left), PACAP/VIP subfamily (top right), PTH subfamily (middle), CRF subfamily (bottom left), and CT subfamily (bottom right). The names of the peptide-bound receptors are shown in parentheses. (C) Binding of non-peptidic ligands in the extracellular half of class B1 GPCRs. The receptor ECD is omitted for clarity. (D) Binding of non-peptidic ligands in the intracellular half of class B1 GPCRs. G protein and fusion protein are omitted for clarity. (E) Surface representations of the peptide-binding pockets of all class B1 GPCRs, with the peptides shown as ribbons. The receptors are colored from Dodger blue for the most hydrophilic regions through white to orange-red for the most hydrophobic regions. Abbreviations: ECD, extracellular domain; NAM, negative allosteric modulator; PAM, positive allosteric modulator; TMD, transmembrane domain.
Figure 3B illustrates that the N-terminal regions of bound peptides generally occupy the orthosteric pockets via extensive interactions with the TMD core, while their C-terminal regions display receptor- and peptide-specific conformations [ 15.
Structural insights into hormone recognition by the human glucose-dependent insulinotropic polypeptide receptor.
]. In the glucagon subfamily, both endogenous hormones (GLP-1, GLP-2, glucagon, GIP, GHRH, and secretin) and peptidic analogs (NNC1702, exendin-P5, peptide 15, peptide 19, ZP3780, tirzepatide, non-acylated tirzepatide, peptide 20, semaglutide, and taspoglutide) all adopt a single straight helix with their N termini inserting into the TMD core and forming both polar and non-polar interactions, while the C termini are recognized by the ECD and ECL1. These peptides use similar residues at different sites in the N-terminal region, including D/E3P (P indicates that the residue belongs to the peptide), F6P, D/E9P, and S11P, that contribute multiple polar interactions with conserved residues such as Y1.43b (class B GPCR numbering in superscript), Y1.47b, R2.60b, and E/T45.52b of the receptor. Notably, ECL1 and the extracellular tip of TM1 have diverse conformations which play an important role in peptide specificity by engaging with the peptide C-terminal region [ 14.
A unique hormonal recognition feature of the human glucagon-like peptide-2 receptor.
]. In the PTH subfamily, two peptides (LA-PTH [ 9.
Structure and dynamics of the active human parathyroid hormone receptor-1.
] and ePTH [ 33.
High-resolution crystal structure of parathyroid hormone 1 receptor in complex with a peptide agonist.
]) exhibit an extended helix with their N termini inserting deeply into the TMD, whereas TIP39 in PTH2R adopts a closed loop for the first four residues at the N terminus and a single amphipathic α-helix for the remaining residues [ 16.
Molecular insights into differentiated ligand recognition of the human parathyroid hormone receptor 2.
]. Such a unique structural feature of TIP39 bound by PTH2R is consistent with TIP39 being selective for PTH2R rather than for PTH1R. In the CRF subfamily, both UCN1 and CRF adopt a similar loop-binding conformation as TIP39 [ 10.
Molecular basis for hormone recognition and activation of corticotropin-releasing factor receptors.
, 11.
Toward a structural understanding of class B GPCR peptide binding and activation.
]. In the PACAP/VIP subfamily, PACAP27 and PACAP38 overlap well and penetrate into the TMD by an almost identical angle and orientation through the N-terminal halves [ 11.
Toward a structural understanding of class B GPCR peptide binding and activation.
, 12.
Cryo-EM structure of an activated VIP1 receptor–G protein complex revealed by a NanoBiT tethering strategy.
, 27.
A distinctive ligand recognition mechanism by the human vasoactive intestinal polypeptide receptor 2.
, 34.
Cryo-EM structure of the human PAC1 receptor coupled to an engineered heterotrimeric G protein.
, 35.
Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism.
], whereas that of the peptide C termini have different orientations and positions resulting from distinctive conformations of the ECD, ECL1, and the extracellular tip of TM1. In the CT subfamily, both CT and CGRP share a large kink that structurally divides the peptide into two parts for recognition by the ECD and TMD, respectively [ 6.
Phase-plate cryo-EM structure of a class B GPCR–G-protein complex.
, 8.
Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor.
, 36.
