Abstract

Neuronal growth regulator 1 (NEGR1) is a glycosylphosphatidylinositol-anchored membrane protein associated with several human pathologies, including obesity, depression, and autism. Recently, significantly enlarged white adipose tissue, hepatic lipid accumulation, and decreased muscle capacity were reported in Negr1-deficient mice. However, the mechanism behind these phenotypes was not clear. In the present study, we found NEGR1 to interact with cluster of differentiation 36 (CD36), the major fatty acid translocase in the plasma membrane. Binding assays with a soluble form of NEGR1 and in situ proximal ligation assays indicated that NEGR1-CD36 interaction occurs at the outer leaflet of the cell membrane. Furthermore, we show that NEGR1 overexpression induced CD36 protein destabilization in vitro. Both mRNA and protein levels of CD36 were significantly elevated in the white adipose tissue and liver tissues of Negr1−/− mice. Accordingly, fatty acid uptake rate increased in NEGR1-deficient primary adipocytes. Finally, we demonstrated that Negr1−/− mouse embryonic fibroblasts showed elevated reactive oxygen species levels and decreased adenosine monophosphate-activated protein kinase activation compared with control mouse embryonic fibroblasts. Based on these results, we propose that NEGR1 regulates cellular fat content by controlling the expression of CD36.

Graphical abstractSupplementary key wordsAbbreviations: AMPK (adenosine monophosphate-activated protein kinase), CD36 (cluster of differentiation 36), FABPpm (plasma membrane fatty acid-binding protein), FATP (fatty acid transporter protein), GA (gastrocnemius), GST (glutathione-S-transferase), HA (hemagglutinin), H2O2 (hydrogen peroxide), IP (immunoprecipitation), LCFA (long-chain fatty acid), MEF (mouse embryonic fibroblast), NEGR1 (neuronal growth regulator 1), p-AMPK (phosphorylated AMPK), PLA (proximity ligation assay), ROS (reactive oxygen species)Obesity is characterized by an abnormal increase in intracellular fat accumulation (1Lipotoxic diseases of nonadipose tissues in obesity.), and it has become the most common global disease (2Obesity as a medical problem.). Exogenous supply of fatty acids seems to be the preferred source for cellular lipids compared with their de novo synthesis in proliferating fibroblasts as well as in HeLa cells (3Yao C.H. Fowle-Grider R. Mahieu N.G. Liu G.Y. Chen Y.J. Wang R. Exogenous fatty acids are the preferred source of membrane lipids in proliferating fibroblasts.), suggesting that the regulation of fatty acid uptake is important for lipid balance. Long-chain fatty acids (LCFAs) not only contribute to cellular metabolic energy generation and storage but also have hormone-like properties that regulate gene expression (4Protein-mediated fatty acid uptake: novel insights from in vivo models.). Several protein groups including cluster of differentiation 36 (CD36)/fatty acid translocase, fatty acid transporter proteins (FATPs), and plasma membrane fatty acid-binding protein (FABPpm) are known to transport LCFAs (5Cellular fatty acid uptake: a pathway under construction., 6Mallick R. Basak S. Duttaroy A.K. Fatty acids and evolving roles of their proteins in neurological, cardiovascular disorders and cancers.). CD36 plays a major role in LCFA uptake in metabolic tissues, including adipose tissues, cardiomyocytes, and skeletal muscle myocytes (7Dynamic role of the transmembrane glycoprotein CD36 (SR-B2) in cellular fatty acid uptake and utilization.), contributing to more than 50% of the rate of fatty acid uptake in these tissues (8Hao J.W. Wang J. Guo H. Zhao Y.Y. Sun H.H. Li Y.F. et al.CD36 facilitates fatty acid uptake by dynamic palmitoylation-regulated endocytosis.).As a scavenger receptor class B-2 protein, CD36 is an integral membrane protein with a hairpin-like topology and two transmembrane regions (7Dynamic role of the transmembrane glycoprotein CD36 (SR-B2) in cellular fatty acid uptake and utilization.). Furthermore, CD36 is associated with membrane rafts, and CD36-mediated fatty acid uptake depends on the integrity of membrane rafts in adipocytes (9Pohl J. Ring A. Korkmaz U. Ehehalt R. Stremmel W. FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires plasma membrane rafts.). CD36 is ubiquitously expressed in diverse mammalian cell types, which include skeletal and cardiac myocytes, macrophages, endothelial cells, adipocytes, and gut epithelial cells (10CD36, a scavenger receptor implicated in atherosclerosis.). Its functions are primarily related to lipid metabolism and innate immunity, and its dysregulation has been reported in various human pathologies, including atherothrombotic diseases, obesity, diabetes, cancer, and Alzheimer’s disease (7Dynamic role of the transmembrane glycoprotein CD36 (SR-B2) in cellular fatty acid uptake and utilization.). Activation of adenosine monophosphate-activated protein kinase (AMPK) is related to the CD36-mediated LCFA uptake pathway and fatty acid utilization in skeletal muscle (11Fentz J. Kjobsted R. Birk J.B. Jordy A.B. Jeppesen J. Thorsen K. et al.AMPKalpha is critical for enhancing skeletal muscle fatty acid utilization during in vivo exercise in mice.).Neuronal growth regulator 1 (NEGR1) consists of three C2-type immunoglobulin domains localized on the extracellular side of plasma membranes. This protein strongly binds membrane lipid rafts via a glycosylphosphatidylinositol anchor. NEGR1 functions as a cell-adhesion molecule that plays an important role in neural cell recognition and neurite outgrowth (12Kim H. Hwang J.S. Lee B. Hong J. Lee S. Newly identified cancer-associated role of human neuronal growth regulator 1 (NEGR1).). Multiple genome-wide association studies have revealed that genetic alterations in NEGR1 are associated with obesity (13Willer C.J. Speliotes E.K. Loos R.J. Li S. Lindgren C.M. Heid I.M. et al.Six new loci associated with body mass index highlight a neuronal influence on body weight regulation.), intellectual disability (14Sniekers S. Stringer S. Watanabe K. Jansen P.R. Coleman J.R.I. Krapohl E. et al.Genome-wide association meta-analysis of 78,308 individuals identifies new loci and genes influencing human intelligence.), schizophrenia (15Schizophrenia Working Group of the Psychiatric Genomics, C
