Uncoupling of rat cerebral cortical alpha 2-adrenoceptors from GTP-binding proteins by N-ethylmaleimide.
Pretreatment of membranes from rat cerebral cortex with N-ethylmaleimide (NEM) decreased [3H]-clonidine binding in a concentration-dependent manner. The Bmax values of high-affinity sites for [3H]clonidine were reduced by 50 microM NEM treatment. Treatment with 500 microM NEM diminished the sum of Bmax of both high- and low-affinity components. GTP, Na+, and Mn2+ exerted little effect on [3H]clonidine binding in NEM-treated membranes. The addition of purified GTP-binding proteins caused an increase in the binding to the membranes pretreated with 50 microM NEM, but did not increase [3H]-clonidine binding in membranes treated with 500 microM NEM. In contrast, NEM pretreatment inhibited islet activating protein (IAP)-catalyzed ADP ribosylation of membrane-bound (41,000-dalton) and purified (39,000/41,000-dalton) GTP-binding proteins. From these results, it is suggested that two or three categories of essential sulfhydryl groups are involved in the coupling between agonist, alpha 2-adrenoceptor, and GTP-binding protein. One is a highly sensitive site to NEM (a concentration range of 1-50 microM), which is probably a cysteine residue, IAP-catalyzed ADP-ribosylating site on the alpha-subunit of GTP-binding protein. Other sites have low sensitivity to NEM (a concentration range of 0.1-1 mM), and are the binding domain of agonist and/or the coupling domain of GTP-binding protein on the alpha 2-adrenoceptor. In addition, Ki-ras p21 protein may lack the capacity to couple with the alpha 2-adrenoceptor.
Kitamura
Y
Y
Department of Pharmacology, Toyama Medical and Pharmaceutical University, Japan.
Nomura
Y
Y
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D002712:Chlorides; D003000:Clonidine / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism* / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D017895:Manganese Compounds; D037342:Pertussis Toxin; D051381:Rats; D011919:Rats, Inbred Strains; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D002712:Chlorides; D017895:Manganese Compounds; D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D008345:Manganese; D012965:Sodium Chloride; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D003000:Clonidine; D005033:Ethylmaleimide; C025340:manganese chloride