GPR37 protein trafficking to the plasma membrane regulated by prosaposin and GM1 gangliosides promotes cell viability

Abstract

The subcellular distribution of the G protein-coupled receptor GPR37 affects cell viability and is implicated in the pathogenesis of parkinsonism. Intracellular accumulation and aggregation of GPR37 cause cell death, whereas GPR37 located in the plasma membrane provides cell protection. We define here a pathway through which the recently identified natural ligand, prosaposin, promotes plasma membrane association of GPR37. Immunoabsorption of extracellular prosaposin reduced GPR37(tGFP) surface density and decreased cell viability in catecholaminergic N2a cells. We found that GPR37(tGFP) partitioned in GM1 ganglioside-containing lipid rafts in the plasma membrane of live cells. This partitioning required extracellular prosaposin and was disrupted by lipid raft perturbation using methyl-β-cyclodextrin or cholesterol oxidase. Moreover, complex formation between GPR37(tGFP) and the GM1 marker cholera toxin was observed in the plasma membrane. These data show functional association between GPR37, prosaposin, and GM1 in the plasma membrane. These results thus tie together the three previously defined components of the cellular response to insult. Our findings identify a mechanism through which the receptor's natural ligand and GM1 may protect against toxic intracellular GPR37 aggregates observed in parkinsonism.

Keywords: Fluorescence Correlation Spectroscopy; G Protein-coupled Receptors (GPCR); Ganglioside; Lipid Raft; Pael-R; Parkinson Disease.

FIGURE 1.

FCS at the apical plasma membrane of N2a cells expressing GPR37^tGFP. A, left panel, confocal image of an N2a cell expressing GPR37^tGFP. For FCS analysis, the observation volume element (drawn to scale) was positioned above the cell nucleus, which has the lowest level of autofluorescence. Scale bar, 5 μm. Right panel, axial fluorescence intensity scan (z-scan) was used to localize the position of the apical plasma membrane. The first maximum reflects the position of the basal membrane, and the second one reflects the position of the apical membrane. B, typical autocorrelation curve obtained after a single 10-s measurement of fluorescence intensity fluctuations at the apical plasma membrane of an N2a cell expressing GPR37^tGFP. The corresponding fluorescence intensity fluctuations are shown in the inset. C, autocorrelation curve reflecting a sporadic observation of a very bright intracellular vesicle traveling through the observation volume element. The corresponding fluorescence intensity fluctuations are shown in the inset. Such individual recordings were excluded from further analysis and did not contribute to the average autocorrelation curves.

FIGURE 2.

Fluorescence intensity fluctuations recorded at the apical plasma membrane of N2a cells expressing GPR37^tGFP are generated by molecular movement. A, autocorrelation curves recorded at varying observation volume sizes show that the amplitudes of the GPR37^tGFP autocorrelation curves decrease as the pinhole size in front of the detector is increased, reflecting an increase in the number of molecules in the observation volume element. B, for varying sizes of the observation volume element, a linear relationship was observed between the number of observed molecules (N) and the (pinhole size). C, autocorrelation curves at two different pinhole sizes show that characteristic times increase with increasing the observation volume size. D, linear relationship was observed between the first characteristic time (τ_D1) and the (pinhole size). The positive intercept suggests that partitioning between domains may occur. E, linear relationship was observed between the second characteristic time (τ_D2) and the (pinhole size). The negative intercept suggests that some confinement by the cytoskeletal protein meshwork may also occur. F, linear relationship between the number of molecules characterized by slow (N₂) and fast (N₁) diffusion was observed for different surface densities of GPR37^tGFP.

FIGURE 3.

WT and GPR37^tGFP-expressing N2a cells both express low levels of endogenous GPR37 and secrete PSAP but respond differently to inhibition of extracellular PSAP. A, Western blot for GPR37 on lysates from WT and GPR37^tGFP-expressing cells showing a weak band at the size corresponding to untagged GPR37 in both cell types and an additional band at the size corresponding to GPR37^tGFP in the GPR37^tGFP-expressing cells. B, Western blot for extracellular PSAP using medium from WT and GPR37^tGFP cells shows that both cell types secrete equal amounts of PSAP (n = 9, p = 0.99). C, estimate of PSAP concentrations in the conditioned medium of differentiated GPR37^tGFP cells based on Western blot analysis. The calibration curve was generated using a standard series of PSAP solutions that were analyzed on the same gel as the conditioned medium. D, immunoprecipitation of PSAP from conditioned cell medium using anti-PSAP (well 1) or normal rabbit IgG (well 2) as a negative isotype control. 40 μg ml⁻¹ anti-PSAP precipitates 83–96% of all PSAP compared with beads alone. Western blot films show three separate immunoprecipitation experiments. E, quantification of GPR37 density at the plasma membrane versus cytoplasm before and after inhibition of extracellular PSAP using anti-PSAP (n = 15 visual fields). Representative images of live cells show distribution of GPR37 with or without anti-PSAP. Data from Western blots and GPR37 density were analyzed by Student's t test. Data from MTT assays were analyzed by two-way analysis of variance followed by t test for pairwise comparisons, ***, p < 0.001 compared with WT. F, MTT assay of WT and GPR37-overexpressing cells untreated or treated with the PSAP-specific antibodies anti-PSAP (antibody: F_(2,138) = 3.6, p < 0.05; cell type: F_(1,138) = 5.0, p < 0.05; interaction: F_(2,138) = 0.9, p = 0.41) or anti-PS769 (antibody: F_(1,60) = 42.4, p < 0.001; cell type: F_(1,60) = 9.2, p < 0.01; interaction: F_(1,60) = 9.6, p < 0.01) (n = 24 in each group). ^###, p < 0.001 compared with WT anti-PS769; *, p < 0.05 compared with WT anti-PSAP.

