Identification of the gene that codes for the σ2 receptor

Abstract

The σ₂ receptor is an enigmatic protein that has attracted significant attention because of its involvement in diseases as diverse as cancer and neurological disorders. Unlike virtually all other receptors of medical interest, it has eluded molecular cloning since its discovery, and the gene that codes for the receptor remains unknown, precluding the use of modern biological methods to study its function. Using a chemical biology approach, we purified the σ₂ receptor from tissue, revealing its identity as TMEM97, an endoplasmic reticulum-resident transmembrane protein that regulates the sterol transporter NPC1. We show that TMEM97 possesses the full suite of molecular properties that define the σ₂ receptor, and we identify Asp29 and Asp56 as essential for ligand recognition. Cloning the σ₂ receptor resolves a longstanding mystery and will enable therapeutic targeting of this potential drug target.

Keywords: NPC1; TMEM97; cholesterol regulation; sigma receptors; sigma-2 receptor.

Fig. 1.

Purification and molecular cloning of the σ₂ receptor. (A) A schematic of the purification of σ₂ from calf liver. The Inset depicts the portion of the ligand that binds σ₂. (B) Single-point ³H DTG-binding assay of membrane preparations from expi293 cells expressing LC-MS/MS hits, shown as means ± SEM for an experiment performed in triplicate. (C) The sequence of TMEM97. Red font indicates regions that were identified by LC-MS/MS. Green cylinders denote predicted transmembrane (TM) helices.

Fig. 2.

Pharmacological validation of TMEM97 as the σ₂ receptor. (A) PC-12 cells treated with Tmem97-targeted or control siRNA were tested to measure Tmem97 mRNA levels (Left) and σ₂ expression by ³H DTG binding (Right). qPCR data are shown as mean ± SD. Radioligand binding data are shown as mean ± SEM and are representative of two independent experiments performed in triplicate. (B) ³H DTG saturation binding on Sf9 insect cell membranes expressing either TMEM97 (red circles) or the negative control β₂ adrenergic receptor (blue squares). (C) ³H DTG competition curves in Sf9 insect cell membranes expressing TMEM97 against the known σ₂ receptor ligands haloperidol (green circles), PB-28 (red squares), (+)-pentazocine (orange hexagons), (+)-SKF-10,047 (blue diamonds), SAS-1121 (purple triangles), and cold DTG (white triangles). (D) ³H DTG competition curves with the known TMEM97 ligands Elacridar (squares, dotted line) and Ro 48-8071 (circles, solid line) in Sf9 insect cell membranes expressing TMEM97 (red) and in MCF-7 cell membranes (blue). For all curves, data points are shown as mean ± SEM and are representative of two independent experiments performed in triplicate.

Fig. 3.

Structural analysis of TMEM97 and mapping of the ligand-binding site. (A) A schematic of TMEM97 in the membrane. Acidic residues chosen for mutagenesis are colored pale red, with Asp29 and Asp56 highlighted in dark red. Membrane helices and TMEM97 topology are according to TMHMM server prediction. (B) Evolutionary coupling map of the top 140 pairs of human TMEM97. Transmembrane helices (TM) and the extracellular domain (EC) are indicated and boxed. (C) Molecular models of TMEM97 generated by evolutionary coupling analysis (29). The top four ranked models are presented. Models are rainbow colored from the N terminus to the C terminus as indicated. The location of the membrane plane was calculated by the PPM server (48) and is shown in gray. Asp29 and Asp56 are shown as red spheres. For clarity a single representative residue pair is presented. (D) Single-point ³H DTG-binding assay of membrane preparations from Expi293 cells expressing TMEM97 single-point mutants. Empty vector is shown as a black bar, wild-type TMEM97 is shows as a white bar, and point mutants are shown as gray bars. Positions 29 and 56, which show abolished binding, are indicated by red arrows. Data are shown as means ± SEM for an experiment performed in triplicate.