Differential solubilization of rat liver σ1 and σ2 receptors: retention of σ2 sites in particulate fractions

Abstract

Rat liver membranes (crude P 2 membranes) were solubilized in 10 mM Tris-HCl, pH 7.4 containing 7 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The soluble fraction was designated the Extract 1. The 105 000 × g pellet was washed once, and then extracted a second time (Extract 2). The various resulting fractions were assayed for sigma (σ) binding characteristics, using [ 3H](+)-pentazocine to label σ 1 sites and [ 3H]1,3-di- o-tolylguanidine (DTG) in the presence of 1 μM dextrallorphan to label σ 2 sites. Both of the extracts and resultant pellets (Pellet 1 and Pellet 2) contained σ 1 and σ 2 receptors, as indicated by the pharmacological profiles upon competition studies. The K d and B max values for σ 1 activity in the original P 2 membranes were 8.3 ± 0.73 nM and 5333 ± 572 fmol/mg protein; K d and B max for σ 2 activity was 19 ± 0.17 nM and 9190 ± 800 fmol/mg protein. There were no changes in the radioligand K d values of the two sites in the subsequent soluble and particulate fractions. However, while the σ 1 and σ 2 B max values in extracts and pellets were generally on the same order as those of P 2 membranes, the actual σ 2 to σ 1 B max ratio varied markedly across the fractions. The ratio of σ 2 σ 1 binding in Extract 1 and Extract 2 was 0.86 and 0.68, respectively, compared to a ratio of 1.7 in the original P 2. However, the ratio in Pellet 2 was 3.8, twice that of the original P 2 membranes. Furthermore, the B max value for σ 1 sites in Pellet 2 did not change, whereas the σ 2 B max increased 1.8 fold relative to the original P 2 membranes. The changes in σ 2 σ 1 binding ratio in extracts were observed using two different assay methods for soluble receptors (retention on polyethyleneimine-coated filters and polyethylene glycol precipitation) and is therefore not an artifact of assay procedure. These data suggest that, relative to σ 1 receptors, σ 2 receptors are more resistant to solubilization and become somewhat enriched in the particulate fractions. This supports the notion that σ 1 and σ 2 receptors are distinct macromolecules and may indicate different modes of association with the cell membrane.