The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization

Abstract

One-third of the approximately 400 nonodorant G protein-coupled receptors (GPCRs) are still orphans. Although a considerable number of these receptors are likely to transduce cellular signals in response to ligands that remain to be identified, they may also have ligand-independent functions. Several members of the GPCR family have been shown to modulate the function of other receptors through heterodimerization. We show that GPR50, an orphan GPCR, heterodimerizes constitutively and specifically with MT(1) and MT(2) melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT(2) did not modify MT(2) function, GPR50 abolished high-affinity agonist binding and G protein coupling to the MT(1) protomer engaged in the heterodimer. Deletion of the large C-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT(1) without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT(1). Pairing orphan GPCRs to potential heterodimerization partners might be of clinical importance and may become a general strategy to better understand the function of orphan GPCRs.

Figure 1

Detection of GPR50 homo- and heterodimers by SDS–PAGE and co-immunoprecipitation. (A) Lysates from HEK 293 cells stably expressing Flag-GPR50-YFP were separated by SDS–PAGE and analysis was performed by Western blot using an anti-GFP antibody. (B–D) Crude membranes were prepared from HEK 293 cells transiently expressing GPR50-YFP alone or with Flag-GPR50 (B), Flag-MT₁ (C) or Myc-MT₂ (D). GPR50-YFP was immunoprecipitated with a monoclonal anti-GFP antibody. Membranes and immnunoprecipitates were then separated by SDS–PAGE and analysis was performed by Western blot using polyclonal anti-Flag (B,C) or anti-Myc (D) antibodies. Similar results were obtained in three additional experiments. mb=membrane; IP=immunoprecipitation, M=monomer; D=dimer.

Figure 2

Constitutive dimerization of GPR50 in living HEK 293 cells. (A) GPR50-Rluc was transiently coexpressed in HEK 293 cells with the indicated C-terminal YFP fusion proteins expressed at comparable amounts as determined by direct fluorescence measurements (20–30 fmol of YFP fusion receptor per mg of protein as estimated from curves correlating YFP fluorescence with the number of ligand-binding sites (Ayoub et al, 2002)). BRET signals generated for the MT₁ homodimer (MT₁-Rluc/MT₁-YFP), when expressed at comparable amounts, were used as internal control. BRET measurements were performed in living cells by adding 5 μM coelenterazine. Data are means±s.e.m. of at least three independent experiments each performed in duplicate. (B–D) BRET donor saturation curves were generated by transfecting transiently HEK 293 cells with a constant DNA amount of GPR50-Rluc and increasing quantities of the indicated YFP-tagged receptors. The BRET, total luminescence and total fluorescence were measured. The curves represent 3–5 individual saturation curves. Curves obtained for the BRET acceptors GPR50-YFP, MT₁-YFP and MT₂-YFP were best fitted with a nonlinear regression equation assuming a single binding site, those obtained for β₂-AR-YFP and CCR5-YFP were best fitted with a linear regression equation.

Figure 3

¹²⁵I-MLT binding to MT₁ and MT₂ in the presence of GPR50. (A, B) HEK 293 cells stably expressing GPR50-YFP (HEK-GPR50) transiently expressed 5–10 fmol of MT₁-Rluc per mg of protein (A) or 10–15 fmol of MT₂-Rluc per mg of protein (B). Expression was monitored with the luciferase assay and by ¹²⁵I-MLT binding (500 pM). (C) HEK 293 cells stably expressing 70–80 fmol of Flag-MT₁ per mg of protein were transfected with untagged GPR50 or C-terminal YFP fusion constructs of GPR50, MT₂ or MT₂C113A. Expression of YFP fusion proteins was monitored by measuring YFP fluorescence, relative expression of GPR50 and GPR50-YFP was detected by Western blot using anti-GRP50-specific antibodies (not shown). Expression of Flag-MT₁ was monitored by flow cytometry using anti-Flag antibodies (not shown) and by ¹²⁵I-MLT binding. (D) Increasing concentrations of GPR50-YFP were expressed in HEK 293 cells stably expressing Flag-MT₁, and YFP fluorescence, ¹²⁵I-MLT binding and MT₁ surface expression were determined. (E) HEK 293 cells transiently expressing MT₁-Rluc in the absence (▪) or presence of GPR50-YFP (▵) were incubated with increasing concentrations of ¹²⁵I-MLT. Data are means±s.e.m. of at least three independent experiments each performed in duplicate (A–C) or are representative of three further experiments (D, E) (^***P<0.001; NS, P>0.05).

Figure 4

Absence of ¹²⁵I-MLT binding to GPR50/MT₁ heterodimers. Membranes from HEK 293 cells transiently expressing 30–40 fmol of Flag-MT₁ alone or in the presence of the indicated receptors were labeled with ¹²⁵I-MLT (500 pM), solubilized and immunoprecipitated with a monoclonal anti-GFP antibody. The amount of precipitated ¹²⁵I-MLT was determined in a γ-counter and precipitates were subsequently separated by SDS–PAGE. The presence of Flag-MT₁ was verified by Western blotting using polyclonal anti-Flag antibodies. Data are means of triplicates that are representative of two further experiments. mb=membranes; IP=immunoprecitate.

