The family of human G protein-coupled receptors (GPCRs) is comprised of about 800 different members, with about 35% of current pharmaceutical drugs targeting GPCRs. However, GPCR structural biology, necessary for structure-guided drug design, has lagged behind that of other membrane proteins, and it was not until the year 2000 when the first crystal structure of a GPCR (rhodopsin) was solved. Starting in 2007, the determination of additional GPCR structures was facilitated by protein engineering, new crystallization techniques, complexation with antibody fragments, and other strategies. More recently, the use of camelid heavy-chain-only antibody fragments (nanobodies) as crystallographic chaperones has revolutionized the field of GPCR structural biology, aiding in the determination of more than 340 GPCR structures to date. In most cases, the GPCR structures solved as complexes with nanobodies (Nbs) have revealed the binding mode of cognate or non-natural ligands; in a few cases, the same Nb has acted as an orthosteric or an allosteric modulator of GPCR signaling. In this review we summarize the multiple ingenious strategies that have been conceived and implemented in the last decade to capitalize on the discovery of nanobodies to study GPCRs from a structural perspective. Significance Statement G protein-coupled receptors (GPCRs) are major pharmacological targets, and the determination of their structures at high resolution has been essential for structure-guided drug design and for insights about their functions. Single domain antibodies (nanobodies) have greatly facilitated the structural determination of GPCRs, by forming complexes directly with the receptors or indirectly through protein partners.

The class F of G protein-coupled receptors (GPCRs) consists of ten Frizzleds (FZD1-10) and Smoothened (SMO). FZDs bind and are activated by secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family and SMO is indirectly activated by the Hedgehog (Hh) family of morphogens acting on the transmembrane protein Patched (PTCH). The advance of our understanding of FZDs and SMO as dynamic transmembrane receptors and molecular machines, which emerged during the past 14 years since the first class F GPCR IUPHAR nomenclature report, justifies an update. This article focuses on the advances in molecular pharmacology and structural biology providing new mechanistic insight into ligand recognition, receptor activation mechanisms, signal initiation and signal specification. Furthermore, class F GPCRs continue to develop as drug targets, and novel technologies and tools such as genetically encoded biosensors and CRISP/Cas9 edited cell systems have contributed to refined functional analysis of these receptors. Also, advances in crystal structure analysis and cryogenic electron microscopy contribute to a rapid development of our knowledge about structure-function relationships providing a great starting point for drug development. Despite the progress questions and challenges remain to fully understand the complexity of the WNT/FZD and Hh/SMO signaling systems. Significance Statement The recent years of research have brought about substantial functional and structural insight into mechanisms of activation of Frizzleds and Smoothened. While the advance furthers our mechanistic understanding of ligand recognition, receptor activation, signal specification and initiation, broader opportunities emerge that allow targeting class F GPCRs for therapy and regenerative medicine employing both biologics and small molecule compounds.

Olfactory receptors are members of class A (rhodopsin-like) family of G protein-coupled receptors (GPCRs). Their expression and function have been increasingly studied in nonolfactory tissues, and many have been identified as potential therapeutic targets. In this manuscript, we focus on the discovery of novel ligands for the olfactory receptor family 51 subfamily E2 (OR51E2). We performed an artificial intelligence-based virtual drug screen of a ∼2.2 million small molecule library. Cell-based functional assay identified compound 80 (C80) as an antagonist and inverse agonist, and detailed pharmacological analysis revealed C80 acts as a negative allosteric modulator by significantly decreasing the agonist efficacy, while having a minimal effect on receptor affinity for agonist. C80 binds to an allosteric binding site formed by a network of nine residues localized in the intracellular parts of transmembrane domains 3, 5, 6, 7, and H8, which also partially overlaps with a G protein binding site. Mutational experiments of residues involved in C80 binding uncovered the significance of the C2406.37 position in blocking the activation-related conformational change and keeping the receptor in the inactive form. Our study provides a mechanistic understanding of the negative allosteric action of C80 on agonist-ctivated OR51E2. We believe the identification of the antagonist of OR51E2 will enable a multitude of studies aiming to determine the functional role of this receptor in specific biologic processes. SIGNIFICANCE STATEMENT: OR51E2 has been implicated in various biological processes, and its antagonists that can effectively modulate its activity have therapeutic potential. Here we report the discovery of a negative allosteric modulator of OR51E2 and provide a mechanistic understanding of its action. We demonstrate that this modulator has an inhibitory effect on the efficacy of the agonist for the receptor and reveal a network of nine residues that constitute its binding pocket, which also partially overlaps with the G protein binding site.