Structure and dynamics of the CGRP receptor in apo and peptide-bound forms.
, 37.
The molecular control of calcitonin receptor signaling.
]. Unlike the N terminus that only affects the interaction with TMD, the peptide C terminus can influence its interaction with both ECD and TMD. Different from the two-domain model for peptide binding, small-molecule ligands have greatly reduced contacts with the receptor. As shown in Figure 3C, five non-peptidic GLP‐1R modulators were structurally observed either in the upper half of the orthosteric pocket (TT-OAD2, LY3502970, PF-06882961, and RGT1381) [ 24.
Differential GLP-1R binding and activation by peptide and non-peptide agonists.
, 25.
Activation of the GLP-1 receptor by a non-peptidic agonist.
, 38.
Structural insights into the activation of GLP-1R by a small molecule agonist.
, 39.
Structural basis for GLP-1 receptor activation by LY3502970, an orally active nonpeptide agonist.
] or at the TM1–TM2 interface (LSN3160440) [ 40.
Structural insights into probe-dependent positive allosterism of the GLP-1 receptor.
]. Distant from the bottom of orthosteric pocket touched by the N terminus (H7P) of GLP-1, four non-peptidic agonists overlapped with the position occupied by residues 10–20 of GLP-1, thus contributing a limited TMD-interacting surface area (~1000 Å2) relative to that of peptides (>2000 Å2) [ 26.
Molecular insights into ago-allosteric modulation of the human glucagon-like peptide-1 receptor.
]. As far as the intracellular half is concerned (Figure 3D), two antagonists (CP-376395 and MK-0893) [ 41.
Extra-helical binding site of a glucagon receptor antagonist.
, 42.
Structure of class B GPCR corticotropin-releasing factor receptor 1.
], two negative allosteric modulators (PF-0637222 and NNC0640) [ 43.
Human GLP-1 receptor transmembrane domain structure in complex with allosteric modulators.
], and one ago-allosteric modulator (compound 2) [ 26.
Molecular insights into ago-allosteric modulation of the human glucagon-like peptide-1 receptor.
] were structurally identified. CP-376395 binds deeply inside the helical bundle and is surrounded by TMs 3, 5, and 6, ~18 Å away from the orthosteric pocket. Compared to the fully active CRF1R structure [ 10.
Molecular basis for hormone recognition and activation of corticotropin-releasing factor receptors.
, 11.
Toward a structural understanding of class B GPCR peptide binding and activation.
], the binding of CP-376395 blocked the sharp kink of TM6 and its subsequent outward movement, thereby acting as an antagonist. MK-0893, PF-0637222, and NNC0640 locate outside the helical bundle of TMs 5–7 of GLP-1R [ 43.
Human GLP-1 receptor transmembrane domain structure in complex with allosteric modulators.
] and GCGR [ 41.
Extra-helical binding site of a glucagon receptor antagonist.
], and are stabilized by multiple hydrogen bonds in a polar cleft between TM6 and TM7, as well as by massive hydrophobic contacts with TM5 and TM6. Consequently, the outward movement of TM6 is hampered. Compound 2, mounted on the lipid-facing surface of the cytoplasmic end of TM6, forms predominantly hydrophobic interactions with the surrounding residues in TM6 such as K346, A350, and K351. Interestingly, the ECD of compound 2-bound GLP‐1R folds down towards the TMD and penetrates into the orthosteric pocket through its N-terminal α-helix to stabilize the active conformation. Obviously, the modulating mechanisms of class B1 receptor-mediated signal transduction by peptidic and non-peptidic ligands are diverse and complex (Figure 3B–E). Identification of uncovered molecular recognition patterns and novel allosteric binding sites would certainly shed light on the design of better therapeutics. G protein couplingClass B1 GPCRs all form a cytoplasmic cavity by three ICLs and the outward movement of TMD involving TMs 2, 3, 5, 6, and 7, allowing anchoring of the α5 helix of Gα. The interface residues are highly conserved and share many charged and hydrophobic interactions, suggesting a common mechanism of G protein engagement (Figure 4A ) [ 15.
Structural insights into hormone recognition by the human glucose-dependent insulinotropic polypeptide receptor.
]. Compared to the class A GPCRs, a strong similarity in Gαs coupling was observed, despite the significant diversity among the amino acid sequences of these receptors [ 5.
Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein.
, 44.
Cryo-EM structures of GPCRs coupled to Gs, Gi and Go.
]. A key difference between the two classes is that class B1 receptors form additional hydrophilic interactions between H8 and Gβ protein (Figure 4A) [ 6.
Phase-plate cryo-EM structure of a class B GPCR–G-protein complex.
]. The hydrogen bond formed by class A receptors and Y391GαH5 was not observed in class B1 GPCRs [ 14.
A unique hormonal recognition feature of the human glucagon-like peptide-2 receptor.
, 45.
Structural insights into the process of GPCR–G protein complex formation.
]. In the GLP-2R–Gαs complex, Y391GαH5 made a hydrogen bond with a water molecule connecting E2813.50b and H2142.50b, a unique phenomenon among class B1 GPCRs [ 14.
A unique hormonal recognition feature of the human glucagon-like peptide-2 receptor.
]. G proteins interact with ICL2 resulting in different contact strengths and locations of the αN helix at the N terminus of Gα. Residue variation in ICL2 that inserts to the cavity formed by the αN α5 helices of Gα leads to different conformations of the base of TM3 (Figures 4B,C) [ 11.
Toward a structural understanding of class B GPCR peptide binding and activation.
]. Comparison of the Gαs- and Gαi-coupled GCGR structures suggests that ICLs have crucial roles in G protein engagement and specificity (Figure 4D) [ 20.
Structural basis of Gs and Gi recognition by the human glucagon receptor.
]. Figure 4Engagement of G proteins by class B1 G protein-coupled receptors (GPCRs). Show full caption (A) General model of the interaction between class B1 GPCRs and Gαs protein (left panel) and conformation comparison between class B1 and β2-adrenergic receptors (β2ARs; right panel). (B) ICL2, TM3, and TM4 sequence alignments of class B1 receptors. (C) Residues that lead to a different conformation at the base of TM3. (D) ICL2 in G protein engagement and specificity. Abbreviations: α, α-helix; H, helix; ICL, intracellular loop; TM, transmembrane helix.
Signal modulationRAMPs are a family of single transmembrane proteins represented by three subtypes (RAMP1, 2, and 3) and can modulate GPCR function in several ways [ 46.
Receptor activity-modifying proteins (RAMPs): new insights and roles.
]. The simplest way is to act as molecular chaperones for class A, B, and C GPCRs [ 47.
]. The functions of RAMPs bound to individual class B1 GPCRs have been reported, covering CLR, CTR, VIP2R, PTH1R, PTH2R, GHRHR, CRF1R, SCTR, GCGR, GIPR, GLP‐1R and GLP‐2R with all three RAMPs [ 32.
Modulating effects of RAMPs on signaling profiles of the glucagon receptor family.
, 48.
RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor.
, 49.
Multiplexed analysis of the secretin-like GPCR–RAMP interactome.
], VIP1R with RAMP2 and RAMP3 [ 49.
Multiplexed analysis of the secretin-like GPCR–RAMP interactome.
], and CRF2R with RAMP3 [ 49.
Multiplexed analysis of the secretin-like GPCR–RAMP interactome.
]. Characterization of these interactions has uncovered diverse functions of RAMPs, including alteration of ligand specificity (Figure 5) exemplified by CT and CLR [ 46.
Receptor activity-modifying proteins (RAMPs): new insights and roles.
]. RAMPs were also shown to alter receptor trafficking, ligand potency, and downstream signaling (Figure 5), as demonstrated for GLP‐1R and GCGR [ 32.
Modulating effects of RAMPs on signaling profiles of the glucagon receptor family.
]. Figure 5Ligand-dependent and constitutive signaling modulation of class B1 G protein-coupled receptors (GPCRs). Show full caption (A) Classic signaling transduction mediated by various ligands, including biased agonists, partial agonists, positive allosteric modulators, negative allosteric modulators, and antagonists; (B) Three constitutive signaling modulation mechanisms reported for class B1 receptors, including splice variants, receptor dimerization, and RAMPs. Signaling modulation is diagrammatically shown in five modes, including: ① a downstream signaling transducer is selectively activated, resulting in varying degrees of biased signaling towards G proteins or β-arrestins; ② the activation potency of a G protein or β-arrestin signaling pathway is improved; ③ the activation potency of a G protein or β-arrestin signaling pathway is reduced; ④ all signaling pathways are suppressed; or ⑤ no downstream signaling effector is activated. Abbreviations: ECL, extracellular loop; ICL, intracellular loop; RAMP, receptor activity-modifying protein.
