Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site

Abstract

The sigma-2 receptor, whose gene remains to be cloned, has been validated as a biomarker for tumour cell proliferation. Here we report the use of a novel photoaffinity probe, WC-21, to identify the sigma-2 receptor-binding site. WC-21, a sigma-2 ligand containing both a photoactive azide moiety and a fluorescein isothiocyanate group, irreversibly labels sigma-2 receptors in rat liver; the membrane-bound protein was identified as PGRMC1 (progesterone receptor membrane component 1). Immunocytochemistry reveals that both PGRMC1 and SW120, a fluorescent sigma-2 receptor ligand, colocalize with molecular markers of the endoplasmic reticulum and mitochondria in HeLa cells. Overexpression and knockdown of the PGRMC1 protein results in an increase and a decrease in binding of a sigma-2 selective radioligand, respectively. The identification of the putative sigma-2 receptor-binding site as PGRMC1 should stimulate the development of unique imaging agents and cancer therapeutics that target the sigma-2 receptor/PGRMC1 complex.

Figure 1. Identification of the putative sigma 2 receptor binding site as PGRMC1 using a novel photoaffinity probe

a, WC-21 contains a FITC group and a photoactive azide (N₃) moiety, and displays high sigma-2 receptor affinity and selectivity; K_i = 8.7 ± 1 nM for sigma-2 receptor and K_i > 4,000 nM for sigma-1 receptor. b, Following UV cross-linking, ligand-labeled proteins were enriched and separated through SDS-PAGE; WC-21 cross-linked proteins can be visualized using an anti-FITC antibody; SS is silver staining and WB is western blot. c, Proteomic studies of the FITC positive band identified two proteins which shared the same genetic name: progesterone receptor membrane component 1 (PGRMC1). The protein structure shows the peptides (bold) detected by mass spectrometry as well as the Cytochrome b5 domain (underlined).

Figure 2. The pharmacological profile of [¹²⁵I]RHM-4 and sigma-2 receptor binding activity in PGRMC1 siRNA and cDNA treated HeLa cells

a, Competitive binding studies of [¹²⁵I]RHM-4 were carried out with the PGRMC1 ligand AG-205, sigma-2 receptor ligands DTG, WC-26, SV119, and siramesine or with the sigma-1 receptor ligand (+)-pentazocine. [¹²⁵I]RHM-4 binding was blocked by AG-205 and the sigma-2 ligands but not by (+)-pentazocine. n = 2, sample in triplicate. b, Typical western blot confirming PGRMC1 protein expression knockdown in PGRMC1 specific siRNA treated HeLa cells relative to nontargeting siRNA treated controls compared to actin (loading control). c, The bar graph shows reduced sigma-2 receptor binding activity in the PGRMC1 knockdown cells; * p<0.01, Student's t-test, n=3. d, Typical western blot confirming increased PGRMC1 protein expression in PGRMC1 transfected HeLa cells relative to vector transfected controls. e, Bar graph showing increased sigma-2 receptor binding activity in PGRMC1 transfected cells; * p<0.01, Student's t-test, n=3. Error bars in a, c, d represent SEM.

Figure 3. Caspase-3 activation and PGRMC1 upregulation induced by the sigma-2 receptor ligand, WC-26

a, Chemical structure of WC-26, a selective sigma-2 receptor ligand. b, Knockdown of PGRMC1 in HeLa cells resulted in decreased caspase-3 activation induced by WC-26. c, WC-26 stimulated PGRMC1 expression in the same manner as AG-205 , a PGRMC1 ligand; * p<0.01, Student's t-test, n=3. Error bars in b represent SEM.

Figure 4. Intracellular co-localization of PGRMC1 and sigma-2 receptors in HeLa cells using confocal microscopy

a, Sigma-2 receptors labeled with SW120 and PGRMC1 partially colocalized with mitochondria markers (b, c) and endoplasmic reticulum markers (d, e) in HeLa cells. For imaging sigma-2 receptors, live cells were incubated with SW120 and either MitoTracker or ER-Tracker and then imaged by confocal microscopy. For imaging PRGMC1, cells were fixed and incubated with goat anti-PGRMC1 antibody followed by a FITC-conjugated secondary antibody (Mito-marker or ER-marker). Antibody stained HeLa cells were then coverslipped and imaged by confocal microscopy. Scale bar represents 20 μm.