Analysis of intracellular signaling cascades mediated by receptor activation and protein phosphorylation in neostriatal

DARPP-32是一种受多巴胺和cAMP调节的磷酸蛋白，分子量为32 kDa，是一种胞浆蛋白，选择性地富含在新纹状体的中等大小的棘神经元中。DARPP-32在Thr34位被cAMP依赖的蛋白激酶(PKA)磷酸化，转化为蛋白磷酸酶(PP)-1的有效抑制物。多巴胺刺激DARPP-32的磷酸化，并通过抑制PP-1调节离子通道和钠泵的功能以及新纹状体神经元的兴奋性。我们研究了多种神经递质和细胞内信号系统对DARPP-32磷酸化的调节机制，得到了以下结果。乙酰胆碱调节DARPP-32的磷酸化：非选择性胆碱能激动剂Calbacholine刺激新纹状体神经元Thr34处的DARPP-32的磷酸化。卡巴胆碱的作用是通过激活M受体介导的。参与…调控的M受体亚型更多的DARPP-32磷酸化，M1或M4，正在研究中。DARPP-32去磷酸化的调节：在新的纹状体神经元中，DARPP-32被钙调神经磷酸酶和PP-2A去磷酸化。这两种磷酸酶协同作用，维持低水平的磷酸Thr34 DARPP-32.3。细胞周期蛋白依赖性蛋白激酶5(CDK5)对DARPP-32的磷酸化作用：在新生纹状体神经元中，Thr75位的DARPP-132Thr75被CDK5/p35复合体磷酸化。Thr75位的磷酸化形式的DARPP-32在体外和新纹状体神经元中作为PKA的抑制因子发挥作用。DARPP-32磷酸化的调节。Thr75被PKA激活：用Forskolin、cAMP类似物或多巴胺D1激动剂SKF81297刺激PKA，可降低新生纹状体神经元中磷酸化Thr75 DARPP-32的水平，从而解除对PKA的抑制并进一步激活PKA。因此，Thr75磷酸化介导的信号级联对PKN/磷酸化Thr34DARPP-32/PP-1和PEA/PKA底物级联起正反馈作用。较少

DARPP-32, a dopamine-and cAMP-regulated phosphoprotein of Mr 32 kDa, is a cytosolic protein that is selectively enriched in medium-sized spiny neurons in neostriatum. DARPP-32 is phosphorylated by cAMP-dependent protein kinase (PKA) at Thr34 residue, converting it into a potent inhibitor of protein phosphatase (PP) -1. Dopamine stimulates the phosphotylation of DARPP-32 and regulates the functions of ion channels and sodium pump and the excitability of neostriatal neurons by inhibiting PP-1. We studied the mechanisms of the regulation of DARPP-32 phosphorylation by various neurotransmitters and intracellular signaling systems, and obtained the following results.1. Regulation of DARPP-32 phosphorylation by acethylcholine : Calbachol, non-selective cholinergic agonist, stimulates the phosphorylation of DARPP-32 at Thr34 in neostriatal neurons. The effect of calbachol was mediated through the activation of muscarinic receptors. Isoform of muscarinic receptors involved in the regulation of … More DARPP-32 phosphorylation, M1 or M4, is under investigation.2. Regulation of DARPP-32 dephosphorylation : Phosph-Thr34 DARPP-32 is dephosphorylated by both calcineurin and PP-2A in neostriatal neurons. These two phosphatases act synergistically to maintain a low level of phospho-Thr34 DARPP-32.3. Phosphorylation of DARPP-32 by cyclin-dependent protein kinase 5 (cdk5): DARPP-l32 at Thr75 is phosphorylated by cdk5/p35 complex in neostriatal neurons. phosphorylated form of DARPP-32 at Thr75 functions as an inhibitor of PKA in vitro and in neostriatal neurons.4. Regulation of DARPP-32 phosphorylation at. Thr75 by PKA : Stimulation of PKA by forskolin, cAMP analogue or dopamine D1 agonist, SKF81297, reduced the level of phospho-Thr75 DARPP-32 in neostriatal neurons, resulting in the removal of the inhibition of PKA and further activation of PKA. Thus, the signaling cascade mediated by Thr75 phosphorylation functions as a positive feedback system for PKN/phospho-Thr34 DARPP-32/PP-1 and PEA/PKA substrate cascades. Less