Biological insights from 108 schizophrenia-associated genetic loci.), depression (16Hyde C.L. Nagle M.W. Tian C. Chen X. Paciga S.A. Wendland J.R. et al.Identification of 15 genetic loci associated with risk of major depression in individuals of European descent.), and Alzheimer’s disease (17Ni H. Xu M. Zhan G.L. Fan Y. Zhou H. Jiang H.Y. et al.The GWAS risk genes for depression may be actively involved in Alzheimer's Disease.). We recently reported alterations in the affective behavior of Negr1−/− mice (18Noh K. Lee H. Choi T.Y. Joo Y. Kim S.J. Kim H. et al.Negr1 controls adult hippocampal neurogenesis and affective behaviors.), implying that NEGR1 is closely related to CNS function.Although NEGR1 is highly expressed in the brain (19Walley A.J. Jacobson P. Falchi M. Bottolo L. Andersson J.C. Petretto E. et al.Differential coexpression analysis of obesity-associated networks in human subcutaneous adipose tissue.), it is also expressed in several peripheral tissues, such as subcutaneous adipose tissues and skeletal muscles (HumanProtein Atlas database, https://www.proteinatlas.org). In addition, NEGR1 is found in different cell types, including adipocytes, myocytes, and endothelial cells, as well as the cells within the nervous system (the Genotype-Tissue Expression database [GTEx] portal, https://www.gtexportal.org). We originally identified human NEGR1 as a commonly downregulated gene in various human tumor tissues (12Kim H. Hwang J.S. Lee B. Hong J. Lee S. Newly identified cancer-associated role of human neuronal growth regulator 1 (NEGR1).). Subsequent binder analysis unexpectedly revealed the role of NEGR1 in intracellular cholesterol trafficking and lipid storage (20Kim H. Chun Y. Che L. Kim J. Lee S. Lee S. The new obesity-associated protein, neuronal growth regulator 1 (NEGR1), is implicated in Niemann-Pick disease Type C (NPC2)-mediated cholesterol trafficking.). In a recent study, Negr1−/− mice also presented substantial enlargement of white adipose tissues (WATs) with increased cell size, further supporting the role of NEGR1 in intracellular lipid transport (21Joo Y. Kim H. Lee S. Lee S. Neuronal growth regulator 1-deficient mice show increased adiposity and decreased muscle mass.). Increased hepatic fat accumulation and skeletal muscle atrophy have also been observed in NEGR1-deficient mice (21Joo Y. Kim H. Lee S. Lee S. Neuronal growth regulator 1-deficient mice show increased adiposity and decreased muscle mass.). In the present study, NEGR1 was found to interact with CD36, increasing our understanding of the role of these proteins in intracellular lipid trafficking and related human diseases.Materials and methodsAnimals, cell culture, and cloningAll animals, including Negr1+/+ and Negr1−/− C57BL6 mice (20Kim H. Chun Y. Che L. Kim J. Lee S. Lee S. The new obesity-associated protein, neuronal growth regulator 1 (NEGR1), is implicated in Niemann-Pick disease Type C (NPC2)-mediated cholesterol trafficking.), were kept on 12 h light/dark cycles in a controlled environment at 22–24°C and 55% humidity. All animal procedures were approved by the Seoul National University Institutional Animal Care and Use Committee. Mouse embryonic fibroblasts (MEFs), 3T3-L1, and 293T cells (21Joo Y. Kim H. Lee S. Lee S. Neuronal growth regulator 1-deficient mice show increased adiposity and decreased muscle mass.) were maintained in DMEM (Welgene, Gyeongsan, South Korea) supplemented with 10% FBS (Atlas Biologicals, Fort Collins, CO). SKOV-3 cells were cultured in RPMI 1640 medium (Welgene).The open reading frame of gene CD36 was obtained by PCR amplification of the total RNA extracted from 293T cells and subcloned into pKH-3HA (22A new kinetochore component CENP-W interacts with the polycomb-group protein EZH2 to promote gene silencing.) or pcDNA3-3FLAG (21Joo Y. Kim H. Lee S. Lee S. Neuronal growth regulator 1-deficient mice show increased adiposity and decreased muscle mass.) plasmids. Glutathione-S-transferase (GST)-fused deletion constructs of the CD36 extracellular domain were generated using plasmid pEBG (20Kim H. Chun Y. Che L. Kim J. Lee S. Lee S. The new obesity-associated protein, neuronal growth regulator 1 (NEGR1), is implicated in Niemann-Pick disease Type C (NPC2)-mediated cholesterol trafficking.). To generate the pcDNA4-FLAG-NEGR1 construct, we subcloned NEGR1 into the pcDNA4/TO vector (Invitrogen, Carlsbad, CA), using restriction enzymes AflII and XbaI and a 3×FLAG sequence inserted between the signal sequence (positions 1–39) and the remaining NEGR1 gene sequence (positions 40–314). To obtain SKOV-3-FLAG-NEGR1 stable cells, pcDNA4-FLAG-NEGR1 was transfected into SKOV-3 cells and selected with zeocin (50 μg/ml; Invitrogen).Histological analysis and immunofluorescence microscopy

