Is the Neuropeptide PEN a Ligand of GPR83?

G protein-coupled receptor 83 (GPR83) is a class A G protein-coupled receptor with predominant expression in the cerebellum and proposed function in the regulation of food intake and in anxiety-like behavior. The neuropeptide PEN has been suggested as a specific GPR83 ligand. However, conflicting reports exist about whether PEN is indeed able to bind and activate GPR83. This study was initiated to evaluate PEN as a potential ligand of GPR83. Employing several second messenger and other GPCR activation assays as well as a radioligand binding assay, and using multiple GPR83 plasmids and PEN peptides from different sources, no experimental evidence was found to support a role of PEN as a GPR83 ligand.

Nanomolar range of FAM237B can activate receptor GPR83.

Our recent study confirmed that the mature neuropeptide FAM237A, also known as neurosecretory protein GL (NPGL), is an efficient agonist for GPR83. The paralog FAM237B was previously reported as a weak agonist for GPR83. In the present study, we prepared mature human FAM237B via an intein-fusion approach and demonstrated that it could cause a significant activation effect at the nanomolar range (1‒10 nM) in a NanoBiT-based ß-arrestin recruitment assay. Thus, FAM237B appears to be another endogenous agonist for GPR83 and future in vivo studies will be required to confirm this.

FAM237A, rather than peptide PEN and proCCK56-63, binds to and activates the orphan receptor GPR83.

G protein-coupled receptor 83 (GPR83) is primarily expressed in the brain and is implicated in the regulation of energy metabolism and some anxiety-related behaviours. Recently, the PCSK1N/proSAAS-derived peptide PEN, the procholecystokinin-derived peptide proCCK56-63, and family with sequence similarity 237 member A (FAM237A) were all reported as efficient agonists of GPR83. However, these results have not yet been reproduced by other laboratories and thus GPR83 is still officially an orphan receptor. The peptide PEN and proCCK56-63 share sequence similarity; however, they are completely different from FAM237A. To identify its actual ligand(s), in the present study we developed NanoLuc Binary Technology (NanoBiT)-based ligand-binding assay, fluorescent ligand-based visualization, and NanoBiT-based ß-arrestin recruitment assay for human GPR83. Using these assays, we demonstrated that mature human FAM237A could bind to GPR83 with nanomolar range affinity, and could activate this receptor and induce its internalization with nanomolar range efficiency in transfected human embryonic kidney 293T cells. However, we did not detect any interaction of PEN and proCCK56-63 with GPR83 using these assays. Thus, our results confirmed that FAM237A is an efficient agonist of GPR83, but did not support PEN and proCCK56-63 as ligands of this receptor. Clarification of their pairing paves the way for further functional studies of the brain-specific receptor GPR83 and the so far rarely studied neuropeptide FAM237A in the future.

Gpr83 Tunes Nociceptor Function, Controlling Pain.

The function of peripheral nociceptors is frequently tuned by the action of G protein-coupled receptors (GPRs) that are expressed in them, which contribute to pain alteration. Expanding new information on such GPRs and predicting their potential outcomes can help to construct new analgesic strategies based on their modulations. In this context, we attempted to present a new GPR not yet acknowledged for its pain association. Gpr83 exhibits relatively high expressions in the peripheral nervous system compared to other tissues when we mined and reconstructed Gene Expression Omnibus (GEO) metadata, which we confirmed using immunohistochemistry on murine dorsal root ganglia (DRG). When Gpr83 expression was silenced in DRG, neuronal and behavioral nociception were all downregulated. Pathologic pain in hind paw inflammation and chemotherapy-induced peripheral neuropathy were also alleviated by this Gpr83 knockdown. Dependent on exposure time, the application of a known endogenous Gpr83 ligand PEN showed differential effects on nociceptor responses in vitro. Localized PEN administration mitigated pain in vivo, probably following Gq/11-involved GPR downregulation caused by the relatively constant exposure. Collectively, this study suggests that Gpr83 action contributes to the tuning of peripheral pain sensitivity and thus indicates that Gpr83 can be among the potential GPR targets for pain modulation.

A Pilot Screen of a Novel Peptide Hormone Library Identified Candidate GPR83 Ligands.

The identification of novel peptide hormones by functional screening is challenging because posttranslational processing is frequently required to generate biologically active hormones from inactive precursors. We developed an approach for functional screening of novel potential hormones by expressing them in endocrine host cells competent for posttranslational processing. Candidate preprohormones were selected by bioinformatics analysis, and stable endocrine host cell lines were engineered to express the preprohormones. The production of mature hormones was demonstrated by including the preprohormones insulin and glucagon, which require the regulated secretory pathway for production of the active forms. As proof of concept, we screened a set of G-protein-coupled receptors (GPCRs) and identified protein FAM237A as a specific activator of GPR83, a GPCR implicated in central nervous system and regulatory T-cell function. We identified the active form of FAM237A as a C-terminally cleaved, amidated 9 kDa secreted protein. The related protein FAM237B, which is 64% homologous to FAM237A, demonstrated similar posttranslational modification and activation of GPR83, albeit with reduced potency. These results demonstrate that our approach is capable of identifying and characterizing novel hormones that require processing for activity.

The wonderful and masterful G protein-coupled receptor (GPCR): A focus on signaling mechanisms and the neuroendocrine control of fertility.

