G Protein-coupled Receptor 83 Research Articles

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.

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 β-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.

The extracellular N-terminal domain of G-protein coupled receptor 83 regulates signaling properties and is an intramolecular inverse agonist.

BackgroundRecently, the orphan G-protein coupled receptor 83 (GPR83) was identified as a new participant in body weight regulation. This receptor is highly expressed in the hypothalamic arcuate nucleus and is regulated in response to nutrient availability. Gpr83 knock-out mice are protected from diet-induced obesity. Moreover, in a previous study, we designed and characterized several artificial constitutively activating mutations (CAMs) in GPR83. A particular CAM was located in the extracellular N-terminal domain (eNDo) that is highly conserved among GPR83 orthologs. This suggests the contribution of this receptor part into regulation of signaling, which needed a more detailed investigation.FindingsIn this present study, therefore, we further explored the role of the eNDo in regulating GPR83-signaling and demonstrate a proof-of-principle approach in that deletion mutants are characterized by a strong increase in basal Gq/11-mediated signaling, whilst none of the additionally characterized signaling pathways (Gs, Gi, G12/13) were activated by the N-terminal deletion variants. Of note, we detected basal GPR83 MAPK-activity of the wild type receptor, which was not increased in the deletion variants.ConclusionsFinally, the extracellular portion of GPR83 has a strong regulatory function on this receptor. A suppressive - inverse agonistic - effect of the eNDo on GPR83 signaling activity is demonstrated here, which also suggests a putative link between extracellular receptor activation and proteolytic cleavage. These new insights highlight important aspects of GPR83-regulation and might open options in the development of tools to modulate GPR83-signaling.

The orphan receptor GPR83 regulates systemic energy metabolism via ghrelin-dependent and -independent mechanisms

The G-protein coupled receptor 83 (GPR83) is an orphan G-protein coupled receptor (GPCRs) that is highly expressed in thymus and brain. Within the brain GPR83 expression can especially be found in hypothalamic nuclei that are known to play an important role in body weight regulation. We explore a putative role for GPR83 in energy metabolism. By doing this we demonstrate that hypothalamic expression of GPR83 is decreased in diet-induced obese (DIO) mice compared to lean mice. Additionally, hypothalamic expression is reduced following fasting and increased upon re-feeding. Via in situ hybridization it could be shown that GPR83 is co-localized with the ghrelin receptor (GHSR1a). In vitro studies could confirm GPR83/GHSR1a heterodimerization. Functional characterization of the heterodimer suggests that GPR83 leads to a reduced activation of GHSR1a by its ligand ghrelin. In agreement, studies in GPR83-knockout mice show that loss of GPR83 potentiates the effect of both, central and peripheral ghrelin treatment, on food intake and adiposity. GPR83-knockout mice have normal body weight and glucose tolerance when fed a regular chow diet. Glucose intolerance and obesity were not observed in response to high-fat diet despite relative hyperphagia. In summary, our data suggest that GPR83 acts as a modulator of ghrelin action in response to nutrient availability. In addition to these findings GPR83 seems to play a more complex role in the regulation of systemic metabolism.

G-Protein Coupled Receptor 83 (GPR83) Signaling Determined by Constitutive and Zinc(II)-Induced Activity

The G-protein coupled receptor 83 (GPR83) is an orphan G-protein coupled receptor for which the natural ligand(s) and signaling pathway(s) remain to be identified. Previous studies suggest a role of GPR83 in the regulation of thermogenesis and the control of circulating adiponectin. The aim of this study was to gain insights into the molecular underpinnings underlying GPR83 signaling. In particular, we aimed to assess the underlying G-protein activated signaling pathway of GPR83 and how this pathway is affected by mutational activation and zinc(II) challenge. Finally, we assessed the capacity of GPR83 for homodimerization. Our results show for the first time that mouse (m) GPR83 has high basal Gq/11 activity without affecting Gi or Gs signaling. Furthermore, we found that, under physiological conditions, zinc(II) (but not calcium(II) and magnesium(II)) potently activates mGPR83, thus identifying zinc(II) as an endogenous molecule with agonistic capability to activate mGPR83. In line with the observation that zinc(II)-ions activate mGPR83, we identified a cluster of ion-binding sensitive amino acids (e.g. His145, His204, Cys207, Glu217) in an activation sensitive receptor region of mGPR83. The occurrence of a constitutive activating mutant and a zinc(II)-binding residue at the N-terminal part corroborate the importance of this region in mGPR83 signal regulation. Finally, our results indicate that mGPR83 forms homodimers, which extend the current knowledge and molecular facets of GPR83 signaling.

