The possibility that glycogen synthase kinase 3 (GSK3) could modulate α1A-adrenergic receptor (α1A-AR) function and regulation was tested employing LNCaP and HEK293 cells transfected to express the enhanced green fluorescent protein-tagged human α1A-AR. Receptor phosphorylation and internalization, intracellular free calcium, α1A-AR-GSK3 colocalization, and coimmunoprecipitation were studied. The effects of the pharmacological GSK3 inhibitor, SB-216763, and the coexpression of a dominant-negative mutant of this kinase, as well as the signaling, desensitization, and internalization of receptors with S229, S258, S352, and S381 substitutions for alanine or aspartate, were also determined. SB-216763 inhibited agonist- and phorbol myristate acetate (PMA)-mediated α1A-AR phosphorylation, reduced oxymetazoline-induced desensitization, and magnified that induced by PMA. Agonists and PMA increased receptor-GSK3 colocalization and coimmunoprecipitation. Expression of a dominant-negative GSK3 mutant reduced agonist- but not PMA-induced receptor internalization. α1A-AR with the GSK3 putative target sites mutated to alanine exhibited reduced phosphorylation and internalization in response to agonists and increased PMA-induced desensitization. Agonist-induced, but not PMA-induced, receptor-β arrestin intracellular colocalization was diminished in cells expressing the GSK3 putative target sites mutated to alanine. Our data indicated that GSK3 exerts a dual action on α1A-AR participating in agonist-mediated desensitization and internalization and avoiding PMA-induced desensitization.

中文翻译：

---

糖原合酶激酶3调节α1A-肾上腺素受体的作用和调节。

糖原合酶激酶3（GSK3）可以调节α1A-肾上腺素能受体（α1A-AR）的功能和调节的可能性已通过转染表达增强的绿色荧光蛋白标签的人α1A-AR的LNCaP和HEK293细胞进行了测试。研究了受体的磷酸化和内在化，细胞内游离钙，α1A-AR-GSK3的共定位和共免疫沉淀。药理学GSK3抑制剂SB-216763的影响，以及该激酶的显性负突变体的共表达，以及用S229，S258，S352和S381取代丙氨酸或天冬氨酸，也被确定。SB-216763抑制了激动剂和佛波肉豆蔻酸酯乙酸盐（PMA）介导的α1A-AR磷酸化，减少了甲氧咪唑啉引起的脱敏，并放大了PMA诱导的结果。激动剂和PMA增加受体GSK3的共定位和免疫共沉淀。显性阴性GSK3突变体的表达减少了激动剂，但没有PMA诱导的受体内在化。具有GSK3假定靶位点突变为丙氨酸的α1A-AR表现出减少的磷酸化和内在化作用，对激动剂有反应，并增加了PMA诱导的脱敏作用。在表达突变为丙氨酸的GSK3假定靶位点的细胞中，激动剂诱导但不是PMA诱导的受体-β抑制蛋白细胞内共定位减少。我们的数据表明，GSK3对α1A-AR发挥双重作用，参与激动剂介导的脱敏和内在化并避免PMA引起的脱敏。显性阴性GSK3突变体的表达减少了激动剂，但没有PMA诱导的受体内在化。具有GSK3假定靶位点突变为丙氨酸的α1A-AR表现出减少的磷酸化和内在化作用，对激动剂有反应，并增加了PMA诱导的脱敏作用。在表达突变为丙氨酸的GSK3假定靶位点的细胞中，激动剂诱导但不是PMA诱导的受体-β抑制蛋白细胞内共定位减少。我们的数据表明，GSK3对α1A-AR发挥双重作用，参与激动剂介导的脱敏和内在化并避免PMA引起的脱敏。显性阴性GSK3突变体的表达减少了激动剂，但没有PMA诱导的受体内在化。具有GSK3假定靶位点突变为丙氨酸的α1A-AR表现出减少的磷酸化和内在化作用，对激动剂有反应，并增加了PMA诱导的脱敏作用。在表达突变为丙氨酸的GSK3假定靶位点的细胞中，激动剂诱导但不是PMA诱导的受体-β抑制蛋白细胞内共定位减少。我们的数据表明，GSK3对α1A-AR发挥双重作用，参与激动剂介导的脱敏和内在化并避免PMA引起的脱敏。GSK3假定的目标位点突变为丙氨酸的α1A-AR表现出减少的磷酸化和内在化对激动剂的反应，并增加了PMA引起的脱敏。在表达突变为丙氨酸的GSK3假定靶位点的细胞中，激动剂诱导但不是PMA诱导的受体-β抑制蛋白细胞内共定位减少。我们的数据表明，GSK3对α1A-AR发挥双重作用，参与激动剂介导的脱敏和内在化并避免PMA引起的脱敏。GSK3假定的目标位点突变为丙氨酸的α1A-AR表现出减少的磷酸化和内在化对激动剂的反应，并增加了PMA引起的脱敏。在表达突变为丙氨酸的GSK3假定靶位点的细胞中，激动剂诱导但不是PMA诱导的受体-β抑制蛋白细胞内共定位减少。我们的数据表明，GSK3对α1A-AR发挥双重作用，参与激动剂介导的脱敏和内在化并避免PMA引起的脱敏。