G protein dependent alterations in [125I]iodocyanopindolol and�cyanopindolol binding at 5-HT1B binding sites in rat brain membranes

Abstract

Several manipulations that affect G protein/receptor coupling also alter the binding of [125I]iodocyanopindolol ([125I]ICYP) and [corrected] +/- cyanopindolol (+/- CYP) to rat brain 5-HT1B binding sites in radioligand binding assays. Inclusion of 5 mM MgSO4 in these assays results in a small but significant increase in the affinity of [125I]ICYP (from KD = 0.046 nM to KD = 0.037 nM). In contrast, 100 microM Gpp(NH)p, GTP, or GDP reduce [125I]ICYP affinity (KD = 0.056 nM with GTP) while ATP and GMP are less effective. +/- CYP affinity for 5-HT1B sites labeled by [3H]dihydroergotamine [( 3H]DE) also displays a small but significant reduction (from Ki = 1.4 nM to Ki = 3.5nM) by the inclusion of 100 microM GTP. Pre-treatment of the brain membranes with N-ethylmaleimide (NEM) in concentrations known to inactivate many G proteins reduces 5-HT1B specific binding of [125I]ICYP. The NEM induced reduction in [125I]ICYP binding can be reversed by reconstitution with purified exogenous G proteins (Go and Gi), demonstrating directly that high affinity binding of [125I]ICYP to 5-HT1B sites is dependent on G proteins. The effects of Mg2+ ion, guanine nucleotides, NEM and G protein reconstitution on [125I]ICYP and +/- CYP binding are all hallmarks of agonist binding to G protein linked receptors. The effect of GTP, however, is quantitatively much less for the binding of these pindolol derivatives than for the binding of 5-HT, a presumed full agonist at 5-HT1B sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of Solubilized Opioid Receptors: Reconstitution and Uncoupling of Guanine Nucleotide‐Sensitive Agonist Binding

“…This effect was specific for guanine nucleotides; CTP and ATP were extremely weak inhibitors. This order of potencies is similar to that seen with respect to the inhibition of agonist binding of other receptors thought to be linked to G proteins (Ariani et al, 1989;Siciliano et al, 1990). These nucleotides also inhibit the hydrolysis of GTP by purified Gi protein in the same order of potency (Milligan and Nee, 1985).…”

Prenatal cocaine produces deficits in serotonin mediated neuroendocrine responses in adult rat progeny: Evidence for long‐term functional alterations in brain serotonin pathways

“…5-HT1, receptors. 5-HTlB receptors were measured according to a modification of the procedure by Ariani et al (1989). Briefly, 5-HT1, sites were measured in a 0.5 ml assay by incubating 1.5 mg wet wt.…”

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‐Hydroxytryptamine1A Receptor

“…and 5-HTID sites in rat brain membranes can be reversed by membrane supplementation with purified exogenous G proteins (Go and Gi) (Stratford et al, 1988;Ariani et al, 1989).…”

“…Therefore, it can be concluded that NaTT oxidation did not affect the same -SH groups as NEM. Because the latter alkylating agent preferentially inactivates an -SH group on the G protein (Stratford et al, 1988;Ariani et al, 1989; see also Table 3), NaTT oxidation might concern -SH groups within the 5-HTIA receptor binding subunit. However, reconstitution experiments with purified R[5-HTIA] and G protein separately treated by NEM and NaTT are necessary to prove definitively the existence of essential -SH groups on both receptor components.…”