Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli

Ramachandran, Subramaniam; Lu, Hongliang; Prabhu, Usha; Ruoho, Arnold E.

doi:10.1016/j.pep.2006.07.019

Cited by 41 publications

(86 citation statements)

References 33 publications

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“…The structures of ligand-bound S1R observed by X-ray crystallography are homotrimeric, which was surprising in light of previous studies suggesting that an even-number of subunits was likely. For example, only ~50 % of S1R preparations were observed to bind (+)-pentazocine [60], and the photoreactive-probe 4-NCCP12 derivatizes no more than 50 % of the receptor molecules [33]. The proteins in these binding studies were recombinantly expressed and purified from E. coli, and it remains possible that on average about half of the proteins are fully folded under those conditions.…”

Section: Oligomerizationmentioning

confidence: 99%

A Review of the Human Sigma-1 Receptor Structure

Ossa

Schnell

Roldan

2017

Advances in Experimental Medicine and Biology

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The Sigma-1 Receptor (S1R) is a small, ligand-regulated integral membrane protein involved in cell homeostasis and the cellular stress response. The receptor has a multitude of protein and small molecule interaction partners with therapeutic potential. Newly reported structures of the human S1R in ligand-bound states provides essential insights into small molecule binding in the context of the overall protein structure. The structure also raises many interesting questions and provides an excellent starting point for understanding the molecular tricks employed by this small membrane receptor to modulate a large number of signaling events. Here, we review insights from the structures of ligand-bound S1R in the context of previous biochemical studies and propose, from a structural viewpoint, a set of important future directions.

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Section: Oligomerizationmentioning

confidence: 99%

A Review of the Human Sigma-1 Receptor Structure

Ossa

Schnell

Roldan

2017

Advances in Experimental Medicine and Biology

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“…Pure guinea pig -1 receptor was prepared as reported previously (Ramachandran et al, 2007). In brief, the maltose binding protein (MBP)-guinea pig -1 receptor fusion protein with a Factor Xa cleavage site linking the MBP and -1 receptor and a six-histidine tag on the C terminus of -1 receptor was expressed in Escherichia coli.…”

Section: Radiosynthesis Of [mentioning

confidence: 99%

“…In addition,-1 receptor binding site peptides that were specifically derivatized by [ 125 I]IAF upon photolysis have been identified both for the membrane-bound -1 receptor and the pure guinea pig -1 receptor (Ramachandran et al, 2007) using cleavage strategies with EndoLys-C and cyanogen bromide.…”

mentioning

confidence: 99%

Identification of Regions of the σ-1 Receptor Ligand Binding Site Using a Novel Photoprobe

Pal

Hajipour²,

Fontanilla³

et al. 2007

Mol Pharmacol

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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

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“…Radioligand Binding Assays-The ligand binding properties of the pure WT (Cys 94 ) and mutant (C94A,V190C) -1 receptors were determined using methods described previously with slight modifications in times and temperatures (45,48,49). The pure receptors (40 ng/well) were incubated with varying concentrations of (ϩ)-[ 3 H]pentazocine at 30°C for 1 h. Binding was carried out in 50 mM Tris-HCl (pH 8.0) in a total volume of 100 l. After incubation, the receptors were harvested on a 0.5% polyethyleneimine-treated Whatman GF/B filters using a Brandel Cell Harvester (Brandel, Gaithersburg, MD).…”

Section: Radiosynthesis Of Methanesulfonothioic Acid [ 125 I]iabm-[mentioning

confidence: 99%

“…Both the wild type (Cys 94 ) and mutant (C94A,V190C) constructs were transformed into BL21(DE3) separately for protein expression and purification as described previously (48). Briefly, the E. coli biomass was collected by centrifugation at 3000 rpm for 30 min, sonicated for 15 min at 4°C.…”

mentioning

confidence: 99%

Juxtaposition of the Steroid Binding Domain-like I and II Regions Constitutes a Ligand Binding Site in the σ-1 Receptor

Pal

Chu

Ramachandran

et al. 2008

Journal of Biological Chemistry

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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...

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Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli

Cited by 41 publications

References 33 publications

A Review of the Human Sigma-1 Receptor Structure

A Review of the Human Sigma-1 Receptor Structure

Identification of Regions of the σ-1 Receptor Ligand Binding Site Using a Novel Photoprobe

Juxtaposition of the Steroid Binding Domain-like I and II Regions Constitutes a Ligand Binding Site in the σ-1 Receptor

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