For visualizing target proteins in tissue sections, small sections of various tissues were fixed overnight in 4% paraformaldehyde and embedded in paraffin. Tissue sections were immunostained with the appropriate primary antibodies in PBS, followed by incubation with the fluorescent-linked secondary antibodies, FITC-labeled anti-mouse IgG antibody (Sigma-Aldrich, St. Louis, MO) or Cy3 anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Immunofluorescence microscopy was conducted as previously described (23Cheon Y. Yoo A. Seo H. Yun S.Y. Lee H. Lim H. et al.Na/K-ATPase beta1-subunit associates with neuronal growth regulator 1 (NEGR1) to participate in intercellular interactions.). Briefly, cells grown on coverslips were either untreated or permeabilized with 0.1% Triton X-100 in PBS for 10 min. After incubation with the appropriate primary antibodies, cells were treated with fluorescent-linked secondary antibodies. Alexa Fluor 594 anti-human IgG antibody (Invitrogen) was used to detect the Fc-fusion protein. To visualize intracellular lipid droplets, fixed cells were stained with BODIPY 493/503 (2 μM; Thermo Fisher Scientific, Waltham, MA) for 10 min. Imaging was performed on an Olympus BX51 (Tokyo, Japan) microscope and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).Gene expression analysis and immunoblotting

To compare gene expression between the peripheral tissues of Negr1−/− and Negr1+/+ C57BL6 mice, liver, skeletal muscle, and epididymal adipose tissues were obtained (n = 4–8). To analyze CD36 mRNA expression, total RNA was extracted from tissue samples using a NucleoSpin RNA Extraction kit (Macherey-Nagel, Düren, Germany) and converted into complementary DNA using a SuperScript III Reverse Transcription kit (Invitrogen). Quantitative real-time RT-PCR was performed on a CFX connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) with specific primers for CD36 (forward: 5-GCATGAGAATGCCTCCAAACA-3; reverse: 5-CGGAACTGTGGGCTCATTG-3); GAPDH was used as reference for normalization.