In addition to heterodimerization with RAMPs, it was reported that some class B1 receptors also form homo- and heterodimers resulting in differentiated effects on signaling (Figure 5). SCTR has been shown to interact with VIP1 or 2 receptors, causing intracellular trapping of the hetero-oligomers and providing a mechanism for dominant negative inhibition of VIP action by SCTR in cells expressing both receptors [ 50.
Constitutive formation of oligomeric complexes between family B G protein-coupled vasoactive intestinal polypeptide and secretin receptors.
]. Receptors that form functional dimers are also thought to modulate each other allosterically, creating a new receptor type with unique pharmacological properties [ 51.
Allostery at G protein-coupled receptor homo- and heteromers: uncharted pharmacological landscapes.
], exemplified by the GLP‐1R–GIPR heterodimer [ 52.
Lateral allosterism in the glucagon receptor family: glucagon-like peptide 1 induces G-protein-coupled receptor heteromer formation.
]. It follows that homodimerization might present a potential mechanism of signaling bias. For instance, disruption of SCTR dimerization was found to impair cAMP responses [ 53.
Transmembrane segment IV contributes a functionally important interface for oligomerization of the class II G protein-coupled secretin receptor.
], whereas disruption of GLP‐1R led to complete loss of intracellular calcium mobilization [ 54.
Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery.
], indicative of biased signaling profiles. Molecules that specifically target either the homodimer or heterodimer are in high demand to better understand the pharmacological significance of such processes. Diversity in cell signaling options caused by receptor dimerization could be further enhanced by the generation of GPCR splice variants (SVs) [ 55.
Alternative mRNA splicing of G protein-coupled receptors.
]. Approximately 50% of GPCRs have more than one exon and thus have a possibility to undergo alternative splicing [ 55.
Alternative mRNA splicing of G protein-coupled receptors.
]. Such splice variants differ in their sequences and therefore in ligand binding, surface expression, receptor trafficking, and signal transduction [ 56.
Alternative splicing of intrinsically disordered regions and rewiring of protein interactions.
]. Alternative splicing in class B1 GPCRs [ 57.
Intrinsically disordered proteins link alternative splicing and post-translational modifications to complex cell signaling and regulation.
] could lead to the expression of isoforms with (i) alternative N termini, as demonstrated for GHRHR [ 58.
Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers.
], CRF1R [ 59.
A variant of the human corticotropin-releasing factor (CRF) receptor: cloning, expression and pharmacology.
], CRF2R [ 60.
Molecular identification and analysis of a novel human corticotropin-releasing factor (CRF) receptor: the CRF2gamma receptor.
], PAC1R [ 61.
N-terminal splice variants of the type I PACAP receptor: isolation, characterization and ligand binding/selectivity determinants.
], CTR [ 62.
Headless splice variant acting as dominant negative calcitonin receptor.
], and SCTR [ 63.
Dominant negative action of an abnormal secretin receptor arising from mRNA missplicing in a gastrinoma.
]. In each case, the N-terminal truncation impaired or abolished ligand binding and/or preferentially activated alternative downstream signaling pathways, thus contributing to biased signaling [ 64.
Constitutive signal bias mediated by the human GHRHR splice variant 1.
]; (ii) variable sizes of ECLs or ICLs affecting ligand binding [ 14.
A unique hormonal recognition feature of the human glucagon-like peptide-2 receptor.
, 65.
The extracellular surface of the GLP-1 receptor is a molecular trigger for biased agonism.
] or G-protein coupling [ 66.
Differential responses of corticotropin-releasing hormone receptor type 1 variants to protein kinase C phosphorylation.
, 67.
Sustained activation of Erk1/2 MAPK and cell growth suppression by the insert-negative, but not the insert-positive isoform of the human calcitonin receptor.
]; (iii) alternative C termini with distinct signaling and/or receptor-internalization properties [ 68.
