Innamorati G, Sadeghi H, Birnbaumer M
Departments of Anesthesiology and Physiology, Molecular Biology Institute, UCLA School of Medicine, Los Angeles, California 90095, USA.
J Biol Chem. 1998 Mar 20;273(12):7155-61. doi: 10.1074/jbc.273.12.7155.
The V1a arginine vasopressin receptor (V1aR) expressed in HEK 293 cells was phosphorylated after binding to arginine vasopressin (AVP). The phosphate was incorporated very rapidly into the protein but remained attached for a very short time despite the continuous presence of hormone. The extent of phosphorylation depended upon the concentration of AVP suggesting the involvement of G-protein-coupled receptor kinases. Protein kinase C (PKC) contributed to V1aR phosphorylation as demonstrated by the fact that inhibition of the kinase decreased the amount of phosphate incorporated into the receptor. However, PKC activity was not responsible for the transient nature of V1aR phosphorylation. The hormone-free receptor could be phosphorylated by phorbol ester-activated PKC. Although the phosphorylation was transient, the phosphate groups incorporated remained on the receptor protein longer than those incorporated after AVP treatment. PKC phosphorylation of unoccupied V1aR was not sufficient to promote sequestration. Vasopressin also promoted sequestration of about 80% of the surface receptor, but measurements of the rate of accumulation of inositol phosphates in the sustained presence of the ligand did not reveal a significant desensitization of coupling to phospholipase C activity. The addition of a V1aR antagonist inhibited the sustained accumulation of inositol phosphates establishing that the sustained stimulation of PLC was mediated by receptors located on the cell surface. The transient character of V1aR phosphorylation seemed intrinsic to the receptor protein rather than a consequence of signaling within the cell, and receptor sequestration appeared to be responsible for the desensitization observed in HEK 293 cells.
在HEK 293细胞中表达的V1a精氨酸加压素受体（V1aR）与精氨酸加压素（AVP）结合后发生磷酸化。磷酸基团能非常迅速地掺入蛋白质中，但尽管激素持续存在，它在蛋白质上附着的时间却非常短。磷酸化程度取决于AVP的浓度，这表明G蛋白偶联受体激酶参与其中。蛋白激酶C（PKC）对V1aR磷酸化有作用，这一事实表明抑制该激酶会减少掺入受体的磷酸基团数量。然而，PKC活性并非V1aR磷酸化具有短暂性的原因。无激素的受体可被佛波酯激活的PKC磷酸化。尽管这种磷酸化是短暂的，但掺入的磷酸基团在受体蛋白上保留的时间比AVP处理后掺入的磷酸基团更长。未被占据的V1aR的PKC磷酸化不足以促进受体隔离。加压素还能促进约80%的表面受体发生隔离，但在配体持续存在的情况下对肌醇磷酸积累速率的测量并未显示与磷脂酶C活性的偶联有明显脱敏现象。添加V1aR拮抗剂可抑制肌醇磷酸的持续积累，这表明对PLC的持续刺激是由位于细胞表面的受体介导的。V1aR磷酸化的短暂特性似乎是受体蛋白固有的，而非细胞内信号传导的结果，并且受体隔离似乎是HEK 293细胞中观察到的脱敏现象的原因。