FIGURE 4.

GPR37 interacts with PSAP-derived PS-TX14(A). A, confocal microscopy shows partial co-localization of GPR37^tGFP (green) with PS-TX14(A)^TAMRA (red) in live cells. PSAP co-localizes with GPR37 at the plasma membrane and in intracellular endosomes/organelles. B, FCS for PS-TX14(A)^TAMRA at the plasma membrane compared with the bulk medium 200 μm above the cell. In the bulk medium, the diffusion time of PS-TX14(A)^TAMRA was τ_D = 73 μs. At the plasma membrane, 93% of the molecules showed fast diffusion, τ_D1 = 130 μs, and 7% diffused at τ_D2 = 14 ms. C, FCCS for GPR37^tGFP and PS-TX14(A)^TAMRA at the plasma membrane (same cell as in B) shows a significant cross-correlation with 90% of the complexes diffusing at τ_D1 = 410 μs and 10% at τ_D2 = 43 ms.

FIGURE 5.

Interactions of PS-TX14(A)^TAMRA with GPR37^tGFP are specific. A, autocorrelation curves (left panel) recorded in the bulk medium (blue) and on the surface of WT N2a cells (red) show that neither the diffusion nor the concentration of PS-TX14(A)^TAMRA are significantly altered at the plasma membrane of WT N2a cells, indicating that PS-TX14(A)^TAMRA does not bind nonspecifically to the plasma membrane of WT N2a cells. Representative images (right panel) show limited binding of PS-TX14(A)^TAMRA to membranes of WT N2a cells with low expression levels of GPR37. B, top left panel, FCCS measurements showed no cross-correlation between GPR37^tGFP and β-End^TAMRA. Bottom left panel, cross-correlation curves confirming interactions between PS-TX14(A)^TAMRA and GPR37^tGFP reflect specific binding (orange), as compared with lack of cross-correlation between β-End^TAMRA and GPR37^tGFP signal (brown). Right panel, representative images show absence of co-localization between β-End^TAMRA and GPR37^tGFP. C, autocorrelation and cross-correlation curves for GPR37^tGFP and PS-TX14(A)^TAMRA using the sequential illumination mode at a switching rate of 100 μs.

FIGURE 6.

GPR37 co-localizes with lysosomes but not with mitochondria. A, representative images of GPR37 and LysoTracker® showing co-localization in a majority of GPR37-containing intracellular vesicles. B, representative images of GPR37 and MitoTracker® showing no co-localization.

FIGURE 7.

Inhibition of extracellular PSAP decreases membrane localization of GPR37 to GM1-dense lipid rafts. Representative images are shown for co-localization of GPR37 and CTxB in live cells at baseline (A) or after inhibition of extracellular PSAP (B). C, average Pearson's correlation of pixel intensity, as well as Manders' coefficient for fraction of green pixels overlapping red, was calculated. For each group, calculations were made from ROIs comprising the plasma membrane of each cell in 30 randomly selected fields of vision, n_untreated = 69 cells and n_anti-PSAP = 70 cells. A marked reduction of the Pearson's correlation confirms reduced accumulation of GPR37 in GM1-dense membrane microdomains after inhibition of extracellular PSAP.

FIGURE 8.

Lipid rafts disruption with mβCD alters distribution and trafficking of GPR37, PS-TX14(A)^TAMRA, and the complex between GPR37^tGFP and PS-TX14(A)^TAMRA at the plasma membrane. A, comparison of FCCS curves for GPR37^tGFP and CTxB from untreated, mβCD-treated, or cholesterol oxidase-treated cells shows a significant cross-correlation (i.e. complex formation) between GPR37^tGFP and CTxB in control cells with 85% of the complexes diffusing at τ_D1 = 0.5 ms and 15% at τ_D2 = 0.2 s. This cross-correlation was largely disrupted by mβCD and cholesterol oxidase. B, FRAP measurements for GPR37^tGFP were performed on live cells (n = 23 cells for each treatment). Measurements were made on the same cell before and after treatment. Either mβCD or cholesterol oxidase increased GPR37^tGFP fluorescence recovery compared with untreated cells, and the average amplitude of the FRAP curves at t = 350 s was significantly higher after mβCD or cholesterol oxidase treatment than before (**, p < 0.01; ***, p < 0.001, Student's t test). C, comparison of FCCS curves for GPR37^tGFP and PS-TX14(A)^TAMRA averaged from three separate untreated, mβCD-treated, or cholesterol oxidase-treated cells shows that cholesterol depletion shifts the curve toward shorter diffusion times. In untreated cells, 75 ± 15% of the complexes diffused at τ_D1 = 1.2 ± 0.6 ms and 25 ± 15% at τ_D2 = 330 ± 200 ms. In mβCD-treated cells, 80 ± 12% of the complexes diffused at τ_D1 = 550 ± 250 μs and 20 ± 12% at τ_D = 29 ± 15 ms. In cholesterol oxidase-treated cells, 89 ± 5% of the complexes diffused at τ_D1 = 170 ± 40 μs and 11 ± 5% at τ_D2 = 15 ± 9 ms.