Sigma receptors are arguably the most mysterious signaling
Sigma receptors are arguably the most mysterious signaling proteins. While the S1R was mischaracterized as an opioid receptor (Martin et al., 1976) until its gene sequence became available (Hanner et al., 1996), the S2R has evaded many attempts to decipher its genetic coding. For example, mass spectrometry data suggested histones or associated proteins as S2R ligand binding proteins (Colabufo et al., 2006), but they differ from the S2R in membrane association. The difficulty in unveiling the S2R molecular identity may stem from the fact that it resides in lipid rafts and is not readily detergent-extractable without compromising its functional integrity (Gebreselassie and Bowen, 2004). Moreover, the abundance of this protein in membranes prepared from mammalian tissues is extremely low (e.g. 0.1μg/mg total protein, Ruoho et al., unpublished data).
The S2R has been historically defined as a binding site with high-affinity (20–80nM) for DTG (1,3-di-o-tolylguanidine) and haloperidol (Vilner et al., 1995; Hellewell et al., 1994; Matsumoto et al., 2014). Its molecular size was shown to be 18–21kDa by [3H]-Azido-DTG photoaffinity labeling (Hellewell and Bowen, 1990; Bowen et al., 1989). To readily “visualize” the S2R, our group developed a highly sensitive photoaffinity probe, [125I]-iodoazido-fenpropimorph ([125I]-IAF) (Pal et al., 2007). In accordance with the [3H]-Azido-DTG photolabeling data (Hellewell and Bowen, 1990; Hellewell et al., 1994), the [125I]-IAF photolabeled S2R band appears at ~18kDa on a SDS gel. Importantly, this S2R band is protected by DTG and haloperidol but not (+)-pentazocine, a specific S1R ligand. Using this chemical biology method we have been able to discriminatively detect the S2R and S1R and their specificity for novel sigma receptor ligands (Pal et al., 2007; Fontanilla et al., 2009; Fontanilla et al., 2008).
A recent finding by Xu et al. identified Pyridoxal isonicotinoyl hydrazone receptor membrane component-1 (PGRMC1) as the S2R (Xu et al., 2011). While this discovery is expected to open broad-spectrum opportunities in research and/or the development of therapeutic interventions given the functional importance of the S2R, critical questions remain. First, the apparent molecular weight of PGRMC1 on SDS gels is 25kDa rather than 18–21kDa as previously reported for the S2R (Pal et al., 2007; Hellewell and Bowen, 1990). Our earlier studies using [125I]-IAF showed only two DTG (or haloperidol)-protectable photolabeled bands on the SDS gel, namely the S2R and the S1R. We did not detect an ~25-kDa band that is consistent with PGRMC1 (Ruoho et al., 2013; Fontanilla et al., 2009; Pal et al., 2007), not even with PGRMC1 overexpressed (Chu et al., 2015; Ruoho et al., 2013). Second and more importantly, a high-affinity (20–80nM) binding with DTG or haloperidol is the signature of the S2R, but the DTG (or haloperidol) binding affinity for PGRMC1 has never been reported. It is worth noting that in spite of this ambiguity, perception of PGRMC1 as the S2R has become increasingly accepted among researchers working on PGRMC1 (Mir et al., 2013; Bali et al., 2013; Izzo et al., 2014). It is therefore important to clarify whether PGRMC1 is truly the S2R. In this study, we aimed to explicitly answer two key questions: 1) is the S2R a splice variant of PGRMC1 and 2) does PGRMC1 bind with high affinity to DTG and haloperidol — the signature of the S2R.
Discussion As a potential target for tumor diagnosis and treatment, the S2R has been the subject of many studies, but its identity has remained a mystery. While PGRMC1 was recently reported to be the S2R (Xu et al., 2011), inconsistency remains in their key characteristics. In order to resolve the ambiguity of genetic and pharmacological relationships between PGRMC1 and the S2R, we first determined whether PGRMC1 and the S2R are derived from the same gene. Through CRISPR/Cas9-mediated genome-editing (Cong et al., 2013), we knocked out PGRMC1 in a motor neuron-like cell line (NSC34). We found that [125I]-IAF photolabeling of the S2R and [3H]-DTG/S2R binding characteristics remained unchanged in the PGRMC1 knockout versus the wild type control. These data indicate that PGRMC1 and S2R are genetically two different proteins. Second, we determined the inhibition constant (KI) of DTG and haloperidol for binding to PGRMC1 using [3H]-progesterone at its reported KD of 35nM for PGRMC1 (Peluso et al., 2008). The affinities of DTG and haloperidol for PGRMC1 in rat liver membranes were found to be 472±420μM and 350±19μM, respectively. These DTG and haloperidol KI values for PGRMC1 are >3 orders of magnitude higher than their KI values for the S2R in the same membranes (20–80nM) (Vilner et al., 1995; Hellewell et al., 1994; Matsumoto et al., 2014). This striking difference indicates that PGRMC1 does not have the signature pharmacological characteristics that have been associated with the S2R in the literature (i.e. tight binding to DTG and haloperidol) (Bowen et al., 1989; Hellewell and Bowen, 1990; Hellewell et al., 1994). Therefore, PGRMC1 is a non-S2R binding site in mammalian tissues.