Figure 5

GPR50 antagonizes MT₁ signaling. HEK 293 cells transiently expressing MT₁-Rluc alone (▪) or with GPR50-YFP (Δ) were stimulated with increasing concentrations of melatonin (A) or S20098 (B) and inositol phosphate levels were determined. MT₁-Rluc expression levels were determined by luminescence measurements (inset). Data are means±s.e.m. of three independent experiments each performed in duplicate. A nonlinear regression equation assuming a single binding site was used to fit the data (GraphPad Prism software).

Figure 6

Downregulation of endogenously expressed GPR50 in hCMEC/D3 cells promotes MT₁ function. (A) GPR50 and MT₁ transcripts from hCMEC/D3 cells were reverse transcript and amplified by PCR. No amplification was observed when experiments were performed in the absence of reverse transcriptase (not shown). (B) HEK 293 cells stably expressing GPR50-YFP were transfected with GPR50-specific siRNA duplexes (100 nM) and GPR50-YFP fluorescence was measured by flow cytometry 48 h post-transfection. (C–E) Effect of control siRNA and GPR50-specific siRNA duplexes on GPR50 mRNA levels (C), ¹²⁵I-MLT binding (500 pM) (D), and forskolin-stimulated cAMP accumulation (E) in hCMEC/D3 cells. Stimulation with 1 μM forskolin alone (black bars) or in the presence of 1 μM melatonin (white bars) or 1 μM S20098 (hatched bars) (30 min). NT, nontransfected; bp, base pairs. Data are means±s.e.m. of at least three independent experiments each performed in triplicate (^***P<0.001; ^*P<0.05).

Figure 7

Subcellular localization of MT₁ and GPR50. (A, B) Confocal images of HEK 293 cells stably expressing Flag-MT₁ or GPR50-YFP. (C–E) Localization of Flag-MT₁ and GPR50-YFP in HEK 293 cells transiently coexpressing both receptors. Flag-MT₁ was detected by immunodetection after permeabilization and GPR50-YFP by measuring the YFP fluorescence. (F, G) ELISA quantification of Flag-MT₁ (F) and Flag-GPR50 (G) surface expression in the absence (black bars) and presence of melatonin (white bars). Data are means±s.e.m. of at least three independent experiments each performed in duplicate.

Figure 8

The inhibitory effect of GPR50 on MT₁ function in the GPR50/MT₁ heterodimer depends on the C-terminus of GPR50. (A, B) HEK 293 cells stably expressing 70–80 fmol of Flag-MT₁ per mg of protein were coexpressed with the indicated proteins. Expression of comparable amounts of YFP fusion proteins was verified by measuring YFP fluorescence (not shown). Expression of the Cter was monitored by Western blot using anti-GPR50 antibodies. (A) Surface expression of Flag-MT₁ was determined by flow cytometry using anti-Flag antibodies (white bars) and by radioligand binding using a saturating concentration of ¹²⁵I-MLT (black bars) (^***P<0.001 compared to NT condition). (B) Cells were stimulated for 30 min with 1 μM forskolin alone (black bars) or in the presence of 1 μM melatonin (white bars) or 1 μM S20098 (hatched bars) and cAMP levels were determined (^**P<0.01; NS, P>0.05 compared with corresponding NT condition). (C) GPR50ΔCter-Rluc was transiently coexpressed in HEK 293 cells with the indicated C-terminal YFP fusion proteins expressed at comparable amounts as determined by direct fluorescence measurements (20–30 fmol of YFP fusion receptor per mg of protein as estimated from curves correlating YFP fluorescence with the number of ¹²⁵I-MLT-binding sites (Ayoub et al, 2002)). BRET measurements were performed in living cells by adding 5 μM coelenterazine. All data are means±s.e.m. of at least three independent experiments each performed in duplicate. NT, nontransfected.

Figure 9

Binding of the GPR50/MT₁ heterodimer to G_i proteins and β-arrestin. (A) Membranes from HEK 293 cells transiently expressing 5–10 fmol of MT₁-YFP alone or in the presence of the indicated receptors were incubated with melatonin and solubilized. Equivalent quantities of YFP fusion proteins were immunoprecipitated, separated by SDS–PAGE and analyzed by Western blot. Data are representative of three experiments. (B) Dynamics of the interaction between MT₁ and β-arrestin. BRET signals were monitored during 30 min after the addition of melatonin (1 μM) in HEK 293 cells coexpressing MT₁-Rluc (MT₁) and YFP-β-arrestin (βArr1) with or without Flag-GPR50 (3 μg vector). (C) Graphic representation of mean melatonin-induced and basal BRET signals measured 15–30 min after melatonin addition. Values are depicted from curves presented in (B) and from additional curve generated from cells transfected with 1 μg of GPR50 expression vector. Data are means±s.e.m. of at least three independent experiments each performed in duplicate.