Mutually Opposite Signal Modulation by Hypothalamic Heterodimerization of Ghrelin and Melanocortin-3 Receptors

Interaction and cross-talk of G-protein-coupled receptors (GPCRs) are of considerable interest because an increasing number of examples implicate a profound functional and physiological relevance of homo-or hetero-oligomeric GPCRs. The ghrelin (growth hormone secretagogue receptor (GHSR)) and melanocortin-3 (MC3R) receptors are both known to have orexigenic effects on the hypothalamic control of body weight. Because in vitro studies indicate heterodimerization of GHSR and MC3R, we investigated their functional interplay. Combined in situ hybridization and immunohistochemistry indicated that the vast majority of GHSR-expressing neurons in the arcuate nucleus also express MC3R. In vitro coexpression of MC3R and GHSR promoted enhanced melanocortin-induced intracellular cAMP accumulation compared with activation of MC3R in the absence of GHSR. In contrast, agonist-independent basal signaling activity and ghrelin-induced signaling of GHSR were impaired, most likely due to interaction with MC3R. By taking advantage of naturally occurring GHSR mutations and an inverse agonist for GHSR, we demonstrate that the observed enhanced MC3R signaling capability depends directly on the basal activity of GHSR. In conclusion, we demonstrate a paradigm-shifting example of GPCR heterodimerization allowing for mutually opposite functional influence of two hypothalamic receptors controlling body weight. We found that the agonistin dependent active conformation of one GPCR can determine the signaling modalities of another receptor in a heterodimer. Our discovery also implies that mutations within one of two interacting receptors might affect both receptors and different pathways simultaneously. These findings uncover mechanisms of important relevance for pharmacological targeting of GPCR in general and hypothalamic body weight regulation in particula

Concentration-response curve of zinc(II)- stimulation at mGPR83.

<p>HEK293 cells were transiently transfected with the empty expression vector pcDps (mock) or pcDps encoding the wild type <i>Gpr83</i>. Two days after transfection, stimulation with 1 nM – 1 mM ZnCl₂ was carried out, cells were lysed and IP₃-accumulation was measured in a reporter gene assay. The hTSHR stimulated with 100 mU/ml bTSH functioned as assay control [data not shown, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-VanSande1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-Allgeier1" target="_blank">[32]</a>. The EC₅₀ value of the wild type mGPR83 is 10.2 ± 1.4 µM zinc(II) and was obtained from the concentration-response curve (1 nM – 1 mM Zn(II)) using GraphPad Prism. First asterisk indicates a significant increase in IP₃ formation in comparison to basal wild type. Second asterisks indicate significance in comparison to the first asterisk. Data were evaluated from a minimum of 3 independent experiments, each performed at least in triplicates and calculated fold over the mock transfection, with 24718.3 ± 3958.7 relative light units, set to 1. Shown data represent mean ± SEM. * p<0.05, ** p<0.01 (unpaired t-test, two-tailed).</p

mGPR83 signals via the Gq/11 pathway revealed by basal signaling activity, CAMs and zinc(II)-stimulation.

<p>HEK293 cells were transiently transfected with the empty expression vector pcDps (mock) or pcDps carrying the wild type <i>Gpr83</i> or the <i>Gpr83 C304W</i> mutant, respectively. Two days after transfection, stimulation with ZnCl₂ (1 nM – 1 mM; stimulation curve in Fig. 2; black column: 100 µM) was carried out and cells were lysed. IP₃-accumulation was measured in a reporter gene assay. The hTSHR stimulated with 100 mU/ml bTSH functions as assay control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-VanSande1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-Allgeier1" target="_blank">[32]</a>. Data were assessed from a minimum of 3 independent experiments, each performed at least in triplicates and represent mean ± SEM calculated fold over basal mock transfection with 19775.4 ± 2259.9 relative light units, set to 1. *** p<0.001 (unpaired t-test, two-tailed).</p

Structural mGPR83 homology model with sensitive positions for constitutive receptor activation and zinc(II)-stimulation.