For immunoblotting, tissue samples were homogenized in a lysis buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 50 mM NaF, 1 mM sodium vanadate, 1% NP-40, and 0.1% SDS) and centrifuged at 12,000 g for 30 min to remove insoluble materials. Specific antibodies were used to visualize each protein: anti-β-actin, anti-FLAG, and anti-NEGR1 (Sigma-Aldrich); antihemagglutinin (HA) and anti-CD36 (Santa Cruz Biotechnology, Dallas, TX); anti-GAPDH (Cusabio, Baltimore, MD); and anti-AMPK and antiphosphorylated AMPK (p-AMPK) (Cell Signaling Technologies, Beverley, MA).

Subcellular fractionation and binding assayLipid raft fractionation was performed using OptiPrep™ iodixanol (Sigma-Aldrich) (12Kim H. Hwang J.S. Lee B. Hong J. Lee S. Newly identified cancer-associated role of human neuronal growth regulator 1 (NEGR1).). Briefly, cell lysates were adjusted to 32% OptiPrep™ and sequentially overlaid with 24% and 20% iodixanol solutions. After centrifugation at 76,000 g for 18 h at 4°C, the fraction collected from the top was designated as no. 1. After tissue lysates were adjusted to 32% OptiPrep™ and overlaid with 24% and 20% iodixanol solutions, endosomal fractionation was carried out using centrifugation at 76,000 g for 1 h at 4°C. Plasma membranes were isolated using a Minute™ Plasma Protein Isolation kit (Invent Biotechnologies, Plymouth, MN) according to the manufacturer’s instructions.Immunoprecipitation (IP) was performed following a previously described method (24CENP-W inhibits CDC25A degradation by destabilizing the SCF(beta-TrCP-1) complex at G2/M.), with slight modifications. Cells were lysed in a buffer (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM PMSF, 50 mM NaF, 1 mM Na3VO4, and 0.02% NaN3) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Then, samples were incubated with 0.75 μg of appropriate antibodies for 3 h at 4°C before incubation with Protein A Sepharose beads (GE Healthcare, Piscataway, NJ). GST-pulldown assays were performed using 1 μg of appropriate antibody or Glutathione-Sepharose 4B beads (GE Healthcare) as described in a previous study (20Kim H. Chun Y. Che L. Kim J. Lee S. Lee S. The new obesity-associated protein, neuronal growth regulator 1 (NEGR1), is implicated in Niemann-Pick disease Type C (NPC2)-mediated cholesterol trafficking.).In situ proximity ligation assay

The proximity ligation assay (PLA) was performed on fixed SKOV-3-NEGR1-FLAG cells using Duolink PLA technology reagents (Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, fixed cells were first incubated with anti-CD36 and anti-FLAG antibodies for 2 h and then with PLA probes (anti-mouse MINUS and anti-rabbit PLUS, respectively) and Alexa Fluor 488 phalloidin (Invitrogen) for 1 h. After incubation with a ligation solution for 30 min and an amplification solution for 2 h, the cells were mounted in a 4′,6-diamidino-2-phenylindole-containing solution. Imaging was performed using an Olympus BX51 microscope or a TCS SP5 AOBS confocal microscope equipped with a 63× inverted NX oil lens (Leica Microsystems GmbH, Wetzlar, Germany).

Measurement of fatty acid uptake and cellular reactive oxygen species levelTo perform the fatty acid uptake assay, primary adipose cells were isolated from epididymal adipose tissue as described previously (21Joo Y. Kim H. Lee S. Lee S. Neuronal growth regulator 1-deficient mice show increased adiposity and decreased muscle mass.). After seeding in 96-well plates, cells were incubated with serum-free DMEM for 1 h. Fatty acid uptake was assessed using a Free Fatty Acid Uptake Assay kit (catalog no.: ab176768; Abcam, Cambridge, UK) according to the manufacturer’s instructions. Fluorescence signals were measured using a fluorescence microplate reader (Varioskan LUX; Thermo Fisher Scientific) at 485/515 nm.

