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Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex.

ABSTRACT: CRISPR/Cas gene studies were conducted in HeLa cells where either PGRMC1, TMEM97 or both proteins were removed via gene editing. A series of radioligand binding studies, confocal microscopy studies, and internalization of radiolabeled or fluorescently tagged LDL particles were then conducted in these cells. The results indicate that PGRMC1 knockout (KO) did not reduce the density of binding sites for the sigma-2 receptor (?2R) radioligands, [125I]RHM-4 or [3H]DTG, but a reduction in the receptor affinity of both radioligands was observed. TMEM97 KO resulted in a complete loss of binding of [125I]RHM-4 and a significant reduction in binding of [3H]DTG. TMEM97 KO and PGRMC1 KO resulted in an equal reduction in the rate of uptake of fluorescently-tagged or 3H-labeled LDL, and knocking out both proteins did not result in a further rate of reduction of LDL uptake. Confocal microscopy and Proximity Ligation Assay studies indicated a clear co-localization of LDLR, PGRMC1 and TMEM97. These data indicate that the formation of a ternary complex of LDLR-PGRMC1-TMEM97 is necessary for the rapid internalization of LDL by LDLR.

SUBMITTER: Riad A

PROVIDER: S-EPMC6238005 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex.

Riad Aladdin A Zeng Chenbo C Weng Chi-Chang CC Winters Harrison H Xu Kuiying K Makvandi Mehran M Metz Tyler T Carlin Sean S Mach Robert H RH

Scientific reports 20181115 1

CRISPR/Cas gene studies were conducted in HeLa cells where either PGRMC1, TMEM97 or both proteins were removed via gene editing. A series of radioligand binding studies, confocal microscopy studies, and internalization of radiolabeled or fluorescently tagged LDL particles were then conducted in these cells. The results indicate that PGRMC1 knockout (KO) did not reduce the density of binding sites for the sigma-2 receptor (σ2R) radioligands, [<sup>125</sup>I]RHM-4 or [<sup>3</sup>H]DTG, but a red ...[more]

PMID: 30443021

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Project description:BackgroundThere are limited effective drugs that can reach the brain to target brain tumors, in particular glioblastoma, which is one of the most difficult cancers to be cured from. Because the overexpression of the sigma-2 receptor is frequently reported in glioma clinical samples and associated with poor prognosis and malignancy, we herein studied the anti-tumor effect of the sigma-2 receptor agonist PB221 (4-cyclohexyl-1-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperidine) on an anaplastic astrocytoma tumor model based on previous encouraging results in pancreatic cancer and neuroblastoma SK-N-SH cells.MethodsThe expression of the sigma-2 receptor, transmembrane protein 97 (TMEM97), in ALTS1C1 and UN-KC6141 cell lines was measured by RT-PCR and quantitative RT-PCR. The binding of sigma-2 receptor fluorescent ligands PB385 (6-[5-[3-(4-cyclohexylpiperazin-1-yl)propyl]-5,6,7,8-tetrahydronaphthalen-5-yloxy]-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)hexanamine) and NO1 (2-{6-[2-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-3,4-dihydroisoquinolin-1(2H)-one-5-yloxy]hexyl}-5-(dimethylamino)isoindoline-1,3-dione) was examined by flow cytometry and the fluorescent plate reader. The antitumor activity of PB221 was initially examined in the murine brain tumor cell line ALTS1C1 and then in the murine pancreatic cell line UN-KC6141. The potential therapeutic efficacy of PB221 for murine brain tumors was examined by in vitro migration and invasion assays and in vivo ectopic and orthotopic ALTS1C1 tumor models.ResultsThe IC50 of PB221 for ALTS1C1 and UN-KC6141 cell lines was 10.61 ± 0.96 and 13.13 ± 1.15 μM, respectively. A low dose of PB221 (1 μM) significantly repressed the migration and invasion of ALTS1C1 cells, and a high dose of PB221 (20 μM) resulted in the apoptotic cell death of ALTS1C1 cells. These effects were reduced by the lipid antioxidant α-tocopherol, but not by the hydrophilic N-acetylcysteine, suggesting mitochondrial oxidative stress is involved. The in vivo study revealed that PB221 effectively retarded tumor growth to 36% of the control tumor volume in the ectopic intramuscular tumor model and increased the overall survival time by 20% (from 26 to 31 days) in the orthotopic intracerebral tumor model.ConclusionsThis study demonstrates that the sigma-2 receptor agonist PB221 has the potential to be an alternative chemotherapeutic drug for brain tumors with comparable side effects as the current standard-of-care drug, temozolomide.

Project description:Sigma-2 receptor/transmembrane protein 97 (TMEM97) is upregulated in cancer cells compared to normal cells. Traditional sigma-2 receptor agonists induce apoptosis and autophagy, making them of interest in cancer therapy. Recently, we reported a novel metabolically stimulative function of the sigma-2 receptor, showing increased 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and stimulation of glycolytic hallmarks. 6-Substituted analogs of the canonical sigma-2 receptor antagonist, 6-acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one (SN79), produce both metabolically stimulative and cytotoxic effects. Here, we compare the activities of two related compounds: 6-amino-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one (CM571), the 6-amino derivative of SN79, which binds with high affinity to both sigma-1 and sigma-2 receptors, and 1,3-bis(3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-2-oxo-2,3-dihydrobenzo[d]oxazol-6-yl)thiourea (MAM03055A), a homo-bivalent dimer of CM571. MAM03055A resulted from the degradation of 3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-6-isothiocyanatobenzo[d]oxazol-2(3H)-one (CM572), the cytotoxic 6-isothiocyanato SN79 derivative. MAM03055A exhibited high affinity and strong preference for sigma-2 receptors (sigma-1 Ki = 3371 nM; sigma-2 receptor Ki = 55.9 nM). Functionally, MAM03055A treatment potently induced cell death in SK-N-SH neuroblastoma, MDA-MB-231 breast, and both SW48 and SW480 colorectal cancer cell lines, causing proapoptotic BH3 interacting-domain death agonist (BID) cleavage in SK-N-SH cells. Conversely, CM571 induced metabolic stimulation. CM571 bound reversibly to both receptors, while MAM03055A bound pseudo-irreversibly to sigma-2 receptors and caused residual cytotoxic activity after acute exposure and removal of the compound from the media. Interestingly, MAM03055A induced a time-dependent loss of sigma-2 receptor/TMEM97 protein from cells, whereas monomer CM571 had no effect on receptor levels. These results suggest that monovalent and bivalent sigma-2 receptor ligands in this series interact differently with the receptor, thus resulting in divergent effects.

Dataset Information

Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex.

Publications

Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex.

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