Heterodimerization of hypothalamic G-protein-coupled receptors involved in weight regulation

Abstract

Background: Melanocortin 3 and 4 receptors (MC3R and MC4R) are known to play an essential role in hypothalamic weight regulation. In addition to these two G-protein-coupled receptors (GPCRs), a huge number of other GPCRs are expressed in hypothalamic regions, and some of them are involved in weight regulation. So far, homodimerization was shown for a few of these receptors. Heterodimerization of unrelated receptors may have profound functional consequence but heterodimerization of GPCRs involved in weight regulation was not reported yet.

Methods: A selective number of hypothalamically expressed GPCRs were cloned into a eukaryotic expression vector. Cell surface expression was demonstrated by an ELISA approach. Subcellular distribution was investigated by confocal laser microscopy. A sandwich ELISA and fluorescence resonance energy transfer (FRET) were used to determine protein-protein interaction.

Results: Via sandwich ELISA and FRET approach we could demonstrate a robust interaction of the MC4R with GPR7, both of which are expressed in the hypothalamic nucleus paraventricularis. Moreover, we determined a strong interaction of MC3R with the growth hormone secretagogue receptor expressed in the nucleus arcuatus.

Conclusion: Identification GPCR heterodimerization adds to the understanding of the complexity of weight regulation and may provide important information to develop therapeutic strategies to treat obesity.

Fig. 1

a Influence on food intake, expression and G-protein coupling of hypothalamic GPCRs involved in body weight regulation. b Schematic illustration of major nuclei of the hypothalamus

Fig. 2

Cellular localization and cell surface expression of the analyzed GPCRs a HEK293 cells were transiently transfected with expression plasmids encoding MC3R, GHSR, NPY2R, µ-OPR or MC4R, GPR7, 5HTR_1B, CB1R carboxyterminally fused with yellow fluorescent protein (YFP). Cells were imaged by confocal laser scanning microscopy 24 h after transfection. Excitation and emission wavelengths for YFP were 488 nm and 515–550 nm. Confocal images of typical cells are shown. b To investigate cell surface expression COS-7-cells were transiently transfected by N-terminal HA-tagged GPCRs. 48 h after transfection the extracellular HA-epitope was detected with a biotin-labelled antibody and peroxidase-labelled streptavidin. Values are mean ± SEM of at least four independent experiments each performed in hexapletts. One way ANOVA and Dunnett tests were conducted for the difference of the positive control rM3R (rat muscarinic receptor, rM3R) and the different GPCRs (* p < 0.01).

Fig. 3

Investigation of dimerization of a MC3R or b MC4R with other GPCRs by sandwich-ELISA. COS-7 cells were co-transfected with differentially tagged GPCR constructs, solubilized overnight and incubated in FLAG-antibody-coated 96-well plates. Dimerization was measured via the N-terminally tagged HA epitope as increase in optical density. MC3R-C-Flag was co-transfected with GHSR-, NPY2R – or µ-OPR-N-HA (orange), while MC4R-C-Flag was co-transfected with GPR7- or 5HTR_1B (blue). As a negative control CB1R was co-expressed with equal amounts of MC3R- and MC4R-C-Flag (beige). The homomeric combination of MC3R-C-Flag and MC3R-N-HA like MC4R-C-Flag and MC4R-N-HA served as positive control (open bars). The mean absorption (492 nm / 620 nm) is shown as percentage of the FLAG-tagged MC3R or MC4R alone of three independent experiments. One way ANOVA and Dunnett tests were performed to determine the significance of the heterodimerization in contrast to the MC3R or MC4R homodimer (**p < 0.01, *p < 0.05).

Fig. 4

FRET analysis of living HEK293 cells transiently co-transfected with a MC3R and GHSR, NPY2R or µ-OPR, or b MC4R and GPR7 or 5HTR_1B. HEK293 cells were co-transfected with plasmids encoding MC3R(oronge)- or MC4R(blue)-YFP and GHSR- NPY2R-, µ-OPR-, GPR7- or 5HTR_1B-CFP. Cotransfection of CB1R with even amounts of MC3R or MC4R served as negative control (beige). As positive control the homodimer of MC3R and MC4R was used (open bars). To prove the significance of heterodimerization compared to the MC3R or MC4R homodimer one way ANOVA and Dunnett tests were performed (**p < 0.01, *p < 0.05). Emission light of a characteristic wavelength is detectable. FRET efficiencies (E) were calculated from the relative increase in CFP emission and the decrease in YFP emission during selective photobleaching at 512 nm. The depicted data represent means ± SEM of 4–6 single cells of one representative measurement.

Fig. 5

Subcellular localization after cotransfection HEK293 cells were transiently co-transfected with expression plasmids encoding MC3R-YFP or MC4R-YFP and GHSR, NPY2R, µ-OPR or MC4R, GPR7, 5HTR_1B, CB1R coupled to CFP. Cells were imaged by confocal laser scanning microscopy 24 h after transfection. CFP-tagged receptors were visualized by excitation at 458 nm, whereas YFP-tagged GPCRs were excitated at 488 nm. Emission wavelengths were detected by using a LP505 filter. Representative cells are shown.