Cellular reactive oxygen species (ROS) level was measured using a DCFDA (2',7'-dichlorofluorescein diacetate)/H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) Cellular ROS Assay kit (catalog no.: ab113851; Abcam) as per the manufacturer’s instructions. If required, cells were preincubated with hydrogen peroxide (H2O2) or oleic acid for 24 h before the assay. Fluorescence signals were measured at 485/515 nm.

ResultsIncreased CD36 expression in the adipose tissues of Negr1−/− miceOur previous study reported that epididymal WAT was enlarged in Negr1−/− mice compared with WT mice (21Joo Y. Kim H. Lee S. Lee S. Neuronal growth regulator 1-deficient mice show increased adiposity and decreased muscle mass.). To confirm this, we determined the gonadal WAT weight of 10-week-old male mice. Negr1−/− mice showed approximately 1.4-fold increase in WAT weight (P = 0.026) compared with WT mice (Fig. 1A). To investigate which fatty acid transporter is involved in the transport of LCFAs across the cell membrane of adipose tissues, we examined the expression levels of three main transporters, namely CD36, FABPpm, and FATP1, in the gonadal WAT of WT and Negr1−/− mice. When quantified using GAPDH as a reference gene, the expression level of CD36 was much higher than that of FABPpm and FATP1, which suggested that CD36 may play an important role in transporting LCFAs in adipose tissues (Fig. 1B). Furthermore, CD36 expression increased significantly (1.3-fold, P = 0.0007) in Negr1−/− mice compared with WT mice, whereas FABPpm and FATP1 showed no difference (Fig. 1B).

Fig. 1CD36 expression was upregulated in the WAT of Negr1−/− mice. A: Epididymal WATs were dissected from 10-week-old Negr1+/+ and Negr1−/− C57BL6 mice (n = 8), and each fat pad weight was determined. The figure shows mean ± SD of individual weights. Bar represents 10 mm. B: Quantitative RT-PCR was performed to measure the mRNA expression levels of CD36, FABPpm, and FATP1 using total RNA obtained from gonadal WAT (n = 8). Relative expression was calculated using GAPDH as a reference gene. C: CD36 gene expression was measured by quantitative RT-PCR using WAT, liver, heart, brain, and skeletal muscle (SkM) tissue samples of 10-week-old mice (n = 4–7). D: Relative CD36 expression in brain tissues of Negr1−/− and WT mice. All experiments were performed in triplicates, and the data are presented as the mean ± SD. ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001. ns, not significant.

To investigate CD36 expression in other tissues, quantitative RT-PCRs were performed on the mRNAs of WAT, liver, heart, brain, and gastrocnemius (GA) skeletal muscle tissues. The expression of CD36 was approximately 3-fold higher in WAT than in the liver or GA muscle tissues (Fig. 1C). In NEGR1-knockout mice, CD36 expression increased in liver (1.4-fold) and brain tissues (1.6-fold) but decreased in GA muscle tissue (1.6-fold) (Fig. 1C, D). Overall, our results suggest that the increased WAT weight of Negr1−/− mice may be associated with increased expression of CD36, which is the main LCFA transporter in adipocytes.Interaction of NEGR1 with CD36Based on earlier findings that both NEGR1 and CD36 contain large extracellular regions and are associated with lipid rafts in the plasma membrane (12Kim H. Hwang J.S. Lee B. Hong J. Lee S. Newly identified cancer-associated role of human neuronal growth regulator 1 (NEGR1)., 25Eyre N.S. Cleland L.G. Tandon N.N. Mayrhofer G. Importance of the carboxyl terminus of FAT/CD36 for plasma membrane localization and function in long-chain fatty acid uptake., 26Luiken J.J. Chanda D. Nabben M. Neumann D. Glatz J.F. Post-translational modifications of CD36 (SR-B2): implications for regulation of myocellular fatty acid uptake.), we investigated the possible molecular interaction of NEGR1 and CD36. We first obtained the complementary DNA of CD36 from the total RNA of 293T cells and generated an HA-tagged CD36 construct using plasmid pcDNA3-HA. After pcDNA3-HA-CD36 and pEGFP-C1-NEGR1 (12Kim H. Hwang J.S. Lee B. Hong J. Lee S. Newly identified cancer-associated role of human neuronal growth regulator 1 (NEGR1).) plasmids were transfected into HeLa cells, we performed co-IP using anti-GFP antibodies. Immunoblotting with anti-HA antibody revealed that CD36 was present in the NEGR1-enriched fraction but not in the IgG-enriched control (Fig. 2A). GFP-NEGR1 was coisolated with HA-CD36 (Fig. 2B), suggesting an interaction between NEGR1 and CD36.

