Effects of sulfhydryl-modifying reagents, 3-nitro-2-pyridinesulfenyl compounds, on the coupling between inhibitory receptors and GTP-binding proteins Gi/Go in rat brain membranes

Y Kitamura, S Imai, R Matsueda and Y Nomura

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.

To gain insight into the coupling mechanism of inhibitory receptors, 5- hydroxytryptamine1A receptors and alpha 2-adrenoceptors, with GTP- binding proteins (G proteins) in the central nervous system, we examined the effects of two 3-nitro-2-pyridinesulfenyl compounds, S-(3- nitro-2-pyridinesulfenyl)-L-cysteine [Cys(Npys)] and N-t-butoxy- carbonyl-S-(3-nitro-2-pyridinesulfenyl)-L-cysteine [Boc-Cys(Npys)], on 1) specific binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH- DPAT) (5-hydroxytryptamine1A agonist) and [3H]clonidine (alpha 2- agonist) to rat brain membranes, 2) [35S]guanosine 5'-O-(3- thio)triphosphate (GTP gamma S) binding, and 3) pertussis toxin (islet- activating protein) (IAP)-catalyzed ADP-ribosylation of purified Go (an IAP-sensitive G protein present in abundance in the mammalian brain). Treatment with Cys(Npys) led to decreased [3H]8-OH-DPAT and [3H]clonidine binding, similar to the inhibitory effects of IAP and N- ethylmaleimide (NEM) on such binding. However, further treatment of Cys(Npys)-pretreated membranes with dithiothreitol completely abolished the inhibitory effect of Cys(Npys) on the binding of both ligands. On the other hand, treatment with Boc-Cys(Npys) inhibited the effect of several GTP analogs (GTP gamma S, guanylyl-imidodiphosphate, guanylyl)- (beta, gamma-methylene)-diphosphate, and GTP) on [3H]8-OH-DPAT and [3H]clonidine binding. Dithiothreitol and mercaptoethanol treatment of Boc-Cys(Npys)-pretreated membranes did not lead to a recovery of the effect of GTP analogs on agonist binding. Regardless of the presence or absence of GTP gamma S, agonist binding to Boc-Cys(Npys)-pretreated membranes was decreased by further addition of NEM or Cys(Npys). Cys(Npys) blocked [35S]GTP gamma S binding as well as IAP-catalyzed ADP- ribosylation in purified Go. In contrast, Boc-Cys(Npys) partially inhibited ADP-ribosylation and did not affect [35S]GTP gamma S binding. These results suggested that Cys(Npys) modifies the receptor-coupling domain in G proteins, followed by the uncoupling of inhibitory receptors from G proteins, similar to the effects of NEM and IAP. Boc- Cys(Npys), however, seems to stabilize the coupling state between the receptors and G proteins, thus abolishing the GTP gamma S effect.

Volume 38, Issue 2, pp. 184-191, 08/01/1990
Copyright © 1990 by American Society for Pharmacology and Experimental Therapeutics