Consequences of splice variation on secretin family G protein-coupled receptor function.
]. Especially for the TM7 shortened isoforms, poor membrane expression and potential dominant negative effects on wild-type receptor function were reported for CRF1R, CTR, VIP2R, and PTH1R [ 69.
Structural determinants critical for localization and signaling within the seventh transmembrane domain of the type 1 corticotropin releasing hormone receptor: lessons from the receptor variant R1d.
, 70.
A naturally occurring isoform inhibits parathyroid hormone receptor trafficking and signaling.
, 71.
The delta e13 isoform of the calcitonin receptor forms a six-transmembrane domain receptor with dominant-negative effects on receptor surface expression and signaling.
]; and (iv) severe truncation (soluble isoforms) capable of ligand binding without signal transduction [ 72.
Combinatorial expression of GPCR isoforms affects signalling and drug responses.
], thereby sequestering ligand (Figure 5). Truncated isoforms can also affect the ability of the receptors to interact with each other or with RAMPs (Figure 5). Coexpression of full-length GHRHR and its splice variant 1 (SV1) resulted in a decrease in GHRH binding, indicating a dominant negative effect of SV1 [ 73.
A dominant-negative human growth hormone-releasing hormone (GHRH) receptor splice variant inhibits GHRH binding.
]. Moreover, cAMP responses induced by GHRH in SV1-expressing cells could be enhanced by RAMP3 that has no modulating effect on GHRHR [ 32.
Modulating effects of RAMPs on signaling profiles of the glucagon receptor family.
], suggesting that SV1 altered receptor pharmacology via interaction with RAMP3. Of note, splice variants could retain the wild-type receptor inside the cell via heterodimerization [ 62.
Headless splice variant acting as dominant negative calcitonin receptor.
] and may contribute to non-canonical endosomal signaling by enhancing receptor internalization [ 74.
Signaling at the endosome: cryo-EM structure of a GPCR–G protein–beta-arrestin megacomplex.
]. In the canonical model of class B1 GPCR signaling, β-arrestins terminate cAMP responses by decoupling G proteins and relocating the receptor to endosomes for degradation and ERK1/2 activation [ ]. However, a shift in this paradigm occurred in recent years because a few receptors, such as PTH1R [ 76.
Noncanonical GPCR signaling arising from a PTH receptor–arrestin–Gbetagamma complex.
, 77.
Gq/11-dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR.
] and GLP-1R [ 78.
Glucagon-like peptide-1 receptor-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic beta-cells.
], can elicit endosomal cAMP production rather than promoting degradation after internalization. Concluding remarks and future perspectivesClass B1 GPCRs are implicated in a variety of human diseases, including osteoporosis, migraine, and sexual dysfunction, as well as in metabolic disorders such as obesity and type 2 diabetes [ 79.
Trends in GPCR drug discovery: new agents, targets and indications.
, 80.
Current understanding of the structure and function of family B GPCRs to design novel drugs.
]. They are activated primarily by peptide hormones which account for 27 approved drugs (Table S2) [ 81.
GPCRdb in 2021: integrating GPCR sequence, structure and function.
]. In recent decades, structure-based drug design has employed a variety of GPCRs, and several drug candidates are currently in clinical trials [ 2.
G protein-coupled receptors: structure- and function-based drug discovery.
, 79.
Trends in GPCR drug discovery: new agents, targets and indications.
, 81.
GPCRdb in 2021: integrating GPCR sequence, structure and function.
] (Table S2). The past 5 years have witnessed not only the first two full-length class B1 GPCR structures but also the complete structural determination of all 15 members of this class. Overall, they contain an ECD defined by three pairs of conserved disulfide bonds and a canonical TMD possessing seven TM helices [ 82.
Differential requirement of the extracellular domain in activation of class B G protein-coupled receptors.
]. The ECD is responsible for high-affinity and specific interaction with peptide hormones [ 19.
Structure and mechanism for recognition of peptide hormones by class B G-protein-coupled receptors.
]. Crystal structures of their ECDs display a common fold of a three-layered α–β–β/α structure for binding of peptide ligands, most of which adopt a single helix and dock into the central groove of the ECD [ 83.
Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor.
, 84.