Downregulation of GPR83 in the hypothalamic preoptic area reduces core body temperature and elevates circulating levels of adiponectin

The G protein-coupled receptor 83 (GPR83) was recently demonstrated in warm sensitive neurons (WSN) of the hypothalamic preoptic area (POA) that participate in temperature homeostasis. Thus, we investigated whether GPR83 may have a role in regulating core body temperature (CBT) by reducing its expression in the POA. Dissipation of energy in the form of heat is the primary mode of energy expenditure in mammals and can ultimately affect energy homeostasis. Thus, we also measured the level of important regulators of metabolism. Downregulation of GPR83 was obtained by lentiviral short-hairpin RNAs (shGPR83) vectors designed and selected for their ability to reduce GPR83 levels in vitro. Mice received POA injection of shGPR83 or non-silencing vectors and were monitored for CBT, motor activity, food intake body weight and circulating levels of IGF-1, insulin, leptin and adiponectin. Down-regulation of GPR83 in the POA resulted in a small (0.15°C) but significant reduction of CBT during the dark/active cycle of the day. Temperature reduction was followed by increased body weight gain independent of caloric intake. shGPR83 mice also had increased level of circulating adiponectin (31916±952pg/mL vs. 23474±1507pg/mL, P<.01) while no change was observed for insulin, IGF-1 or leptin. GPR83 may participate in central thermoregulation and the central control of circulating adiponectin. Further work is required to determine how GPR83 can affect POA WSN and what are the long term metabolic consequences of its down-regulation.

Inflammation in vivo is modulated by GPR83 isoform-4 but not GPR83 isoform-1 expression in regulatory T cells

Most recently, we have described the G-protein coupled receptor 83 (GPR83), which is highly expressed by CD4(+)CD25(+) regulatory T cells (Tregs) to be involved in the induction of CD4(+)Foxp3(+) Tregs in the course of an ongoing immune response. Four GPR83 isoforms have been described. Here, we have shown that GPR83 isoform-4, which differs from GPR83 isoform-1 by 20 additional aminoacids in the second cytoplasmatic loop, is predominantly expressed by Tregs. Interestingly, GPR83 isoform-4 but not GPR83 isoform-1 retrovirally transduced T cells were able to interfere with inflammatory responses in vivo. Re-analysis of GPR83 transduced T cells revealed that this in vivo acquisition of suppressive activity was associated with the induction of Treg-associated molecules including Foxp3 in GPR83 isoform-4 but not GPR83 isoform-1 transduced CD4(+) T cells under inflammatory conditions. Our results suggest that the 20 additional aminoacids within GPR83 isoform-4 are involved in Treg induction during inflammatory immune responses.

G Protein-Coupled Receptor 83 Is Dispensable for the Development and Function of Regulatory T Cells

Global analyses of gene expression in regulatory T (Treg) cells, whose development is critically dependent upon the transcription factor Foxp3, have provided many clues as to the molecular mechanisms these cells employ to control immune responses and establish immune tolerance. Through these studies, G protein-coupled receptor 83 (GPR83) was found to be expressed at high levels in Treg-cell populations. However, its function remained unclear. Recently, it has been suggested that GPR83 is involved in the induction of Foxp3 expression in the peripheral nonregulatory Foxp3- CD4 T cells. To examine a role for GPR83 in Treg-cell biology, we generated and characterized GPR83-deficient mice. We have shown that GPR83 abolition does not result in measurable pathology or changes in the numbers or function of Foxp3+ Treg cells. Furthermore, while in vitro analysis suggested a potential involvement of GPR83 in transforming growth factor beta-dependent Foxp3 induction, there was no difference in the ability of nonregulatory GPR83-deficient and nondeficient Foxp3- T cells to acquire Foxp3 expression in vivo. Collectively, our results demonstrate that GPR83 is dispensable for Treg-cell development and function.