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.2020 Jun 9;117(23):13105-13116.
doi: 10.1073/pnas.1917906117. Epub 2020 May 26.

In vivo mapping of a GPCR interactome using knockin mice

Affiliations

In vivo mapping of a GPCR interactome using knockin mice

Jade Degrandmaison et al. Proc Natl Acad Sci U S A..

Abstract

With over 30% of current medications targeting this family of proteins, G-protein-coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.

Keywords: G-protein–coupled receptors; GPCR interactome; mass spectrometry; mouse model; δ-opioid receptor.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Generation of FLAG-DOPr-KI mice. (A) Schematic representation of the generation of FLAG-DOPr-KI mice obtained by breeding FLAG-DOPr-KO mice with Zp3-Cre mice. In KO mice, a sequence encoding the FLAG tag epitope was introduced immediately after the START codon, in the 5′-end of the DOPr coding sequence. A translational STOP cassette flanked by two loxP sites was also inserted in the 5′-untranslated region (5′-UTR) of theOPRD1 gene to disable the expression of FLAG-DOPr. Breeding with Zp3-Cre mice enables the excision of the STOP cassette, therefore allowing the expression of FLAG-DOPr instead of the endogenous DOPr in all tissues normally expressing DOPr. Genotypes were confirmed by PCR using various combinations of primers (B–D). For each genotype, primer locations are indicated with black arrowheads (B), and the expected lengths of the fragments generated by PCR are shown (C). The complete sequence of each primer can be found inSI Appendix, Table S5. (D) The PCR amplification using pDOPr-01 and 02 (Upper) distinguishes between WT (150 bp), homozygous transgenic (174 bp), and heterozygous (HET) (150 and 174 bp) mice, while the amplification using the specific sequence encoding the FLAG tag epitope resulted in a band of 331 bp for all genotypes except WT (pDOPr-03 and 05;Middle). The presence of an intact STOP cassette is shown by a band of 520 bp in KO and HET(KO) mice (pDOPr-04 and 05,Bottom).
Fig. 2.
Fig. 2.
Expression and distribution of FLAG-DOPr in the CNS of KI mice. (A) Autoradiography of125I-DLT binding on sagittal and coronal brain sections of WT, KI, and KO mice. (B) Pharmacological properties (Kd andBmax) of DOPr (either WT or FLAG-DOPr) expressed in WT (red squares), KI (blue circles), and KO mice (orange triangles) were evaluated with saturation binding assays on brain membranes using125I-DLT. In the absence of residual specific binding in KO mice,Kd andBmax were not calculated. Results are the mean ± SEM of four independent experiments, each performed in triplicates.
Fig. 3.
Fig. 3.
Functional activation of FLAG-DOPr in KI mice. SNC80 (10 mg/kg; s.c.)-induced locomotor activity was assessed in WT, (A) KI (WT,n = 5; KI,n = 6; *P = 0.0102, **P = 0.0032, andφP = 0.0170; one-way ANOVA followed by Tukey’s post hoc test), and (B) KO mice (WT,n = 5–7; KO,n = 6; **P = 0.0038, ***P = 0.0004, andφφP = 0.0056; one-way ANOVA followed by Tukey’s post hoc test). DLT II (1 µg; i.t.)-induced antihyperalgesic effects was evaluated in WT, KO, and (C) KI mice using the Hargreaves test and the CFA model of inflammation (WT,n = 11 and ***P = 0.0005; KI,n = 7 and ***P = 0.0004; two-way ANOVA followed by Tukey’s post hoc test compared to KO [n = 11]) or (D) KO mice intrathecally injected with the recombinant adenoassociated virus AAV2/9-CBA-Cre (WT,n = 11 and ****P < 0.0001; KO + AAV2/9-CBA-Cre,n = 8 and *P = 0.0330; two-way ANOVA followed by Tukey’s post hoc test compared to KO [n = 8]). (E andF) The integrity of the G-protein coupling of DOPr (and FLAG-DOPr) was evaluated using the35S-GTPγS binding assay on coronal sections of olfactory bulb (20 µm) from WT, KI, and KO mice.35S-GTPγS binding density is shown in color scale (E). Quantification of binding signal was carried out on olfactory bulb sections (WT,n = 7 and ***P = 0.0004; KO,n = 4 andP = 0.1457; KI,n = 4 and *P = 0.0182; one-sample Wilcoxon test) from three independent experiments. Data are expressed as fold increase in GTPγS binding for nontreated (basal) and DLT II-treated sections (activated). Nonspecific binding was assessed in the presence of 10 µM GTPγS. n.s., not significant.
Fig. 4.
Fig. 4.