Molecular recognition of parathyroid hormone by its G protein-coupled receptor.
]. Ligand binding to the ECD orients the hormone N terminus to insert into the TMD and induces conformational changes involving breakage of the hydrophobic lock and polar core formed in part by TM6 [ 85.
Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.
], leading to outward movement of this helix in an manner analogous to that seen in class A GPCRs. The crystal structures of isolated and inactive TMDs of several class B1 GPCRs exhibit a compressed helical fold [ 42.
Structure of class B GPCR corticotropin-releasing factor receptor 1.
]. For GLP‐1R, structures also include those of the receptor bound to small-molecule agonists and allosteric modulators [ 24.
Differential GLP-1R binding and activation by peptide and non-peptide agonists.
, 25.
Activation of the GLP-1 receptor by a non-peptidic agonist.
, 26.
Molecular insights into ago-allosteric modulation of the human glucagon-like peptide-1 receptor.
, 38.
Structural insights into the activation of GLP-1R by a small molecule agonist.
, 40.
Structural insights into probe-dependent positive allosterism of the GLP-1 receptor.
]. Together, these structures provide a comprehensive understanding of the potency and specificity of peptide hormones to their cognate receptors, as well as of the basis of signal transduction from ligand binding at the ECD across the TMD to the intracellular side. They also offer tangible information about peptide-binding pocket topology and properties that are crucial for rational design of a new generation of peptidic and/or non-peptidic ligands targeting these receptors. Important findings include the unique loop conformation of TIP39 bound to PTH2R that determines its subtype selectivity, the deeply inserted N-terminal α-helix of VIP2R that directly interacts with PACAP27, and the ago-allosteric modulation of compound 2 at GLP‐1R. The discovery of novel binding sites of both positive and negative allosteric modulators and elucidation of their mechanisms of action provide new directions for modifying receptor functionalities beyond conventional orthosteric ligands for the development of better therapeutic agents with reduced adverse effects. These and other key issues in the field are summarized in the outstanding questions (see Outstanding questions). Future studies would be directed towards receptor structures in complex with antibodies and novel small-molecule modulators to facilitate structure-based drug discovery for a variety of important diseases. Outstanding questions What is the structural basis of the distinct functionalities between pairs of closely related GPCRs, such as GLP-1R versus GLP-2R, PTH1R versus PTH2R, and CRF1R versus CRF2R? If this can be clarified, how does this relate to the difference between the native peptides (e.g., GLP-1 vs GLP-2)? Is there any commonality among these receptor and ligand pairs? Can major differences in terms of ligand-binding affinities and receptor-activation efficacies between peptide and small-molecule modulators be structurally explained? What is the minimum requirement for a small-molecule ligand to show in vivo activity? What is the advantage of a small-molecule agent in competing with orally available protein drugs targeting the same receptor? Do any structural insights support the use of non-peptidic therapeutics? Should both be evaluated head-to-head regarding their safety, efficacy, and long-term cardiovascular benefits in the case of GLP-1R agonists? Why do splice variants only exist for some class B1 GPCRs such as GIPR and GHRHR? Is their presence related to the functions of the receptors? What is the relationship between different splice variants and RAMPs? Which plays the dominant role when both are present on the same cell surface? What is the difference between ligand-mediated and constitutive biased signaling? Is there any additive effect when both signaling bias mechanisms are combined? Can splice variants be directly targeted for drug development? If the 'two-domain' binding mechanism is fundamental to class B1 GPCR activation, is there a possibility to design small molecules that circumvent this requirement while possessing potent activity, particularly in vivo? Is there any structural evidence of GPCR basal activity or self-activation without ligand? Can the N terminus of a receptor ECD exert such an action? What is physiological and/or pathological significance of basal activity? AcknowledgmentsThis work was supported in part by National Natural Science Foundation of China grants 81872915 (M-W.W.), 82073904 (M-W.W.), 32071203 (L.H.Z), 81773792 (D.Y.), 81973373 (D.Y.) and 21704064 (Q.Z.); the