Fig. 2NEGR1 interacts with CD36. A: Co-IP between transiently expressed HA-CD36 and GFP-NEGR1. After HeLa cells were transfected with HA-CD36 and GFP-NEGR1 plasmids, cell lysates were subjected to IP with anti-GFP antibody. ∗IgG bands. B: Reciprocal IP using anti-HA antibody. C: IP was performed with SKOV-3-FLAG-NEGR1 stable cells using anti-FLAG antibody. Coisolated CD36 was visualized with anti-CD36 antibody. D: Binding assay using soluble NEGR1-Fc protein. SKOV-3 cells were incubated for 2 h with the conditioned medium of 293T cells expressing NEGR1-Fc or Fc control. Then, cells were immunostained with anti-human Fc and anti-CD36 antibodies. Bar represents 20 μm. E: In situ PLA was performed with SKOV-3-FLAG-NEGR1 cells using Duolink PLA technology. After treatment with anti-CD36 and anti-FLAG antibodies for 2 h, cells were incubated with PLA probes (anti-mouse MINUS and anti-rabbit PLUS, respectively) for 1 h. Cellular actin polymers were stained with Alexa Fluor 488 phalloidin. Bar represents 10 μm.

Next, we examined NEGR1 and CD36 molecular interaction at the endogenous level using HeLa cell lysates. As no interaction was observed, possibly because of the low IP efficiency of the anti-NEGR1 antibody, we performed IP using the SKOV-3-FLAG-NEGR1 stable cells. When IP was performed with an anti-FLAG antibody, highly glycosylated forms of CD36 were observed in the NEGR1 fraction by immunoblotting with anti-CD36 antibody (Fig. 2B).To clearly demonstrate that NEGR1-CD36 interaction can occur at the extracellular surface of cell membranes, we performed an in situ binding assay using the human Fc-fused secreted form of NEGR1 (23Cheon Y. Yoo A. Seo H. Yun S.Y. Lee H. Lim H. et al.Na/K-ATPase beta1-subunit associates with neuronal growth regulator 1 (NEGR1) to participate in intercellular interactions.). The culture medium of 293T cells transfected with NEGR1-Fc or Fc control plasmids was collected and provided to SKOV-3 cells for incubation for 2 h. After this period, cells were coimmunostained with anti-human Fc antibody (red) and CD36 antibody (green) (Fig. 2C). Whereas the Fc signal was barely observed in control cells treated with Fc-containing medium (fourth row, Fig. 2C), strong Fc signals were visualized in the NEGR1-Fc-treated cells, which overlapped CD36 signals well. These results suggest NEGR1 can interact with CD36 at the cell surface.To verify the NEGR1-CD36 interaction, an in situ PLA was performed using SKOV-3-FLAG-NEGR1 cells (Fig. 2D). Cells were incubated with anti-FLAG and anti-CD36 antibodies after cell permeabilization, and phalloidin staining was used to examine cell morphology. While no signals were observed in cells treated with a single antibody, clear fluorescent signals were detected in cells treated with both antibodies, indicating NEGR1 and CD36 exist in close proximity. In addition, strong fluorescent signals were observed when PLA was performed under nonpermeabilized conditions (Supplemental Fig. S1) demonstrating that NEGR1-CD36 interaction occurs in the cell membrane.Determination of binding regions of NEGR1 and CD36 proteinsNEGR1 has a relatively simple structure containing three consecutive C2-type Ig-like domains. In contrast, the extracellular region of CD36 contains only the CLESH (CD36, lysosomal integral membrane protein-2 [LIMP-2], Emp sequence homology; residues 93–155) and proline-rich (243–375) domains (27Nergiz-Unal R. Rademakers T.