High throughput screening at novel striatum orphan GPCRs identified by human transcriptomic profiling

G protein-coupled receptors (GPCRs) are pivotal in biological processes and represent a significant class of drug targets, with 516 approved drugs acting on 121 GPCRs. Many GPCRs, particularly orphan receptors, remain underexplored, emphasizing the need for innovative investigative tools. Fluorescent ligands provide a powerful means to characterize GPCRs including their functional mechanisms and spatial organization, bridging fundamental research and drug discovery. This review presents recent advances (2018-2024) in fluorescent probe development for Class A GPCRs, analyzing over 120 newly developed probes covering 60 GPCRs. We examine their distribution across receptor subclasses, comparing pre-2018 data with contemporary findings and identifying previously uncharted GPCRs that now have fluorescent ligands. Notably, novel probes have been developed for 12 new receptor subtypes and 6 orphan receptors such as GPR6, GPR52, GPR84, MAS1, MRGPRX2, and MRGPRX4. Advances in GPCR structural biology, driven by cryo-EM and AlphaFold technologies, have significantly enhanced probe development, facilitating the design of selective fluorescent ligands across aminergic, peptidergic, lipid, nucleotide, alicarboxylic, melatonin, protein, and orphan GPCRs. These innovations support a broad range of applications, from single-molecule imaging and in vivo bioimaging to diagnostics and fluorescence-guided surgery. By integrating fluorescence-based approaches with structural and pharmacological insights, this field continues to refine polypharmacology profiling, optimize drug-receptor interactions, and accelerate GPCR-targeted drug discovery.

Glioblastomas represent the most common primary brain tumors, and carry an extremely poor prognosis (survival <1 year). While microarray analyses have identified 4 subgroups of glioblastomas, clearly additional biomarkers are essential for the diagnosis and treatment of these deadly tumors. G protein coupled receptors (GPCRs) represent the most abundant receptor family encoded by the human genome, and are involved in multiple cellular processes, including growth, structure, and migration, making GPCRs attractive targets for the treatment of cancers. Orphan GPCRs are often excluded from microarray analysis, and thus their role in glioblastomas may have been overlooked. We therefore sought to evaluate the expression of orphan GPCRs in glioblastomas using a novel, multiplex PCR‐based orphan GPCR MasterMix technology that we developed to rapidly elucidate the orphan GPCR expression profile of any human tissue or cell type. Using this technology, we have found that tumors tend to exhibit a unique “fingerprint” in terms of their orphan GPCR expression. This technology may be useful for identifying orphan GPCRs that could be used as diagnostic markers or therapeutic targets for these devastating neoplasms.

The orphan receptor GPR68 is expressed in the hypothalamus and is involved in the regulation of feeding

G protein-coupled receptors (GPCRs), the largest family of targets for approved drugs, are rarely targeted for cancer treatment, except for certain endocrine and hormone-responsive tumors. Limited knowledge regarding GPCR expression in cancer cells likely has contributed to this lack of use of GPCR-targeted drugs as cancer therapeutics. We thus undertook GPCRomic studies to define the expression of endoGPCRs (which respond to endogenous molecules such as hormones, neurotransmitters and metabolites) in multiple types of cancer cells. Using TaqMan qPCR arrays to quantify the mRNA expression of ∼340 such GPCRs, we found that human chronic lymphocytic leukemia (CLL) cells/stromal cells associated with CLL, breast cancer cell lines, colon cancer cell lines, pancreatic ductal adenocarcinoma (PDAC) cells, cancer associated fibroblasts (CAFs), and PDAC tumors express 50 to >100 GPCRs, including many orphan GPCRs (which lack known physiologic agonists). Limited prior data exist regarding the expression or function of most of the highly expressed GPCRs in these cancer cells and tumors. Independent results from public cancer gene expression databases confirm the expression of such GPCRs. We propose that highly expressed GPCRs in cancer cells (for example, GPRC5A in PDAC and colon cancer cells and GPR68 in PDAC CAFs) may contribute to the malignant phenotype, serve as biomarkers and/or may be novel therapeutic targets for the treatment of cancer.

Adropin, a peptide secreted by the liver, has been reported to be a novel mediator of energy metabolism. Clinical studies have shown an association between adropin levels and pathological markers of metabolic disease. Our lab has found that intra‐arterial administration of adropin enhances glucose disposal despite decreases in plasma insulin levels in healthy male rats. These data support a role for adropin in the regulation of glucose homeostasis; however, the mechanism has yet to be elucidated, partly due to the elusiveness of the adropin receptor. In endothelial cell cultures adropin stimulated downstream signaling pathways typically associated with G protein coupled receptor (GPCR) activation (Lovren 2010). Therefore, we hypothesized that adropin signals via a GPCR and in particular one of the known ~135 orphan GPCRs. To test this hypothesis, we first identified three adropin responsive cell lines [human gastric carcinoma (KATOIII), human coronary artery endothelial cells (HCAEC) and mouse fibroblasts (3T3‐L1)] using cfos mRNA expression as a reporter of cellular activation. We then applied the “Deductive Ligand‐Receptor Matching Strategy” developed in our lab (Yosten 2012) to generate an orphan GPCR expression profile of each cell line and identified five adropin receptor candidates; GPR151, TAAR9, GPR160, GPR19, and GPR63. In preliminary experiments, knockdown of one of these orphan receptors using siRNA abrogated adropin‐induced cfos mRNA expression in KATOIII cells, identifying this orphan receptor to be our top candidate adropin receptor. Identification of the cognate adropin receptor will facilitate our current attempts to identify the sites and mechanisms of action of this potent, liver‐derived hormone.

