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

Abstract

Background: Recently, 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.

Findings: In 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.

Conclusions: Finally, 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.

Figure 1

Sequence comparison of GPR83 othologs and designed GPR83 variants A) Alignment of N-terminal amino acids of GPR83 orthologs for comparison and identification of sequence conservation. Regions of conservation can be recognized by the overlapping colors. High conservation is also indicative for a specific fold and/or function. It is evident that especially the second half of the N-terminal tail (between positions 36–65) is highly conserved among the compared variants. Different colors of amino acids indicate their biophysical properties: green – hydrophobic, blue – positively charged, red – negatively charged. The alignment was visualized using BioEdit. B) Schematic representation of GPR83 deletion mutants: The experimentally deleted parts are highlighted: a. deletion 18–35, b. deletion 36–65, c. deletion 18–65. The position of the signal peptide is indicated as SP and the hemagglutinin tag with HA.

Figure 2

Cell surface expression levels of different N-terminal GPR83 deletion mutants compared to wild type GPR83 which was set to 100% (absorption (492/620): 0.31 ± 0.01). The Gpr83 variants were detected using an HA-ELISA system as previously reported [12]. Untagged GPR83 served as the negative control. Data were assessed from a minimum of three independent experiments, each performed at least in triplicate, and are represented as mean + SEM. ***p ≤ 0.001 (unpaired t-test, two-tailed).

Figure 3

Different N-terminal GPR83 deletion mutants were functionally characterized and compared with the wild type GPR83 (A/B) or an empty vector control (pcDps, B/C/D). Shown are Gq/11-activation in A), MAPK-activation in B), G12/13-activation in C) and Gs/Gi-signaling properties in D). Wild type GPR83 serves as a positive control for Gq/11-signaling (A). The TSH-stimulated TSHR serves as a positive control for MAPK- and G12/13-signaling (B/C). Forskolin-stimulation shows Gs-activity of the used cell line by activating adenylyl cyclase. A decreased value of forskolin-stimulation in comparison to the empty vector control would indicate inhibitory Gi-activity. Data were evaluated from a minimum of three independent experiments, each performed at least in triplicate. Data were calculated as fold over the empty vector control, set to 1 (A: 4828.67 ± 1165.71, B: 1334800 ± 326986.69, C: 2095559.89 ± 447919.48 relative light units; D: 2.03 ± 0.15 nM cAMP). Data represent mean + SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (unpaired t-test, two-tailed).

Figure 4

Structural scheme of GPR83 and variants with indicated findings and derived hypotheses. A) In wild type GPR83, the N-terminal domain (eNDo) is stabilized intramolecularly by side-chain interactions between specific amino acids (red broken lines). The eNDo might have a defined structural fold, which is indicated by high amino acid conservation throughout GPR83 orthologs (Figure 1). The eNDo most likely interacts with the extracellular loops (E’s 1–3) and these interactions probably maintain the basal state. B) In contrast, deletion of residues 18–35, a partial deletion, increases the inverse agonistic effect of the remaining eNDo residues on GPR83 and suppresses basal activity, by presumptively stabilizing the inactive conformation. C) Deletion of residues 35–65 of the eNDo leads to slight receptor activation, possibly due to the partial loss of intramolecular interactions with the extracellular loops. D) The GPR83 becomes highly active following removal of the entire N-terminal domain. Ctt = C-terminal tail, I = intracellular loop, E = extracellular loop.