The third intracellular loop of G-protein-coupled receptors (GPCRs) shows remarkable diversity in sequence and overall length. Sadler and colleagues recently demonstrated that this domain acts as an \'autoregulator\' of receptor activity and that its length contributes to receptor/G-protein coupling selectivity. These observations may prove useful for developing novel therapeutics.

The neurotransmitter γ-hydroxybutyric acid (GHB) is suggested to be involved in neuronal energy homeostasis processes, but the substance is also used as a recreational drug and as a prescription medication for narcolepsy. GHB has several high-affinity targets in the brain, commonly generalized as the GHB receptor. However, little is known about the structural and functional properties of GHB receptor subtypes. This opinion article discusses the literature on the putative structural and functional properties of the GHBh1 receptor subtype. GHBh1 contains 11 transmembrane helices and at least one intracellular intrinsically disordered region (IDR). Additionally, GHBh1 shows a 100% overlap in amino acid sequence with the Riboflavin (vitamin B2) transporter, which opens the possibility of a possible dual-function (transceptor) structure. Riboflavin and GHB also share specific neuroprotective properties. Further research into the GHBh1 receptor subtype may pave the way for future therapeutic possibilities for GHB.

The B cell and T cell antigen receptors (BCR and TCR) share a common architecture in which variable dimeric antigen-binding modules assemble with invariant dimeric signaling modules to form functional receptor complexes. In the TCR, a highly conserved T cell receptor αβ (TCRαβ) transmembrane (TM) interface forms a rigid structure around which its three dimeric signaling modules assemble through well-characterized polar interactions. Noting that the key features stabilizing this TCRαβ TM interface also appear with high evolutionary conservation in the TM sequences of the membrane immunoglobulin (mIg) heavy chains that form the BCR\'s homodimeric antigen-binding module, we asked whether the BCR contained an analogous TM structure. Using an unbiased biochemical and computational modeling approach, we found that the mouse IgM BCR forms a core TM structure that is remarkably similar to that of the TCR. This structure is reinforced by a network of interhelical hydrogen bonds, and our model is nearly identical to the arrangement observed in the just-released cryo-electron microscopy (cryo-EM) structures of intact human BCRs. Our biochemical analysis shows that the integrity of this TM structure is vital for stable assembly with the BCR signaling module CD79AB in the B cell endoplasmic reticulum, and molecular dynamics simulations indicate that BCRs of all five isotypes can form comparable structures. These results demonstrate that, despite their many differences in composition, complexity, and ligand type, TCRs and BCRs rely on a common core TM structure that has been shaped by evolution for optimal receptor assembly and stability in the cell membrane.

