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Uncoupling of Rat Cerebral Cortical α 2 ‐Adrenoceptors from GTP‐Binding Proteins by N ‐Ethylmaleimide
Author(s) -
Kitamura Yoshihisa,
Nomura Yasuyuki
Publication year - 1987
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1987.tb02452.x
Subject(s) - gtp' , n ethylmaleimide , g protein , chemistry , binding site , membrane , agonist , biophysics , gtp binding protein regulators , biochemistry , receptor , biology , enzyme
Pretreatment of membranes from rat cerebral cortex with N ‐ethylmaleimide (NEM) decreased [ 3 H]clonidine binding in a concentration‐dependent manner. The B max values of high‐affinity sites for [ 3 H]clonidine were reduced by 50 μM NEM treatment. Treatment with 500 μM NEM diminished the sum of B max of both high‐and low‐affinity components. GTP, Na + , and Mn 2+ exerted little effect on [ 3 H]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 μM NEM, but did not increase [ 3 H]clonidine binding in membranes treated with 500 μM 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, α 2 ‐adrenoceptor, and GTP‐binding protein. One is a highly sensitive site to NEM (a concentration range of 1–50 μM ), which is probably a cysteine residue, IAP‐catalyzed ADP‐ribosylating site on the α‐subunit of GTP‐binding protein. Other sites have low sensitivity to NEM (a concentration range of 0.1–1 m M ), and are the binding domain of agonist and/or the coupling domain of GTP‐binding protein on the α 2 ‐adrenoceptor. In addition, Ki‐ ras p21 protein may lack the capacity to couple with the α 2 ‐adrenoceptor.