Receptors, once considered a class of opioid receptors, are now regarded as a unique class of receptors that contain binding sites for a wide range of ligands, including the drug 1-N(2Ј,6Ј-dimethylmorpholino)3-(4-t-butylpropylamine) (fenpropimorph), a yeast sterol isomerase inhibitor. Because fenpropimorph has high-binding affinity to the -1 receptor, we have synthesized a series of fenpropimorph-like derivatives with varying phenyl ring substituents and have characterized their binding affinities to the -1 receptor. In addition, we have synthesized a carrier-free, radioiodinated fenpropimorph-like photoaffinity label, 1-N-(2Ј,6Ј-dimethyl-morpholino)-3-(4-azido- The receptor is a unique receptor that, for the past 30 years, has been persistently enigmatic. receptors were initially proposed to be subtypes of opioid receptors based on work performed by Martin and colleagues (1976) to study the actions of antipsychotic drugs. In these experiments, they proposed the existence of a -opioid receptor based on the psychomimetic effects of SKF-10047, which could not be explained by -or -opioid receptors (Martin et al., 1976). This hypothesis, however, was later refuted when the receptor was shown to be insensitive to naloxone, a common opioid receptor antagonist (Iwamoto, 1981;Su, 1982;Vaupel, 1983). As a result, receptors were reclassified as unique nonopioid and nonphencyclidine binding sites present in the central nervous system and peripheral organs that are distinct from other known neurotransmitter or hormone receptors (Quirion et al., 1992).To date, two subtypes of the receptor have been identified, the -1 and -2 receptors, which are distinguishable by their pharmacology, function, and molecular mass. The -1 receptor was first cloned from guinea pig liver in 1996 and subsequently from other sources, including human placental choriocarcinoma cells (Kekuda et al., 1996), human brain (Prasad et al., 1998), rat brain (Seth et al., 1998;Mei and Pasternak, 2001), and mouse brain (Pan et al., 1998). The -2 receptor, however, has yet to be cloned. The -1 receptor has 223 amino acids and shares 30% identity and 67% similarity with a yeast sterol C8 -C7 isomerase, which is involved in cholesterol synthesis (Moebius et al., 1997). Unlike this yeast sterol isomerase, however, the -1 receptor does not have any sterol isomerase activity , and it shares no sequence homology with any known mammalian proteins, including the mammalian C8 -C7 sterol isomerase. This work was supported by the National Institutes of Health grant R01-MH065503 (to A.E.R.).Article, publication date, and citation information can be found at

Sigma-1 receptors are associated with Alzheimer's disease, major depressive disorders, and schizophrenia. These receptors show progrowth/antiapoptotic properties via their chaperoning functions to counteract ER (endoplasmic reticulum) stress, to block neurodegeneration, and to regulate neuritogenesis. The sigma-1 receptor knock out mouse offered an opportunity to assess possible mechanisms by which the Sigma-1 receptor modulates cellular oxidative stress. Nuclear magnetic resonance (NMR) metabolomic screening of the WT (wild type) and sigma-1 KO (knockout) livers was performed to investigate major changes in metabolites that are linked to oxidative stress. Significant changes in protein levels were also identified by two-dimensional (2D) gel electrophoresis and mass spectrometry. Increased levels of the antioxidant protein peroxiredoxin 6 (Prdx6), and the ER chaperone BiP (GRP78) compared to WT littermates were detected. Oxidative stress was measured in WT and sigma-1 KO mouse liver homogenates, in primary hepatocytes and in lung homogenates. Furthermore, sigma-1 receptor mediated activation of the antioxidant response element (ARE) to upregulate NAD(P)H quinone oxidoreductase 1 (NQO1) and superoxide dismutase 1 (SOD1) mRNA expression in COS cells was shown by RT PCR. These novel functions of the sigma-1 receptor were sensitive to well-known sigma ligands via their antagonist/agonist properties.

The sigma1 receptor is distinguished for its ability to bind various pharmacological agents including drugs of abuse such as cocaine and methamphetamine. Some endogenous ligands have been identified as putative sigma1 receptor regulators. High affinity ligands for the sigma1 receptor contain a nitrogen atom connected to long alkyl chains. We found that long alkyl chain primary amines including endogenous amines belonging to the sphingolipid family such as D-erythro-sphingosine and sphinganine bind with considerable affinity to the sigma1 receptor but not to the sigma2 receptor. The binding of D-erythro-sphingosine to the sigma1 receptor appears to be competitive in nature as assessed against the radioligand [3H]-(+)-pentazocine. Interestingly, the well studied sphingolipid mediator sphingosine-1 phosphate did not bind to the sigma1 or the sigma2 receptor. Sphingosine is converted to sphingosine-1 phosphate by a family of sphingosine kinases that regulate the relative levels of these two bioactive lipids in the cell. The selective binding of sphingosine but not sphingosine-1 phosphate to the sigma1 receptor suggests a mechanism for regulation of sigma1 receptor activity by the sphingosine kinase. We have successfully reconstituted this hypothetical model in HEK-293 cells overexpressing both the sigma1 receptor and sphingosine kinase-1. The data presented here strongly supports sphingosine as an endogenous modulator of the sigma1 receptor.

