The GTP‐Insensitive Component of High‐Affinity [³H]8‐Hydroxy‐2‐(Di‐n‐Propylamino)tetralin Binding in the Rat Hippocampus Corresponds to an Oxidized State of the 5‐Hydroxytryptamine_1A Receptor

Abstract

Previous studies on central 5-hydroxytryptamine1A (5-HT1A) receptors have consistently shown the existence of a GTP-insensitive component of agonist binding, i.e., binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp-insensitive component of [3H]8-OH-DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate-soluble extracts to air; it was reduced by preincubation of solubilized 5-HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short-cross-linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp-insensitive over total [3H]8-OH-DPAT binding. The pharmacological properties of this GppNHp-insensitive component of [3H]8-OH-DPAT binding were similar to those of 5-HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5-HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5-HT1A receptor binding subunit (R[5-HT1A]) and a guanine nucleotide-binding protein (G protein). Moreover, the oxidation of -SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8-OH-DPAT specific binding by N-ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same -SH groups on the R[5-HT1A]-G protein complex. These data suggest that the appearance of GTP-insensitive [3H]8-OH-DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential -SH groups (different from those preferentially inactivated by N-ethylmaleimide) on the R[5-HT1A]-G protein complex.

Heterogeneity of Cortical and Hippocampal 5‐HT1A Receptors: A Reappraisal of Homogenate Binding with 8‐[3H]Hydroxydipropylaminotetralin

“…More important, the reduction of 1 nM [3H]8-OH-DPAT high-affinity binding by Gpp(NH)p confirms that this site corresponds to the known 5-HT,, receptor. In contrast, the other component of 1 nM [3H]8-OH-DPAT binding, or 5-HTIALoW sites, remained insensitive to 100 pM Gpp(NH)p. A similar observation has recently been reported using a sucrose gradient sedimentation procedure (Emerit et al, 1991); these authors as well as Mongeau and coworkers ( 1992) proposed that 5-HT,, receptors exist in different conformational states. However, in the present study, the pharmacological profile, in particular that of the 5-HT antagonist pindolol, which also revealed two binding sites, as well as the experiments with Gpp(NH)p, suggest the existence of a novel 5-HT,, binding site, labeled with 1 nM [3H]8-OH-DPAT, that has a micromolar affinity for 5-HT and that is seemingly not regulated by guanine nucleotides.…”

The Serotonin1A A Receptor: A Representative Member of the Serotonin Receptor Family

“…Black circles represent sites for protein kinase mediated phosphorylation. Adapted and modified from Raymond et al (1999). agents that inhibit ligand binding to the 5-HT 1A receptor (Emerit et al, 1991;.…”

Characterisation of the Binding of [3H]WAY‐100635, a Novel 5‐Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

“…WAY-100635 8 .87 -0 .14 1 .11 ± 0 .17 SDZ-216525 8 .72 ± 0.24 1 .15 (1994) have reported that such sites represent presynaptic 5 -HT,A terminal autoreceptors, whereas Emerit et al . (1990Emerit et al . ( , 1991 have suggested that the formation of disulfide bridges within the 5-HT IA -G protein complex will produce a GTP-insensitive form of the postsynaptic receptor .…”

“…However, only a portion of the [ 3 H]8-OH-DPAT binding sites was converted to the low-affinity state (-60%), with the remainder constituting a GTP-insensitive population . These findings are similar to those reported by several other groups showing GTP and related analogues to inhibit partially high-affinity hippocampal [ 3 H]8-OH-DPAT agonist binding to the 5-HT,A site (Hall et al ., 1985 ;Emerit et al ., 1990Emerit et al ., , 1991Mongeau et al ., 1992) . The affinity and binding capacity of [ 3H]WAY-100635 to rat hippocampal membranes were unaltered by guanyl nucleotides, as its association with the recognition domains on the 5 -HT,A binding site is not influenced by the state of receptor-G protein coupling .…”