Uncoupling of Rat Cerebral Cortical α₂‐Adrenoceptors from GTP‐Binding Proteins by N‐Ethylmaleimide

Abstract

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.

α2-Adrenoceptor-Mediated Presynaptic Modulation of GABAergic Transmission in Mechanically Dissociated Rat Ventrolateral Preoptic Neurons

“…The ␣ 2 -adrenoceptor that mediates the inhibition of GABA transmission to multipolar VLPO neurons are coupled to NEM-sensitive G i /G o proteins, as has also been observed in a number of tissues including the rat cerebral cortex (Kitamura and Nomura 1987) and rat vas deferens (Browne et al 1994). Our results further suggest that ␣ 2 -adrenoceptor activation acts via the AC/cAMP/PKA transduction pathway.…”

On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart.

“…This observation indicates that IAP and NEM affect neither the channel itself nor the interaction between the exogenously applied G protein subunit and the channel (see also Nakajima et al, 1990). Therefore, the functional uncoupling of G~ and the membrane receptor may have progressed time-dependently during incubation of the inside-out patches by IAP and NEM (Katada and Ui, 1982;Ui, 1984;Katada et al, 1986a;Kitamura and Nomura, 1987;Ueda et al, 1990), resulting in loss of activation of IK.ach by GTP and then finally by GTP--,/S (Fig. 3).…”

“…/K.ACh" Since lAP specifically ADP-ribosylates a certain population of G proteins, and NEM alkylates G~ proteins to uncouple them from membrane receptors (Katada and Ui, 1982;Ui, 1984;Katada et al, 1986a;Kitamura andNomura, 1987, Ueda et al, 1990), the failure of channel activation by GTP and GTP-~S may be due to the functional uncoupling of GK and the membrane receptor caused by lAP and NEM.…”

“…IAP specifically ADP-ribosylates a certain population of G proteins, uncoupling them from their associated membrane receptors and preventing the transduction of signals from the receptors (Katada and Ui, 1982;Ui, 1984). At low concentrations, NEM, a sulfhydryl-alkylating agent, also mimics IAP in blocking the IAP-sensitive G proteins (Katada, Kurose, Oinuma, Hoshino, Shinoda, Asanuma, and Ui, 1986a;Kitamura and Nomura, 1987;Ueda, Misawa, Katada, Ui, Takagi, and Satoh, 1990) and inhibits ACh activation of IKACh (Nakajima, Irisawa, and Giles, 1990). Therefore, the inhibitory effects of IAP and NEM on the GTP-TS-induced activation of IKAC h current suggest that the interaction between the agonist-free membrane receptor and G K is essential for the basal turn-on reaction of GK-However, since bacterial toxin-mediated ADP ribosylation alters the amount of various G proteins in the cell membrane (Chang and Bourne, 1989;Watkins, Northup, and Malbon, 1989), loss of IAP substrate from the cardiac cell membrane during the pretreatment of animals with IAP could also cause the decrease in the GTP-~S-induced K + current in IAP-treated atrial myocytes.…”

Activation of Ca(2+)-dependent K+ current by acetylcholine and histamine in a human gastric epithelial cell line.

“…In Fig. 13 B, we examined the effects of NEM (50 I~M), which uncouples the receptors from the PTX-sensitive G proteins (Katada, Kurose, Oinuma, Hoshino, Shinoda, Asanuma, and Ui, 1986;Kitamura and Nomura, 1987;Ueda, Misawa, Katada, Ui, Takagi, and Satoh, 1990), on the ACh induction of IK.ca. After washout of the first application of ACh, NEM was added to the bathing solution for 7 min.…”