Human GnRH deficiency, both clinically and genetically, is a heterogeneous disorder comprising of congenital GnRH deficiency with anosmia (Kallmann syndrome), or with normal olfaction [normosmic idiopathic hypogonadotropic hypogonadism (IHH)], and adult-onset hypogonadotropic hypogonadism. Our understanding of the neural mechanisms underlying GnRH secretion and GnRH signaling continues to increase at a rapid rate and strikingly, the heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) continue to emerge as essential players in these processes. GPCRs were once viewed as binary on-off switches, where in the "on" state they are bound to their Gα protein, but now we understand that view is overly simplistic and does not adequately characterize GPCRs. Instead, GPCRs have emerged as masterful signaling molecules exploiting different physical conformational states of itself to elicit an array of downstream signaling events via their G proteins and the ß-arrestins. The "one receptor-multiple signaling conformations" model is likely an evolved strategy that can be used to our advantage as researchers have shown that targeting specific receptor conformations via biased ligands is proving to be a powerful tool in the effective treatment of human diseases. Can biased ligands be used to selectively modulate signaling by GPCR regulators of the neuroendocrine axis in the treatment of IHH? As discussed in this review, the grand possibility exists. However, while we are still very far from developing these treatments, this exciting likelihood can happen through a much greater mechanistic understanding of how GPCRs signal within the cell.

Uterine Gpr83 mRNA is highly expressed during early pregnancy and GPR83 mediates the actions of PEN in endometrial and non-endometrial cells.

OBJECTIVE: To characterize the expression and signaling of uterine GPR83 in vivo in the nonpregnant and pregnant mouse and in vitro in human endometrial and nonendometrial cells. DESIGN: Controlled laboratory study. SETTING: Not applicable. PATIENTS: Not applicable. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Expression of uterine Gpr83 was determined by quantitative polymerase chain reaction throughout the estrous cycle and during early pregnancy in ovarian-stimulated and non-ovarian-stimulated mice and pregnant and pseudopregnant mice. Expression was also determined in ovariectomized mice after the administration of oil, E2, P4, or E2 + P4 and in stromal cells following 6 days of in vitro decidualization. GPR83 signaling was studied in human endometrial and embryonic kidney cell lines. Cells were treated by PEN, a GPR83 ligand, and PEN-induced extracellular signal-regulated kinase (ERK) phosphorylation was assayed under conditions that blocked Gαq/11 and/or ß-arrestin signaling. RESULTS: Uterine Gpr83 is expressed throughout the estrous cycle and during early pregnancy; expression increases dramatically at the time of uterine receptivity, embryo implantation, and stromal cell decidualization. In the ovariectomized mouse, hormone add-back reveals that Gpr83 expression is highly responsive to the combined treatment of E2 and P4, and studies in the ovarian-stimulated mouse show that expression is also very sensitive to changes in E2 and P4 and is therefore tightly regulated by E2 and P4. At the implantation site, expression is elevated up to D6 of pregnancy and then declines rapidly on D7 and D8, suggesting that if there is any involvement in decidualization, it is likely associated with primary but not secondary stromal cell decidualization. This premise was supported by the observation that stromal cell decidualization in vitro progresses with a decline in Gpr83 expression. In ERα/PR-expressing endometrial Ishikawa cells, GPR83 mediates PEN signals in a Gαq/11-dependent manner, and studies conducted in HEK 293 cells lacking ß-arrestin revealed that GPR83 also signals via a ß-arrestin-dependent manner. When signaling by either one or both pathways is downregulated, cells exhibit a major reduction in responsiveness to PEN treatment, demonstrating that signaling by both pathways is significant. CONCLUSION: We hypothesize that PEN/GPR83 signaling regulates uterine receptivity, embryo implantation, and primary stromal cell decidualization by coupling to Gαq/11- and ß-arrestin-dependent pathways.

Targeting the Recently Deorphanized Receptor GPR83 for the Treatment of Immunological, Neuroendocrine and Neuropsychiatric Disorders.

G-protein coupled receptors (GPCRs) are a superfamily of receptors responsible for initiation of a myriad of intracellular signaling cascades. Currently, GPCRs represent approximately 34% of marketed pharmaceuticals, a large portion of which have no known endogenous ligand. These orphan GPCRs represent a large pool of novel targets for drug development. Very recently, the neuropeptide PEN, derived from the proteolytic processing of the precursor proSAAS, has been identified as a selective, high-affinity endogenous ligand for the orphan receptor, GPR83. GPR83 is highly expressed in the brain, spleen and thymus, indicating that this receptor may be a target to treat neurological and immune disorders. In the brain GPR83 is expressed in regions involved in the reward pathway, stress/anxiety responses, learning and memory and metabolism. However, the cell type specific expression of GPR83 in these regions has only recently begun to be characterized. In the immune system, GPR83 expression is regulated by Foxp3 in T-regulatory cells that are involved in autoimmune responses. Moreover, in the brain this receptor is regulated by interactions with other GPCRs, such as the recently deorphanized receptor, GPR171, and other hypothalamic receptors such as MC4R and GHSR. The following review will summarize the properties of GPR83 and highlight its known and potential significance in health and disease, as well as its promise as a novel target for drug development.