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TMED2 binding restricts SMO to the ER and Golgi compartments

['Giulio Di Minin', 'Institute Of Molecular Health Sciences', 'Department Of Biology', 'Swiss Federal Institute Of Technology Eth Hönggerberg', 'Zurich', 'Markus Holzner', 'Alice Grison', 'Department Of Biomedicine', 'University Of Basel', 'Basel']

Date: 2022-04

Hedgehog (HH) signaling is important for embryonic pattering and stem cell differentiation. The G protein–coupled receptor (GPCR) Smoothened (SMO) is the key HH signal transducer modulating both transcription-dependent and transcription-independent responses. We show that SMO protects naive mouse embryonic stem cells (ESCs) from dissociation-induced cell death. We exploited this SMO dependency to perform a genetic screen in haploid ESCs where we identify the Golgi proteins TMED2 and TMED10 as factors for SMO regulation. Super-resolution microscopy shows that SMO is normally retained in the endoplasmic reticulum (ER) and Golgi compartments, and we demonstrate that TMED2 binds to SMO, preventing localization to the plasma membrane. Mutation of TMED2 allows SMO accumulation at the plasma membrane, recapitulating early events after HH stimulation. We demonstrate the physiologic relevance of this interaction in neural differentiation, where TMED2 functions to repress HH signal strength. Identification of TMED2 as a binder and upstream regulator of SMO opens the way for unraveling the events in the ER–Golgi leading to HH signaling activation.

Funding: GDM was supported by the ETH Zurich Postdoctoral Fellowship Program as well as the Marie Curie Actions for People COFUND Program. WC and LAJM were supported by a grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2015-06699). LJM is a member of the Research Centre of the McGill University Health Centre which is supported in part by FRQS. HR was supported by a grant from NIGMS (R01GM117090). This work was supported by grants from the Swiss National Science Foundation (SNF grants 31003A_152814/1 and 31003A_175643/1) to AW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we discover that SMO sustains embryonic stem cell (ESC) survival in a GLI-independent manner. We use this dependency for a genetic screen in haploid ESCs for factors involved in the earliest events of SMO activation. Identification of the COPI and COPII components TMED2 and TMED10 allowed to uncover a new mechanism of SMO trafficking from the ER–Golgi apparatus in the regulation of HH signaling.

SMO is cotranslationally imported into the endoplasmic reticulum (ER) membrane [ 34 , 35 ]. Transport from the ER through the Golgi to the plasma membrane is highly regulated [ 36 ]. The secretory pathway includes bidirectional transport between the ER and the Golgi through coat protein complex I (COPI)-coated and COPII-coated vesicles [ 37 ]. Retrograde transport is important for quality control of folding and glycosylation of membrane proteins before they reach the plasma membrane. The precise route of SMO to the plasma membrane, as well as interacting partners determining its localization, remains to be understood [ 38 ]. It is thought that SMO reaches the PM passing normally through the Golgi. However, the oncogenic SMO-A1 variant might reach the primary cilium through a different and potentially direct route [ 38 ].

Screens in cell lines for genes involved in the HH response have been based on a transcriptional (GLI mediated) readout of pathway activation [ 26 , 31 – 33 ]. Earlier studies have uncovered details of the assembly and trafficking of the primary cilia. Despite impressive progress in understanding events downstream of SMO that has been made, the molecular mechanism of SMO regulation upstream of the primary cilium remains less well understood.

PTCH1 is a member of the RND family of proton-driven antiporters [ 16 ], which is conserved in all domains of life. PTCH1 shares characteristics with the RND cholesterol transporter Niemann–Pick C1 (NPC1) including a sterol-sensing domain and multiple cholesterol binding sites [ 17 – 22 ]. In its unliganded state, PTCH1 affects SMO distribution and prevents its translocation to the primary cilium [ 23 – 26 ], where activation of GLI transcription factors occurs. However, G protein regulation by SMO is not restricted to the primary cilium [ 27 – 30 ]. The primary cilium is dispensable or even inhibitory for nontranscriptional effects of SMO [ 8 ]. These findings suggest that different effects of SHH signals (GLI mediated or non-GLI mediated) are determined by SMO localization prior to, or cycling through, the primary cilium.