<p>This structual GPR83 homology model is based on the crystal structure of rhodopsin in the inactive state. Highlighted wild type amino acids were depicted from this model for experimental approaches, because they are located extracellularly at the ECLs or at the extracellular ends of the TMHs and those amino acids are known as putative determinants of metal-ion binding motifs: histidine, glutamate, asparagine or cysteine. Arranged in defined spatial arrangements they can interact e.g. with zinc(II)-ions. Interestingly, six side-chain substitutions abolished stimulation by zinc(II) (green sticks) and five substitutions at different positions expressed an increase in constitutive signaling activity of mGPR83 (red sticks). The are spatially clustered in two different regions – red cycle (CAMs) and green (zinc(II)-binding) full cycle. In summary, they are indicating the extracellular region of the mGPR83 as highly sensitive for activation. Cysteine 304 (blue stick) at TMH6 is one of the highly conserved family A GPCR residues and it was reported for several receptors that mutations here are leading almost always to constitutive receptor activation. Indeed, also the mGPR83 Cys304Trp mutation causes a slight ligand independent (constitutive) activation of Gq/11 mediated signaling pathways.</p

Functional characterization of amino acids that could be involved in zinc(II)-binding.

<p>For cell surface expression studies COS-7 cells were transiently transfected with the empty expression vector pcDps (mock), <i>Gpr83</i> wild type or <i>Gpr83</i> mutants. HEK293 cells were used for functional characterization. Data were evaluated from three or four independent experiments, each performed at least in triplicates. IP₃ accumulation performed as reporter gene assay was calculated fold over the basal mock transfection with 24718.3 ± 3958.7 relative light units, set to 1. The hTSHR stimulated with 100 mU/ml bTSH functioned as control for Gq/11 activation [data not shown, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-VanSande1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-Allgeier1" target="_blank">[32]</a>]. Shown data represent mean ± SEM. The mutated aminoacid residues are grouped into extracellular located Ds (Asp) and És (Glu), Hs (His) and one C (Cys) that could be involved in Zn(II)-binding. Asteriks indicate significant higher basal activity in comparison to wild type. ** p<0.01, *** p<0.001 (unpaired t-test, two-tailed); Ntt – N-terminal tail.</p

Homodimerization of mGPR83.

<p>Dimerization studies were performed using sandwich ELISA. COS-7 cells were transiently transfected. As negative control serves the NHA-hGHSR (white column) and as positive control the co-transfection of NHA-tagged hMC3R and FLAG-tagged hGHSR (black column, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-Rediger1" target="_blank">[28]</a>). The light grey column represents the average of the HA- respectively FLAG-tagged mGPR83 in combination with the correspondent tagged rM3R. The dark grey column represents co-transfection of HA-tagged mGPR83 and FLAG-tagged mGPR83. Dimerization was measured via the HA epitope. The mean absorption (492 nm/620 nm) is calculated per 1 mg/ml of protein and shown as percentage of the hMC3R/hGHSR heterodimer (absorption (492/620)/ 1mg/ml of protein: 0.3 ± 0.04). Data were assessed from 3 independent experiments, each performed in triplicates and represent mean ± SEM.</p

The orphan receptor Gpr83 regulates systemic energy metabolism via ghrelin-dependent and ghrelin-independent mechanisms

The G protein-coupled receptor 83 (Gpr83) is widely expressed in brain regions regulating energy metabolism. Here we report that hypothalamic expression of Gpr83 is regulated in response to nutrient availability and is decreased in obese mice compared with lean mice. In the arcuate nucleus, Gpr83 colocalizes with the ghrelin receptor (Ghsr1a) and the agouti-related protein. In vitro analyses show heterodimerization of Gpr83 with Ghsr1a diminishes activation of Ghsr1a by acyl-ghrelin. The orexigenic and adipogenic effect of ghrelin is accordingly potentiated in Gpr83-deficient mice. Interestingly, Gpr83 knock-out mice have normal body weight and glucose tolerance when fed a regular chow diet, but are protected from obesity and glucose intolerance when challenged with a high-fat diet, despite hyperphagia and increased hypothalamic expression of agouti-related protein, Npy, Hcrt and Ghsr1a. Together, our data suggest that Gpr83 modulates ghrelin action but also indicate that Gpr83 regulates systemic metabolism through other ghrelin-independent pathways