Identification of potential DOPr-interacting proteins from brains of FLAG-DOPr-KI mice. (A) Immunoprecipitation of FLAG-DOPr (∼50-kDa band) was performed on forebrain lysates from WT, KI, and KO mice using a M2 mouse monoclonal anti-FLAG antibody immobilized on magnetic beads. Immunoblotting was carried out using a rabbit polyclonal anti-FLAG antibody. Following LC-MS/MS analysis of immunoprecipitated FLAG-DOPr from the forebrain of KI mice, identified DOPr-interacting proteins were classified according to their cellular localization using the PANTHER Classification System online tool (B) and reported molecular functions covering intracellular trafficking (blue), folding (purple), signal transduction (yellow), and proteins belonging to the receptor/channel/transporter families (red) (C). Previously reported DOPr interactors are identified in green (C). In order to validate an endogenous interaction, immunoprecipitation of FLAG-DOPr from brain lysates of KO and KI mice was carried out using a M2 mouse monoclonal anti-FLAG antibody immobilized on magnetic beads and immunoblotting was performed using a rabbit polyclonal anti-VPS41 antibody (D). Further validation of DOPr-interacting proteins was assessed in lysates of HEK293 cells transiently expressing the human FLAG-DOPr and (E) ARF6-HA or (F) HA-Rabaptin-5 using a M2 mouse monoclonal anti-FLAG antibody. Immunoblotting of the receptor was carried out with FLAG-specific rabbit polyclonal antibody (D–F), and ARF6-HA and HA-Rabaptin-5 were immunoblotted using HA-specific HRP-conjugated antibodies. (A andD–F) Blots shown are representative of at least three independent experiments. IB, immunoblotting; IP, immunoprecipitation.
Fig. 5.
Fig. 5.
Identification of the ICL3 and CT of DOPr as the major molecular determinants for the interaction with Rab10. (A) Immunoprecipitation of FLAG-DOPr was performed in HEK293 cells transiently expressing FLAG-DOPr and HA-Rab10 using a M2 mouse monoclonal anti-FLAG antibody. (B) In vitro binding assays were carried out using purified GST or the DOPr intracellular loops (ICLs) or C terminus as GST-fusion proteins incubated with purified recombinant (His)6-HA-Rab10. Sequences of the GST-fused protein constructs are listed inSI Appendix, Table S6. Rab10 binding to the receptor was detected by immunoblotting using HA-specific HRP-conjugated antibody, and the GST fusion proteins present in the binding reaction were detected using an anti-GST polyclonal antibody.
Fig. 6.
Fig. 6.
Rab10 colocalizes with DOPr in Golgi-associated compartments. HEK293 cells transiently transfected with FLAG-DOPr alone (A) or together with GalT-YFP (B) a marker of the Golgi apparatustrans cisternae were fixed and prepared as described inSI Appendix. FLAG-DOPr was labeled with a rabbit polyclonal anti-FLAG antibody and an Alexa Fluor 594 goat anti-rabbit IgG antibody (a, green). Endogenous Rab10 was labeled with a specific mouse monoclonal anti-Rab10 antibody and an Alexa Fluor 647 goat anti-mouse IgG antibody (b, red). GalT-YFP was labeled with a chicken anti-GFP antibody and an Alexa Fluor 488 goat anti-chicken IgY antibody (B,c, blue). In merge panels, colocalization between FLAG-DOPr and Rab10 appears in yellow (A,c, andB,d), and triple colocalization between FLAG-DOPr, Rab10, and GalT-YFP appears in white (B,d). The images shown are single confocal slices and are representative of ∼100 observed cells over three independent experiments. (Scale bars, 10 µm.)
Fig. 7.
Fig. 7.
Rab10 and AS160 regulate DOPr trafficking. (A) HEK293 cells were transiently transfected with FLAG-DOPr alone or with HA-Rab10 for 48 h. Cell surface expression was measured by ELISA with FLAG-specific rabbit polyclonal and alkaline phosphatase-conjugated goat anti-rabbit antibodies. Results are shown as a percentage of cell surface FLAG-DOPr expression when HA-Rab10 is transfected compared with FLAG-DOPr alone set at 100% (n = 4, **P = 0.0100, two-tailed Student’st test). (B–F) HEK293 cells stably expressing FLAG-DOPr were transfected with a control DsiRNA (DsiCTRL), (B–D) DsiRNAs targeting the exon 6 or 3 of the human Rab10 gene (DsiRNA-Rab10-13.2 or DsiRNA-Rab10-13.3, respectively), or (E andF) DsiRNAs targeting the exon 3 or 15 of the human AS160 gene (DsiRNA-AS160-13.1 or DsiRNA-AS160-13.2, respectively) for 72 h along with HA-Rab10 for 48 h. (B andE) Cell surface expression of FLAG-DOPr was measured as described above. Results are shown as a percentage of cell surface receptor expression when DsiRNAs targeting Rab10 or AS160 are transfected compared with the DsiCTRL condition set at 100% (B:n = 5, ****P < 0.0001;E:n = 6, **P = 0.0076 and ****P < 0.0001; one-way ANOVA with Dunnett’s multiple-comparisons test). (C) Total cell lysates were immunoblotted with a specific mouse monoclonal anti-Rab10 antibody to assess the effect of Rab10 DsiRNAs on total expression of Rab10. (F) Following RNA extraction, RT-PCRs were performed to assess the effect of AS160 DsiRNAs on total expression of AS160. Primers used are listed inSI Appendix, Table S5. Densitometry was performed using ImageJ software to quantify relative expression of Rab10 or AS160 normalized with GAPDH expression, and results were analyzed using one-way ANOVA with Dunnett’s multiple-comparisons test (C:n = 3, ***P = 0.0003 andφφφP = 0.0001;F:n = 4, ****P < 0.0001). IB, immunoblotting. (D) Cells were stimulated with SNC80 (1 µM) for up to 60 min, and cell surface expression of FLAG-DOPr was measured by ELISA as mentioned above (n = 4, *P = 0.0285, **P = 0.0017 and ***P = 0.0001; two-way ANOVA with Dunnett’s multiple-comparisons test).
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