National Science and Technology Major Project of China – Key New Drug Creation and Manufacturing Program 2018ZX09735–001 (M-W.W.) and 2018ZX09711002–002–005 (D.Y.); the National Science and Technology Major Project of China – Innovation 2030 for Brain Science and Brain-Inspired Technology 2021ZD0203400 (Q.Z.); the National Key Basic Research Program of China 2018YFA0507000 (M-W.W.); the Ministry of Science and Technology of China 2018YFA0507002 (H.E.X.); Shanghai Municipal Science and Technology Commission Major Project 2019SHZDZX02 (H.E.X.); the Strategic Priority Research Program of Chinese Academy of Sciences XDB37030103 (H.E.X.); Novo Nordisk-CAS Research Fund grant NNCAS-2017–1-CC (D.Y.); Shanghai Science and Technology Development Funds grants 18ZR1447800 (L.H.Z.) and 18431907100 (M-W.W.); the Young Innovator Association of CAS 2018325 (L.H.Z.), and the SA-SIBS Scholarship Program (L.H.Z. and D.Y.). Declaration of interestsThe authors declare no conflicts of interest. Supplemental informationReferences
GlossaryAgonista ligand that binds to and activates a receptor to increase signaling over basal activity (a full agonist shows intrinsic activity and can produce a maximal response, whereas a partial agonist has less intrinsic activity and can only produce a partial response). Ago-allosteric modulator (ago-PAM)a molecule that is capable of acting both as an agonist on its own and as an efficacy enhancer of orthosteric ligands. Allosteric modulatora ligand that enhances or attenuates agonist-mediated receptor response by binding to an allosteric site that is spatially and topographically remote from the orthosteric sits; such molecules are positive allosteric modulators (PAMs) or negative allosteric modulators (NAMs), respectively. Antagonista ligand that binds to a receptor but cannot activate it. It occupies the orthosteric binding site and prevents agonist binding. Cryo-electron microscopy (cryo-EM)an electron microscopy-based technique that can determine protein structures in great detail. Extracellular domain (ECD)a part of a receptor that is on the outer surface of the cell membrane and is responsible for high-affinity and specific ligand binding. Extracellular loop (ECL)the amino acid sequence connecting the seven transmembrane helices in a receptor facing the space outside the plasma membrane. G proteina guanine nucleotide (GTP and GDP)-binding protein composed of Gα, Gβ, and Gγ subunits that is activated upon ligand binding to the receptor. It transduces signals to downstream effectors. The Gα subunit is divided into four families, Gαi/o, Gαs, Gαq/11, and Gα12, which interact with different effector enzymes in a highly specific manner. G protein-coupled receptors (GPCRs)abundant cell-surface proteins that are composed of seven transmembrane α-helixes linked by three ECLs, three ICLs, and the amphipathic helix VIII. Binding of ligands (light, ions, small molecules, lipids, peptides, or proteins) to GPCRs transmits signals to the intracellular compartment and activates pathways primarily coupled to G proteins but also to other mediators such as arrestins, kinases, or lipases. Based on their structural relatedness, GPCRs are divided into five families: class A (rhodopsin), class B1 (secretin), class B2 (adhesion), class C (glutamate), and class F (frizzled/smoothened). Intracellular loop (ICL)the amino acid sequence connecting the seven transmembrane helices in a receptor facing the space inside of the plasma membrane. Orthosteric liganda ligand that interacts with the receptor at orthosteric pocket, a binding site of a natural endogenous ligand. P6.47xxG6.50 motifa highly conserved motif among class B1 GPCRs that is located in the middle of TM6. Receptor activity-modifying protein (RAMP)a protein that regulates signaling and/or trafficking in a receptor-dependent manner and is able to act as a pharmacological switch or chaperone. Splice variant (SV)two or more gene sequence variants that consist of different exons from the same gene that can be transcribe/translated to generate different mRNAs/proteins. Transmembrane domain (TMD)a helical bundle that consists of seven transmembrane helices and connects the extracellular and intracellular loops. It is required for receptor activation and signal coupling to downstream G proteins. Article InfoPublication HistoryPublished online: January 22, 2022 IdentificationDOI: https://doi.org/10.1016/j.tips.2022.01.002 Copyright© 2022 The Author(s). Published by Elsevier Ltd. User LicenseCreative Commons Attribution – NonCommercial – NoDerivs (CC BY-NC-ND 4.0) |How you can reuse PermittedFor non-commercial purposes:
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