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.
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.
Kitamura
Y
Y
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Imai
S
S
Matsueda
R
R
Nomura
Y
Y
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003000:Clonidine / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives; D007202:Indicators and Reagents; D008297:Male; D009574:Nitro Compounds / Q000494:pharmacology*; D037342:Pertussis Toxin; D011725:Pyridines / Q000494:pharmacology*; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects* / Q000378:metabolism; D011985:Receptors, Serotonin / Q000187:drug effects* / Q000378:metabolism; D013438:Sulfhydryl Compounds; D013764:Tetrahydronaphthalenes / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007202:Indicators and Reagents; D009574:Nitro Compounds; D011725:Pyridines; D011942:Receptors, Adrenergic, alpha; D011985:Receptors, Serotonin; D013438:Sulfhydryl Compounds; D013764:Tetrahydronaphthalenes; D010566:Virulence Factors, Bordetella; C050518:t-butyloxycarbonyl-(S-(3-nitro-2-pyridinesulfenyl))cysteine; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; C062289:S-(3-nitro-2-pyridinesulfenyl)cysteine; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D003545:Cysteine; D003000:Clonidine; D005033:Ethylmaleimide