The HH receptor Patched (PTCH) inhibits SMO, and binding of Sonic, Indian, or Desert HH (SHH, IHH, and DHH, respectively) releases this inhibition [ 6 ]. SMO activation can have both transcriptional and nontranscriptional effects. SMO is a G protein–coupled receptor (GPCR) and regulates cAMP levels and cytoskeleton dynamics [ 7 – 9 ], as well as calcium levels affecting cell metabolism in muscle and brown fat [ 10 ]. The transcriptional response to SMO activation is mediated by the GLI family of zinc finger transcription factors. In vertebrates, activation of GLI proteins appears to be limited to a specialized plasma membrane compartment, the primary cilium [ 11 , 12 ]. In mice, a Gli1/Gli2 −/− double mutation or primary cilium deficiencies [ 13 , 14 ] result in milder phenotypes than a Shh/Ihh double mutation [ 15 ] and a Smo mutation [ 15 ]. These phenotypic differences suggest the relevance of GLI independent functions of SMO.

Hedgehog (HH) signaling controls key events in embryonic development, tissue homeostasis, and repair [ 1 – 3 ]. Deregulation of HH signaling by genetic or pharmacologic means causes severe developmental abnormalities, and activation of the HH response is frequently implicated in tumor initiation and dissemination [ 4 ]. Compounds inhibiting the HH signal transducer Smoothened (SMO) are used for therapy of basal cell carcinoma (BCC) and medulloblastoma [ 5 ].

Results

Purmorphamine reduces SMO protein abundance in ESCs To investigate how GLI-independent SMO signaling activity is inhibited by high concentrations of PMP and SAG, we decided to analyze SMO protein level and distribution in ESCs. We stably introduced a cDNA construct for expression of a carboxyl-terminally hemagglutinin (HA) epitope tagged SMO protein (SMO–HA) into Smo−/− cells. We observed that treatment with high concentrations of PMP or SAG led to a decrease in SMO, particularly the higher molecular weight form, using western analysis (Fig 2C). SMO glycosylation in the ER and Golgi causes an increased molecular weight [51]. In PMP-treated cells, these higher molecular weight forms of SMO became undetectable at the plasma membrane (Fig 2D). Our observations suggest that loss of glycosylated SMO from the plasma membrane is correlated with cell death. Notably, it has been shown that SMO glycosylation even if dispensable for GLI regulation is required for modulating G protein activity [52]. We investigated if high expression of SMO would rescue PMP induced cell death. We introduced the SMO–HA construct into wild-type ESCs and obtained high expressing clones (S2L Fig). Importantly, SMO overexpression conferred PMP resistance to mouse ESCs (Fig 2E).

