Sromek S M, Harden T K
Department of Pharmacology and Curriculum in Neurobiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Mol Pharmacol. 1998 Sep;54(3):485-94. doi: 10.1124/mol.54.3.485.
The Gq/phospholipase C-linked human P2Y2 receptor was tagged at its amino terminus with the hemagglutinin A (HA) epitope sequence (P2Y2-HA) and stably expressed in 1321N1 human astrocytoma cells. Neither the pharmacological selectivity nor the signaling properties of the receptor were altered by the presence of the epitope. An enzyme-linked immunosorbent assay was developed to quantify cell surface levels of P2Y2-HA receptors using an anti-HA antibody. Incubation of cells with P2Y2 receptor agonists resulted in a concentration of agonist- and time-dependent decrease in cell surface immunoreactivity. Methodology for indirect immunofluorescence confocal microscopy was developed and applied to demonstrate that the agonist-promoted decreases in cell surface immunoreactivity paralleled increases in intracellular immunoreactivity. Agonist-induced internalization of P2Y2 receptors was demonstrated directly by prelabeling P2Y2-HA receptors with antibody before agonist challenge and then quantifying the movement of receptors from a cell surface to intracellular localization in the presence of agonist. Removal of agonist from the medium resulted in recovery of cell surface immunoreactivity to control levels within approximately 1 hr. Incubation of P2Y2-HA receptor-expressing cells with P2Y2 receptor agonists also resulted in receptor-specific desensitization of nucleotide-promoted inositol phosphate accumulation. This loss of responsiveness occurred more rapidly and to a greater extent than did the agonist-promoted loss of surface receptors. Inhibition of receptor internalization by reduction of temperature to 16 degrees had no effect on the capacity of nucleotides to induce P2Y2 receptor-specific desensitization. These results illustrate that the P2Y2 receptor undergoes agonist-promoted movement to an intracellular compartment. This receptor internalization is not required for agonist-induced desensitization.
将Gq/磷脂酶C偶联的人P2Y2受体在其氨基末端用血凝素A（HA）表位序列进行标记（P2Y2-HA），并在1321N1人星形细胞瘤细胞中稳定表达。表位的存在既未改变受体的药理学选择性，也未改变其信号传导特性。开发了一种酶联免疫吸附测定法，使用抗HA抗体来定量P2Y2-HA受体的细胞表面水平。用P2Y2受体激动剂孵育细胞导致细胞表面免疫反应性呈浓度和时间依赖性降低。开发并应用间接免疫荧光共聚焦显微镜方法来证明激动剂促进的细胞表面免疫反应性降低与细胞内免疫反应性增加平行。通过在激动剂刺激前用抗体预标记P2Y2-HA受体，然后在存在激动剂的情况下定量受体从细胞表面到细胞内定位的移动，直接证明了激动剂诱导的P2Y2受体内化。从培养基中去除激动剂导致细胞表面免疫反应性在约1小时内恢复到对照水平。用P2Y2受体激动剂孵育表达P2Y2-HA受体的细胞也导致核苷酸促进的肌醇磷酸积累的受体特异性脱敏。这种反应性丧失比激动剂促进的表面受体丧失发生得更快且程度更大。将温度降低到16度抑制受体内化对核苷酸诱导P2Y2受体特异性脱敏的能力没有影响。这些结果表明，P2Y2受体经历激动剂促进的向细胞内区室的移动。这种受体内化对于激动剂诱导的脱敏不是必需的。
