Innamorati G, Sadeghi H, Birnbaumer M
Department of Anesthesiology, UCLA School of Medicine 90095, USA.
J Recept Signal Transduct Res. 1999 Jan-Jul;19(1-4):315-26. doi: 10.3109/10799899909036654.
The rate of ligand-induced phosphorylation of the V2 and V1a vasopressin receptors was characterized in HEK 293 cells. Both receptors were phosphorylated predominantly by GRKs, and the V1a receptor was also phosphorylated by protein kinase C regardless of the presence or absence of ligand. Phosphorylation of the V1aR catalyzed by GRKs reached maximal values at the shortest measured time: 15 seconds, and decayed rapidly with a t1/2 of 6 min in the continuous presence of AVP. In agreement with the hypothesis that dephosphorylation must precede receptor recycling to the cell surface, the V1aR returned rapidly to the cell surface after removal of the hormone from the medium. Phosphate incorporation into the V2R proceeded at a slower pace, and the internalized phosphorylated receptor failed to recycle to the cell surface and retained its phosphate for a long time in the presence or absence of ligand. A single mutation in the carboxy terminus of the V2R accelerated de-phosphorylation of the protein and conferred recycling properties to the V2R. These experiments provided molecular evidence for the hypothesis that internalization is required for de-phosphorylation and recycling of reactivated G protein coupled receptors to the cell surface.
在HEK 293细胞中对V2和V1a血管加压素受体的配体诱导磷酸化速率进行了表征。两种受体主要被GRKs磷酸化，并且无论有无配体，V1a受体也被蛋白激酶C磷酸化。GRKs催化的V1a受体磷酸化在最短测量时间（15秒）时达到最大值，并在持续存在AVP的情况下以6分钟的半衰期迅速衰减。与去磷酸化必须先于受体再循环到细胞表面的假设一致，从培养基中去除激素后，V1aR迅速回到细胞表面。V2R的磷酸掺入进行得较慢，内化的磷酸化受体未能再循环到细胞表面，并且在有无配体的情况下都长时间保留其磷酸基团。V2R羧基末端的单个突变加速了该蛋白的去磷酸化，并赋予V2R再循环特性。这些实验为内化是再激活的G蛋白偶联受体去磷酸化和再循环到细胞表面所必需的这一假设提供了分子证据。
