Hess E J, Battaglia G, Norman A B, Creese I
Mol Pharmacol. 1987 Jan;31(1):50-7.
Both in vivo and in vitro treatments with the irreversible protein-modifying reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), were used to investigate rat striatal D1 dopamine receptor/effector interactions. Peripherally administered EEDQ markedly reduced D1 dopamine receptor binding and D1 dopamine receptor-stimulated adenylate cyclase in a dose-dependent manner. However, EEDQ administered in vivo did not result in functional modification of either the guanine nucleotide-regulatory protein (Ns) or the catalytic subunit of striatal adenylate cyclase as assessed via guanine nucleotide- or forskolin-stimulated cAMP production. Interestingly, the loss in D1 dopamine receptor binding did not correlate directly with observed reductions in dopamine-stimulated adenylate cyclase activity; 40% of D1 dopamine receptor binding was lost with no significant reduction in the Vmax of dopamine-stimulated adenylate cyclase activity. Conversely, the reduction by EEDQ of the adenylate cyclase activity stimulated by the partial agonist SKF38393 was reduced in parallel with EEDQ-induced reductions in the D1 dopamine receptor Bmax. However, when SKF38393-stimulated adenylate cyclase activity was potentiated by forskolin, approximately 30% of receptors could be lost with no significant reduction in cAMP production, resembling the pattern observed utilizing the full agonist dopamine. In vivo pretreatment with the specific D1 antagonist, SCH23390, prevented reductions in dopamine-stimulated adenylate cyclase activity and D1 dopamine receptor binding, suggesting that EEDQ acts at the ligand recognition site of the receptor. Unlike in vivo treatment, in vitro EEDQ treatment resulted in dose-dependent decreases in catalytic subunit activity as assessed by forskolin-stimulated cAMP production, indicating that, in vitro, the adenylate cyclase catalytic subunit is vulnerable to EEDQ-induced modification. These data indicate that EEDQ is an effective tool for elucidating the mechanisms and biochemistry of D1 dopamine receptor/effector coupling.
使用不可逆蛋白修饰试剂N - 乙氧羰基 - 2 - 乙氧基 - 1,2 - 二氢喹啉（EEDQ）进行体内和体外处理，以研究大鼠纹状体D1多巴胺受体/效应器相互作用。外周给予EEDQ以剂量依赖性方式显著降低D1多巴胺受体结合和D1多巴胺受体刺激的腺苷酸环化酶。然而，通过鸟嘌呤核苷酸或福司可林刺激的cAMP产生评估，体内给予EEDQ并未导致鸟嘌呤核苷酸调节蛋白（Ns）或纹状体腺苷酸环化酶催化亚基的功能改变。有趣的是，D1多巴胺受体结合的丧失与多巴胺刺激的腺苷酸环化酶活性的观察到的降低并不直接相关；D1多巴胺受体结合丧失40%，而多巴胺刺激的腺苷酸环化酶活性的Vmax没有显著降低。相反，EEDQ对部分激动剂SKF38393刺激的腺苷酸环化酶活性的降低与EEDQ诱导的D1多巴胺受体Bmax降低平行。然而，当福司可林增强SKF38393刺激的腺苷酸环化酶活性时，约30%的受体可能丧失，而cAMP产生没有显著降低，类似于使用完全激动剂多巴胺观察到的模式。用特异性D1拮抗剂SCH23390进行体内预处理可防止多巴胺刺激的腺苷酸环化酶活性和D1多巴胺受体结合的降低，表明EEDQ作用于受体的配体识别位点。与体内处理不同，体外EEDQ处理导致福司可林刺激的cAMP产生评估的催化亚基活性呈剂量依赖性降低，表明在体外，腺苷酸环化酶催化亚基易受EEDQ诱导的修饰。这些数据表明EEDQ是阐明D1多巴胺受体/效应器偶联机制和生物化学的有效工具。