Chapter Two - Orphan GPCRs and Methods for Identifying Their Ligands

Germinal center (GC) reaction greatly contributes to the humoral immune response, which begins in lymph nodes or other secondary lymphoid organs after follicular B cells are activated by T-dependent antigens. The GCs then serve as a platform for follicular B cells to complete clonal expansion and somatic hypermutation and then interact with follicular dendritic cells (FDC) and follicular helper T cells (Tfh). Through the interaction between the immune cells, significant processes of the humoral immune response are accomplished, such as antibody affinity maturation, class switching, and production of memory B cells and plasma cells. Cell positioning during the GC reaction is mainly mediated by the chemokine receptors and lipid receptors, which both belong to G protein-coupled receptors (GPCRs) family. There are some orphan GPCRs whose endogenous ligands are unclear yet contribute to the regulation of GC reaction as well. This review will give an introduction on the ligands and functions of two types of GC-relating GPCRs-chemokine receptors like CXCR4 and CXCR5, as well as emerging de-orphanized GPCRs like GPR183, GPR174, and P2RY8. The roles these GPCRs play in several antibody-mediated autoimmune skin diseases will be also discussed, including systemic lupus erythematosus (SLE), pemphigus, scleroderma, and dermatomyositis. Besides, GPCRs are excellent drug targets due to the unique structure and vital functions. Therefore, this review is aimed at providing readers with a focused knowledge about the role that GPCRs play in GC reaction, as well as in provoking the development of GPCR-targeting agents for immune-mediated diseases besides autoimmune diseases.

Deorphanization of the large group of G protein-coupled receptors (GPCRs) for which an endogenous activating ligand has not yet been identified (orphan GPCRs) has become increasingly difficult. A specialized technique that has been successfully applied to deorphanize some of these GPCRs involves two-electrode voltage-clamp recordings of currents through ion channels, which are activated by GPCRs heterologously expressed in Xenopus oocytes. The ion channels that couple to GPCR activation in Xenopus oocytes can be endogenous calcium-activated chloride channels (CaCCs) or heterologously expressed G protein-coupled inwardly rectifying potassium channels (GIRKs). We will describe a general approach for expression of GPCRs in Xenopus oocytes and characterization of these using electrophysiological recordings. We will focus on the detection of GPCR activation by recordings of currents through CaCCs that are activated by calcium release from the endoplasmic reticulum and thus the G(q) signaling pathway.

The special glycerophospholipids plasmalogens (Pls) are enriched in the brain and reported to prevent neuronal cell death by enhancing phosphorylation of Akt and ERK signaling in neuronal cells. Though the activation of Akt and ERK was found to be necessary for the neuronal cells survival, it was not known how Pls enhanced cellular signaling. To answer this question, we searched for neuronal specific orphan GPCR (G-protein coupled receptor) proteins, since these proteins were believed to play a role in cellular signal transduction through the lipid rafts, where both Pls and some GPCRs were found to be enriched. In the present study, pan GPCR inhibitor significantly reduced Pls-induced ERK signaling in neuronal cells, suggesting that Pls could activate GPCRs to induce signaling. We then checked mRNA expression of 19 orphan GPCRs and 10 of them were found to be highly expressed in neuronal cells. The knockdown of these 10 neuronal specific GPCRs by short hairpin (sh)-RNA lentiviral particles revealed that the Pls-mediated phosphorylation of ERK was inhibited in GPR1, GPR19, GPR21, GPR27 and GPR61 knockdown cells. We further found that the overexpression of these GPCRs enhanced Pls-mediated phosphorylation of ERK and Akt in cells. Most interestingly, the GPCRs-mediated cellular signaling was reduced significantly when the endogenous Pls were reduced. Our cumulative data, for the first time, suggest a possible mechanism for Pls-induced cellular signaling in the nervous system.

Nanobodies as sensors of GPCR activation and signaling.

