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 (σ2R) 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.
Highlights
We report that CRISPR gene editing of TMEM97/σ2R results in a complete reduction of the binding of the σ2R ligand [125I]N-(4-(6,7-dimethoxy3,4-dihydroisoquinolin-2(1 H)-yl)butyl)-2,3-dimethoxy-5-iodo-benzamide ([125I]RHM-4)[23], but only a partial reduction in the specific binding of [3H]DTG to HeLa cell membranes
In order to study whether TMEM97 or/and PGRMC1 is responsible for σ2R binding and what their biological functions are, CRISPR technology was used to knockout TMEM97, PGRMC1 and both TMEM97 and PGRMC1 in HeLa cells to generate four knockout (KO) cell lines, i.e. control, TMEM97 KO, PGRMC1 KO, and TMEM97/PGRMC1 double KO
Western blot showed that TMEM97 or/and PGRMC1 were completely removed in the knockout cell lines (Fig. 1A)
Summary
Yi et al reported that genetic knockout or knockdown of vem-1, the nematode ortholog of PGRMC1, diminished the neurotoxicity of amyloid precursor protein in a nematode model of AD, and this effect could be demonstrated pharmacologically by using σ2 R antagonists[10]. These data suggested a strong association between the σ2R and PGRMC1. We present evidence demonstrating that deletion of either TMEM97/σ2R or PGRMC1 reduces the level of internalization and trafficking of LDL by the LDL receptor Knocking out both proteins in HeLa cells did not have an additive effect on LDL uptake. The residual binding of [3H]DTG was not attributed to binding to the σ1R, and may represent a third, currently uncharacterized sigma receptor
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