Li M, Bermak J C, Wang Z W, Zhou Q Y
Department of Pharmacology, University of California, Irvine, California, USA.
Mol Pharmacol. 2000 Mar;57(3):446-52. doi: 10.1124/mol.57.3.446.
Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.
最近，与G蛋白偶联受体结合的蛋白质已被确定为受体锚定和信号传导的调节因子。在本研究中，肌动蛋白结合蛋白280（ABP - 280）是一种广泛表达的细胞骨架相关蛋白，在调节细胞形态和运动中起重要作用，被发现与多巴胺D(2)受体的第三个细胞质环相关联。这种相互作用的特异性最初是在酵母双杂交筛选中确定的，并通过蛋白质结合得到证实。在缺乏ABP - 280的人黑色素瘤细胞中评估了D(2)受体 - ABP - 280关联的功能意义。D(2)受体激动剂在抑制这些细胞中福斯可林刺激的cAMP产生方面效力较低。D(2)受体激活的最大抑制反应也降低了。进一步的酵母双杂交实验表明，ABP - 280关联严重依赖于D(2)受体第三个细胞质环的羧基结构域，那里有一个潜在的丝氨酸磷酸化位点（S358）。将丝氨酸358替换为天冬氨酸以模拟受体磷酸化的作用。这种突变体（D(2)S358D）与ABP - 280的结合以及与腺苷酸环化酶的偶联受损。PKC激活也导致D(2)受体信号传导减弱，但仅在含有ABP的细胞中，这表明PKC在D(2)-ABP关联中起调节作用。这些结果的一种机制可能源于ABP - 280在D(2)受体聚集中的作用，这是通过对缺乏ABP和富含ABP的细胞进行免疫细胞化学分析确定的。我们的结果提示了一种通过细胞骨架蛋白相互作用调节D(2)受体信号传导的新分子机制。
