Src regulates constitutive internalization and rapid resensitization of a cholecystokinin 2 receptor splice variant
Src regulates constitutive internalization and rapid resensitization of a cholecystokinin 2 receptor splice variant
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DOI：
10.1074/jbc.m506337200
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发表时间：
2005-09-30
影响因子：
4.8
通讯作者：
Hellmich, MR
中科院分区：
文献类型：
--
作者：
Chao, C;Ives, KL;Hellmich, MR
G蛋白偶联受体的第三胞内环结构域调节其脱敏、内化和再敏化。结肠直肠癌和胰腺癌，但不是非恶性组织，表达胆囊收缩素2受体（CCK 2 R）的剪接变体，称为CCK 214 svR，由于内含子4保留，在其第三胞内环结构域中含有额外的69个氨基酸。这种结构改变与Src激酶的激动剂非依赖性活化有关（Olszewska-Pazdrak，B.，汤森，C。M.，小的，和Hellmich，M. R.（2004）J.Biol.Chem.279，40400 - 40404）。本研究的目的是确定内含子4保留和Src激酶在CCK 214 svR脱敏、内化和再敏化中的作用。胃泌素1 - 17（G17）与CCK 2 R和CCK（2 i4 sv）R结合，诱导细胞内Ca ~（2+）（[Ca ~（2+）]（i））增加。激动剂诱导的[Ca 2 +]（i）增加用于评估受体活性。Src激酶活性被抑制转导细胞与逆转录病毒含有显性负突变Src（A430 V）。使用激光扫描共聚焦显微镜监测增强的绿色荧光蛋白标记的受体的亚细胞位置。两种受体变体以相同的速率脱敏;然而，CCK 214 svR比CCK 2 R快5倍。在没有激动剂的情况下，80%的CCK 214 svR位于细胞内区室中。而CCK 2 R则有80%位于细胞膜上。用反向激动剂（YM 022）或显性负性Src的表达处理阻断CCK 214 svR的组成性内化，导致其在质膜上积累。显性负性Src基因的表达减缓了CCK 214 svR再敏感的速度。Src的抑制并不影响G17诱导的任何受体变体的内化。CCK 214 svR的组成性内化通过产生可快速再循环回到质膜的受体的细胞内池来增加其再敏化速率。
The third intracellular loop domain of G protein-coupled receptors regulates their desensitization, internalization, and resensitization. Colorectal and pancreatic cancers, but not the nonmalignant tissue, express a splice variant of the cholecystokinin 2 receptor (CCK2R) called CCK2i4svR that, because of intron 4 retention, contains an additional 69 amino acids within its third intracellular loop domain. This structural alteration is associated with agonist-independent activation of Src kinase (Olszewska-Pazdrak, B., Townsend, C. M., Jr., and Hellmich, M. R. ( 2004) J. Biol. Chem. 279, 40400 - 40404). The purpose of the study was to determine the roles of intron 4 retention and Src kinase on CCK2i4svR desensitization, internalization, and resensitization. Gastrin1 - 17 (G17) binds to both CCK2R andCCK(2i4sv)R and induces intracellular Ca2+ ([Ca2+](i)) increases. Agonist-induced increases in [Ca2+](i) were used to assess receptor activity. Src kinase activity was inhibited by transducing cells with a retrovirus containing a dominant-negative mutant Src (A430V). The subcellular location of enhanced green fluorescent protein-tagged receptors was monitored using laser scanning confocal microscopy. Both receptor variants desensitized at the same rate; however, CCK2i4svR resensitized five times faster than CCK2R. Without agonist, 80% of CCK2i4svR is located in an intracellular compartment. In contrast, 80% of CCK2R was located on the plasma membrane. Treatment with inverse agonist (YM022) or expression of dominant-negative Src blocked the constitutive internalization of CCK2i4svR, resulting in its accumulation on the plasma membrane. Expression of dominant-negative Src slowed the rate of CCK2i4svR resensitization. Inhibition of Src did not affect G17-induced internalization of either receptor variant. Constitutive internalization of CCK2i4svR increases its rate of resensitization by creating an intracellular pool of receptors that can rapidly recycle back to the plasma membrane.