Selatogrel is a potent inhibitor of adenosine diphosphate (ADP) binding to the P2Y12 receptor, preventing platelet activation. We have previously shown that the P2Y12 receptor constitutively activates Gi- and Go-protein-mediated signaling in human platelets. Here, we report that selatogrel acts as an inverse agonist of the P2Y12 receptor. Specifically, using bioluminescence resonance energy transfer2 (BRET2) probes, selatogrel, ticagrelor, and elinogrel were shown to stabilize the inactive form of the Gαi/o-Gβγ complex in cells with recombinant expression of the P2Y12 receptor. In dose-response experiments, while selatogrel exhibited a maximal efficacy similar to ticagrelor, selatogrel was approximately 100-fold more potent than ticagrelor. Quantification of relative cyclic adenosine monophosphate (cAMP) levels in cells expressing the cAMP BRET1 sensor (CAMYEL probe) confirmed that selatogrel completely abolished the constitutive activity of the P2Y12 receptor. In agreement, selatogrel increased basal cAMP levels in human platelets, confirming inverse agonism on the endogenous human platelet P2Y12 receptor. In agreement with the biochemical phenotype of inverse agonism efficacy of selatogrel, the 2.8 Angstrom resolution cocrystal structure of selatogrel bound to the P2Y12 receptor confirmed that selatogrel stabilizes the inactive, basal state of the receptor. Selatogrel bound to pocket 1, spanning helix III to VII. Furthermore, the binding mode of selatogrel, suggesting steric overlap with the proposed binding site of ADP and the ADP analog 2-methylthioadenosine diphosphate (2MeSADP), agrees with the functional characterization of selatogrel preventing platelet activation by blocking ADP binding to the P2Y12 receptor.

Signaling lymphocyte activation molecule family member 9 (SLAMF9) is a cell surface protein of the CD2/SLAM family of leukocyte surface receptors. It is conserved throughout mammals and has roles in the initiation of inflammatory responses and regulation of plasmacytoid dendritic cell function. Through comparison of reference sequences encoding SLAMF9 in human, mouse, and primate sequences, we have determined that the SLAMF9 gene underwent successive mutation events, resulting in the loss of the protein and subsequent recovery of a less stable version. The mutations included a single base pair deletion in the second exon and a change in the splice acceptor site of that same exon. These changes would have had the effect of creating and later repairing a frameshift in the coding sequence. These events took place since the divergence of the human lineage from the chimpanzee-human last common ancestor and represent the first known case of the functional loss and recovery of a gene within the human lineage.

GnRH receptor mutations, Glu2.53(90)Lys and Glu2.53(90)Asp, cause congenital hypogonadotropic hypogonadism. The Glu2.53(90) side-chain has been proposed to form an intramolecular salt-bridge with Lys3.32(121), but conserved intramolecular interaction networks in G protein-coupled receptor crystal structures predict that it interacts with Ser3.35(124) and Trp6.48(280). We investigated interhelical interactions of Glu2.53(90) that stabilise GnRH receptor folding using functional analyses and computational modelling of mutant receptors. The Glu2.53(90)Asp mutant was non-functional, but mutants with hydrophobic amino acids or Arg substituted for Glu2.53(90) were functional, excluding a salt-bridge interaction. The Glu2.53(90)Arg and Trp6.48(280)Arg mutants had decreased affinity for GnRH. Models showed that congenital Glu2.53(90)Lys and Glu2.53(90)Asp mutations disrupt interactions with Ser3.35(124) and Trp6.48(280) respectively, whereas the Glu2.53(90)Arg and Trp6.48(280)Arg mutations preserve intramolecular contacts, but increase distance between the transmembrane helices. Our results show that disruption of interhelical contacts that are conserved in G protein-coupled receptors accounts for the effects of some disease-associated GnRH receptor mutations.

Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning α-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-binding site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues stabilizing the inactive GnRH receptor structure. Active conformation-specific interhelical contacts stabilize an open G protein-binding site. Progress in defining the GnRH-binding site has recently slowed, with evidence that Tyr6.58(290) contacts Tyr5 of GnRH, whereas other residues affect recognition of Trp3 and Gly10NH2. The surprisingly consistent observations that GnRH receptor mutations that disrupt GnRH binding have less effect on \"conformationally constrained\" GnRH peptides may now be explained by crystal structures of agonist-bound peptide receptors. Analysis of GPCR structures provides insight into GnRH receptor function.