Structure and function of G protein-coupled receptors using NMR spectroscopy

The role of orphan G protein-coupled receptors in the modulation of pain: A review

Two papers published this week report both clarifying and somewhat contradictory findings regarding signaling by insect odorant receptors. Odorant receptors (ORs) in mammals are canonical heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) and are often coupled to generation of the second messenger adenosine 3′,5′-monophosphate (cAMP), which can modulate conductance of ion channels. Insect ORs have presented a puzzle, however, as they only vaguely resemble GPCRs and assemble as heterodimers of one variable OR and a common co-receptor called Or83b. Wicher et al . expressed Drosophila receptors Or22a and Or83b in human embryonic kidney cells and used patch clamping to characterize odorant-induced ion currents in the transfected cells. They report that expression of Or83b alone conferred a nonselective cation channel whose conductance was increased by cAMP or guanosine 3′,5′-monophosphate (cGMP). When the authors transfected cells with the Or22a-Or83b pair and also with known mammalian cyclic nucleotide-gated channels, odorant-induced activation of the mammalian channel indicated that the insect OR activated cAMP signaling. Thus, they conclude that the insect ORs themselves form ligand-gated ion channels that sense odorant and amplify signals by G-protein signaling. Sato et al . used similar strategies to analyze various insect ORs. Human cells expressing receptor proteins from fruit fly, mosquito, or silk moth showed a response to odorant that included Ca 2+ influx and increased nonselective cation conductance. Outside-out patch clamp studies from membranes of oocytes or human cells expressing the receptor proteins confirmed that the insect ORs themselves conferred the observed currents. However, Sato et al .’s experiments did not detect odorant-induced generation of cAMP, and pharmacological inhibition of G-protein signaling failed to block odorant-induced currents. As noted in commentary from Chesler and Firestein, resolution of the discrepancies will require further work but may have to do with Sato et al .’s focus on early events (within seconds) and Wicher et al .’s characterization of later events—30 seconds to a minute after receptor stimulation. Insect ORs do not resemble known ion channels at all, so the work suggests there is more to learn about such proteins and raises the question of whether orphan GPCRs in other organisms might also function as ligand-gated ion channels. K. Sato, M. Pellegrino, T. Nakagawa, T. Nakagawa, L. B. Vosshall, K. Touhara, Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature 452 , 1002-1006 (2008). [PubMed] D. Wicher, R. Schäfer, R. Bauernfeind, M. C. Stensmyr, R. Heller, S. H. Heinemann, B. S. Hannson, Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature 452 , 1007-1011 (2008). [PubMed] A. Chesler, S. Firestein, Current views on odour receptors. Nature 452 , 944 (2008). [PubMed]

GPR88 is an orphan G-protein-coupled receptor (GPCR) highly expressed in striatal medium spiny neurons (MSN), also found in cortical neurons at low level. In MSN, GPR88 has a canonical GPCR plasma membrane/cytoplasmic expression, whereas in cortical neurons, we previously reported an atypical intranuclear localization. Molecular size analysis suggests that GPR88, expressed in plasma membrane of MSN or in nuclear compartment of cortical neurons, corresponds to the full-length protein. By transfection of cortical neurons, we showed that GPR88 fluorescent chimeras exhibit a nuclear localization. This localization is contingent on the third intracytoplasmic loop and C-terminus domains, even though these domains do not contain any known nuclear localization signals (NLS). Using yeast two-hybrid screening with these domains, we identified the nuclear proteins ATRX, TOP2B, and BAZ2B, all involved in chromatin remodeling, as potential protein partners of GPR88. We also validated the interaction of GPR88 with these nuclear proteins by proximity ligation assay on cortical neurons in culture and coimmunoprecipitation experiments on cortical extracts from GPR88 wild-type (WT) and knockout (KO) mice. The identification of GPR88 subcellular partners may provide novel functional insights for nonclassical modes of GPCR action that could be relevant in the maturating process of neocortical neurons.

Family C of G-protein coupled receptors (GPCRs) from humans is constituted by eight metabotropic glutamate (mGlu(1-8)) receptors, two heterodimeric gamma-aminobutyric acid(B) (GABA(B)) receptors, a calcium-sensing receptor (CaR), three taste (T1R) receptors, a promiscuous L-alpha-amino acid receptor (GPRC6A), and five orphan receptors. Aside from the orphan receptors, the family C GPCRs are characterised by a large amino-terminal domain, which bind the endogenous orthosteric agonists. Recently, a number of allosteric modulators binding to the seven transmembrane domains of the receptors have also been reported. Family C GPCRs regulate a number of important physiological functions and are thus intensively pursued as drug targets. So far, two drugs acting at family C receptors (the GABA(B) agonist baclofen and the positive allosteric CaR modulator cinacalcet) have been marketed. Cinacalcet is the first allosteric GPCR modulator to enter the market, which demonstrates that the therapeutic principle of allosteric modulation can also be extended to this important drug target class. In this review we outline the structure and function of family C GPCRs with particular focus on the ligand binding sites, and we present the most important pharmacological agents and the therapeutic prospects of the receptors.