The receptors represent unique nonopioid and nonphencyclidine binding sites that are distinct from other known neurotransmitters or hormone receptors (4). Initially, Martin et al. (5) proposed the receptors as opioid receptors based on the psychomimetic effects of N-allyl-normetazocine (SKF-10047), which could not be explained by -or -opioid receptors (5). This hypothesis, however, was later corrected when the receptors were found to be insensitive to a common opioid receptor antagonist, naloxone (6 -8). Two well known subtypes of the receptor have been identified, the -1 receptor and the -2 receptor, which are distinguishable by their ligand binding properties and molecular weights (2, 9). They are ubiquitously expressed in different tissues, such as brain, adrenal gland, testis, ovary, spleen, lung, heart, blood leukocytes, and cancer cells (10 -15).Pharmacological studies have indicated that the -1 receptor is able to bind a wide range of compounds, including opiates (7,16,17), antipsychotics (18), antidepressants (18), antihistamines (16), phencyclidine-like compounds (19), ␤-adrenergic receptor ligands (7, 16), serotonergic compounds (16), cocaine and cocaine analogs (1, 20, 21), cholesterol (22), steroids (23-25), and neuropeptides (25), although intriguingly no known natural specific ligands have been discovered. In addition, -1 receptor knock-out mice are viable and fertile, showing no overt constitutive phenotype (26). However, the broad tissue distribution as well as the ability to bind different classes of ligands allows the -1 receptor to mediate various cellular events, such as modulation of voltage-gated K ϩ channels (27), calcium release (28), regulation of lipid compartmentalization on the endoplasmic reticulum (29), and "ligand-operated" chaperoning activity at "mitochondrion-associated endoplasmic reticulum membranes" (30). Regulation of cocaine effects (31, 32), neuroprotective effects (33), increase in extracellular acetylcholine levels (34), and inhibition of proliferative responses to mitogens (35) have also been ascribed to the -1 receptor. A role in psychostimulant actions has been proposed for the -1 receptor, based on methamphetamine binding (36).The -1 receptor has been cloned from different mammalian species, including guinea pigs (37), humans (38, 39), rats (40, 41), and mice (42), and shares about 90% identity and 95% similarity across these species. However, the -2 receptor has not been cloned yet. The cloned -1 receptor has 223 amino acids and shares 30% identity and 67% similarity with a yeast sterol C8-C7 isomerase (ERG2), which is involved in cholesterol synthesis (43). Interestingly, unlike the yeast sterol isomerase, the -1 receptor does not possess any sterol isomerase activity (37) and, in addition, shares no sequence homology with any known mammalian proteins, including the mammalian C8-C7 sterol isomerase. Hydrophobicity analysis indicates that the receptor and fungal sterol isomerases are topologically similar, each containing three hydrophobic regions. The first hydro...

Radioiodinated photoactivatable photoprobes can provide valuable insights regarding protein structure. Previous work in our laboratory showed that the cocaine derivative and photoprobe 3-[ (125)I]iodo-4-azidococaine ([ (125)I]IACoc) binds to the sigma-1 receptor with 2-3 orders of magnitude higher affinity than cocaine [Kahoun, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393-1397]. Using this photoprobe, we demonstrated the insertion site for [ (125)I]IACoc to be Asp188 [Chen, Y. (2007) Biochemistry 46, 3532-3542], which resides in the proposed steroid binding domain-like II (SBDLII) region (amino acids 176-194) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. An additional photoprobe based on the sigma-1 receptor ligand fenpropimorph, 1- N-(2-3-[ (125)I]iodophenyl)propane ([ (125)I]IAF), was found to label a peptide in both the SBDLII and steroid binding domain-like I (SBDLI) (amino acids 91-109) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. In this report, we describe two novel strategically positioned carrier-free, radioiodinated photoaffinity labels specifically designed to probe the putative "nitrogen interacting region" of sigma-1 receptor ligands. These two novel photoprobes are (-)-methyl 3-(benzoyloxy)-8-2-(4-azido-3-[ (125)I]iodobenzene)-1-ethyl-8-azabicyclo[3.2.1]octane-2-carboxylate ([ (125)I]-N-IACoc) and N-propyl- N-(4-azido-3-iodophenylethyl)-3-(4-fluorophenyl)propylamine ([ (125)I]IAC44). In addition to reporting their binding affinities to the sigma-1 and sigma-2 receptors, we show that both photoaffinity labels specifically and covalently derivatized the pure guinea pig sigma-1 receptor (26.1 kDa) [Ramachandran, S. (2007) Protein Expression Purif. 51, 283-292]. Cleavage of the photolabeled sigma-1 receptor using Endo Lys C and cyanogen bromide (CNBr) revealed that the [ (125)I]-N-IACoc label was located primarily in the N-terminus and SBDLI-containing peptides of the sigma-1 receptor, while [ (125)I]IAC44 was found in peptide fragments consistent with labeling of both SBDLI and SBDLII.