Induction of G protein-independent agonist high-affinity binding sites of D-1 dopamine receptors by beta-mercaptoethanol.
Induction of G protein-independent agonist high-affinity binding sites of D-1 dopamine receptors by beta-mercaptoethanol.
复制标题
β-巯基乙醇诱导 D-1 多巴胺受体的 G 蛋白非依赖性激动剂高亲和力结合位点。
DOI：
10.1021/bi00203a021
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Vachvanichsanong,P
中科院分区：
文献类型：
--
作者：
Sidhu,A;Kimura,K;Vachvanichsanong,P
Revised Manuscript Received July 15, 19949 abstract: We have purified the D-1 dopamine receptor 8200-fold to 78% purity from rat striatal membranes. Critical to this purification was the iV-ethylmaleimide (NEM)-mediated alkylation of all endogenous sulfhydryl groups, except those associated with the D-1 dopamine receptors, which were protected by the D-1 agonist SKF R-38393. Such NEM treatment of Dl receptors abolished all agonist high-affinity binding sites of the receptors, but did not alter the antagonist binding properties. When NEM-treatedD-l receptors were affinity-purified by mercury-agarose columns, the pharmacological properties of these purified receptors were examined, after removal of/3-mercaptoethanol (/3ME), which was used for elution of receptors from the affinity column. Purified Dl receptors displayed typical dopaminergic antagonist binding values; however, agonists bound to the purified receptors with only high-affinity binding values, despite the prior absence of high-affinity sites in crude soluble extracts of NEM-treated receptors. The agonist high-affinity binding of purified D-1 receptors was insensitive to modulation by the GTP analog Gpp (NH) p and occurred in the absence of any G proteins. These Gpp (NH) p-insensitive high-affinity sites appeared to be induced by/3ME, since similar high-affinity binding was also restoredby/3ME to crude soluble and membranebound receptors, which had been pretreated with NEM. The ability of Dl dopamine receptors to bind with high-affinity to agonists in the absence of functionally active G proteins may be an intrinsic property of the reduced state of Dl dopamine receptors.Dysfunctional dopaminergic neurotransmissions have been implicated in several neuropathological conditions, including schizophrenia, Parkinson’s and Huntington’s diseases, drug (cocaine and amphetamine) addiction, hypertension, and hyperprolactemia. At the molecular level, the effects of dopamine are mediated through two receptor subtypes, D-1 and D-2, which are broadly classified according to different pharmacological and physiological properties (Kebabian & Caine, 1979). Recently, as many as five genetically distinct dopamine receptors havebeen identified through molecular cloning techniques (O’Dowd, 1993). D-1 dopamine receptors have been shown to stimulate adenylate cyclase (Kebabian & Caine, 1979), phospholipase C (Felder et al., 1989; Undie & Friedman, 1990), and protein kinase C (Bertorello & Aperia, 1989), to inhibit Na+/K+-ATPase stimulation (Bertorello et al., 1990), and to activate the arachidonic acid cascade system (Piomelli et al., 1991). The mechanism (s) bywhich Dl