TMED2 is involved in SMO retention at the ER–Golgi compartments Our findings suggested a role of the TMED2–SMO complex as an early step controlling HH signaling activation. In order to explore this possibility, we differentiated Tmed2−/− ESCs expressing SMO–HA to NPCs and analyzed the effects on SMO distribution. The Tmed2 mutation did not affect the overall morphology of NPCs (S9A–S9D Fig). Small differences were observed in agreement with the literature [59,60] including accumulation of the ERP72 marker in localized regions of the ER (S9A Fig). Treatment of NPCs with a low activating concentration of PMP led to translocation of SMO from internal to external compartments (Fig 7A). The perinuclear SMO staining detected in untreated NPCs diminished after chemical activation of SMO. Stimulation with SHH also promoted a similar SMO translocation but not to the extent of that induced by PMP. 3D-STORM experiments indicated that the mutation of Tmed2 phenocopied the effects of SHH ligand on SMO localization (Fig 7B). Ciliary accumulation of SMO after SHH or PMP treatment was detected in few cells. This is likely explained by cell proliferation in NPC cultures that prevents the formation of clear cilia. Next, we analyzed the effects of the Tmed2 mutation on SMO distribution performing dual color 3D-STORM imaging. We observed a consistent reduction of the area in the ER and LE that was occupied by SMO in Tmed2−/− cells compared to control cells (Fig 7C, 7E, and 7F, S10A–S10D Fig). T reduction was even more pronounced in the Golgi (Fig 7D, S10C Fig). In Tmed2 mutant cells, colocalization of SMO with RCAS1 was 3-fold lower thhean in control cells (Fig 7F). Notably, treatment with SHH induced a comparable relocalization of SMO as the Tmed2 mutation. SHH treatment reduces the SMO pool in the ER, LE, and Golgi compartments. Besides these similarities, a difference also emerged. In Tmed2 mutant cells, we detected a global decrease of SMO in the ER, whereas in cells treated with SHH, SMO depletion was more pronounced in ER peripheral domains (Fig 7C, S10B Fig). Perinuclear ER regions are enriched in cisternal domains associated with ribosomes and polysomes and therefore involved in protein synthesis [69]. We believe that the detected difference reflects the short-term effect of the SHH treatment, compared to a constitutive loss of Tmed2. According to this interpretation, SHH treatment first mobilizes the peripheral pool of SMO. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 7. TMED2 regulates SMO abundance at the PM in a SHH-dependent manner. (A, B) Activation of HH signaling promotes SMO cellular redistribution. (A) FM images of SMO–HA in WT NPCs treated for 24 hours with PMP or SHH and in Tmed2−/− cells. Scale bar = 10 μm. (B) Three-dimensional color distribution of HA–SMO in 3D-STORM experiments performed in WT NPCs treated with SHH for 24 hours and in Tmed2−/− NPCs. Scale bar = 5 μm. (C–F) SHH treatment and Tmed2 mutation promotes SMO trafficking form ER–Golgi compartments. Dual color 3D-STORM analysis showing ERP72 (C), RCAS1 (D) and RAB7 (E) distribution in green and the SMO colocalizing domains in gray. (F) Plot showing percentage of SMO–HA colocalizing events normalized to marker distribution upon SHH treatment and Tmed2 mutation. Asterisks denote statistical significance for difference from the WT untreated samples. (G) Tmed2 regulates SMO abundance at the PM. Cells were treated with NHS-SS-Biotin (Biotin) to label PM proteins, and with SHH as indicated. Western analysis of PM proteins and input before purification (1/50 of pull-down) are shown. (H) Percentage of cells expressing OLIG2 (left) and NKX2.2 (right) relative to total cell count in neuralized EBs. Neuralized EBs derived from WT cells and from ESCs overexpressing SMO–HA with and without a Tmed2 mutation were treated with or without SHH. Asterisks denote statistical significance for difference between indicated samples. Individual EBs are plotted (n = 10). (I) SHH treatment disrupts the SMO–TMED2 complex in NPCs. Western analysis of co-immunopurification of endogenous TMED2 with HA–SMO (top) and input (1/25 of IP, bottom) in NPCs expressing N-term HA tagged SMO. Cells were treated with recombinant SHH for the indicated amount of time. Actin is shown as loading control. (J) Summary of the proposed mechanism for TMED2-regulated SMO secretion from the ER–Golgi compartment. The data underlying all the graphs shown in the figure are included in the S1 Data file. EB, embryoid body; EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; ESC, embryonic stem cell; FM, fluorescence microscopy; HA, hemagglutinin; HH, hedgehog; LE, late endosome; NPC, neural progenitor cell; NT, xxx; PM, plasma membrane; PMP, purmorphamine; SHH, Sonic hedgehog; SMO, Smoothened; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001596.g007

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