Appleyard S M, Celver J, Pineda V, Kovoor A, Wayman G A, Chavkin C
Neurobiology Program, University of Washington, Seattle, Washington 98195-7280, USA.
J Biol Chem. 1999 Aug 20;274(34):23802-7. doi: 10.1074/jbc.274.34.23802.
We used the Xenopus oocyte expression system to examine the regulation of rat kappa opioid receptor (rKOR) function by G protein receptor kinases (GRKs). kappa agonists increased the conductance of G protein-activated inwardly rectifying potassium channels in oocytes co-expressing KOR with Kir3.1 and Kir3.4. In the absence of added GRK and beta-arrestin 2, desensitization of the kappa agonist-induced potassium current was modest. Co-expression of either GRK3 or GRK5 along with beta-arrestin 2 significantly increased the rate of desensitization, whereas addition of either beta-arrestin 2, GRK3, or GRK5 alone had no effect on the KOR desensitization rate. The desensitization was homologous as co-expressed delta opioid receptor-evoked responses were not affected by KOR desensitization. The rate of GRK3/beta-arrestin 2-dependent desensitization was reduced by truncation of the C-terminal 26 amino acids, KOR(Q355Delta). In contrast, substitution of Ala for Ser within the third intracellular loop [KOR(S255A,S260A, S262A)] did not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuated desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations did not. These results suggest that co-expression of GRK3 or GRK5 and beta-arrestin 2 produced homologous, agonist-induced desensitization of the kappa opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR.
我们利用非洲爪蟾卵母细胞表达系统来研究G蛋白偶联受体激酶（GRKs）对大鼠κ阿片受体（rKOR）功能的调节作用。κ激动剂可增加在共表达KOR与Kir3.1和Kir3.4的卵母细胞中G蛋白激活的内向整流钾通道的电导。在未添加GRK和β抑制蛋白2的情况下，κ激动剂诱导的钾电流脱敏作用较弱。GRK3或GRK5与β抑制蛋白2共表达可显著提高脱敏速率，而单独添加β抑制蛋白2、GRK3或GRK5对KOR脱敏速率无影响。脱敏是同源性的，因为共表达的δ阿片受体诱发的反应不受KOR脱敏的影响。GRK3/β抑制蛋白2依赖性脱敏速率因C末端26个氨基酸的截短即KOR(Q355Delta)而降低。相反，在第三个细胞内环内将丝氨酸替换为丙氨酸[KOR(S255A,S260A,S262A)]并没有降低脱敏速率。在C末端区域，KOR(S369A)替换显著减弱了脱敏作用，而KOR(T363A)和KOR(S356A,T357A)点突变则没有。这些结果表明，GRK3或GRK5与β抑制蛋白2共表达通过一种需要rKOR丝氨酸369磷酸化的机制产生κ阿片受体同源性、激动剂诱导的脱敏作用。
