Selective coupling of purified alpha-subunits of pertussis toxin-substrate GTP-binding proteins to endogenous receptors in rat brain membranes treated with N-ethylmaleimide.
The membrane fraction prepared from rat brain was incubated with 0.5 mM N-ethylmaleimide (NEM) for 10 min. 3H-labelled agonist binding to muscarinic, A1-adenosine, opiate and alpha 2-adrenergic receptors was markedly inhibited by this NEM treatment of membranes, which interfered with the subsequent ADP-ribosylation of endogenous G-proteins by pertussis toxin. This indicated that the toxin target cysteine residues of the G-protein were modified by NEM. The NEM-induced inhibition of agonist bindings was mostly reversed by reconstitution of the alpha-subunits of purified Gi or Go into the membranes. The NEM-induced inhibition, together with the reversal by the G alpha reconstitution, was due to changes in the relative number of high- to low-affinity receptors solely without change in the total (high- plus low-affinity) receptor number. Thus, in NEM-treated membranes endogenous G-proteins become uncoupled from receptors, which were coupled to either Gi alpha or Go alpha. Reconstitution of NEM pre-treated membranes showed that Go acted in preference to Gi in interaction with muscarinic receptors and vice versa in interaction with three other types of receptor. The possible involvement of Go in mediating phospholipase C activation and Gi in mediating adenylate cyclase inhibition is discussed.
Shinoda
M
M
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Katada
T
T
Ui
M
M
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004745:Enkephalins / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D037342:Pertussis Toxin; D051381:Rats; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D010566:Virulence Factors, Bordetella / Q000378:metabolism*
D037361:Adenylate Cyclase Toxin; D004745:Enkephalins; D011956:Receptors, Cell Surface; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide