Chao Celia, Ives Kirk L, Goluszko Elizabeth, Kolokoltsov Andrey A, Davey Robert A, Townsend Courtney M, Hellmich Mark R
Department of Surgery, University of Texas Medical Branch, Galveston, Texas 77555, USA.
J Biol Chem. 2005 Sep 30;280(39):33368-73. doi: 10.1074/jbc.M506337200. Epub 2005 Aug 3.
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 CCK(2i4sv)R 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 CCK(2i4sv)R desensitization, internalization, and resensitization. Gastrin1-17 (G17) binds to both CCK2R and CCK(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, CCK(2i4sv)R resensitized five times faster than CCK2R. Without agonist, 80% of CCK(2i4sv)R 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 CCK(2i4sv)R, resulting in its accumulation on the plasma membrane. Expression of dominant-negative Src slowed the rate of CCK(2i4sv)R resensitization. Inhibition of Src did not affect G17-induced internalization of either receptor variant. Constitutive internalization of CCK(2i4sv)R increases its rate of resensitization by creating an intracellular pool of receptors that can rapidly recycle back to the plasma membrane.
G蛋白偶联受体的第三个细胞内环结构域调节其脱敏、内化和再敏化。结直肠癌和胰腺癌组织而非非恶性组织表达胆囊收缩素2受体（CCK2R）的一种剪接变体，称为CCK(2i4sv)R，由于内含子4保留，其第三个细胞内环结构域内含有额外的69个氨基酸。这种结构改变与Src激酶的非激动剂依赖性激活有关（奥尔谢夫斯卡-帕兹德拉克，B.，汤森，C.M.，小，和赫尔米奇，M.R.（2004年）《生物化学杂志》279，40400 - 40404）。该研究的目的是确定内含子4保留和Src激酶在CCK(2i4sv)R脱敏、内化和再敏化中的作用。胃泌素1 - 17（G17）与CCK2R和CCK(2i4sv)R都结合并诱导细胞内Ca2 +（[Ca2 +]i）增加。激动剂诱导的[Ca2 +]i增加用于评估受体活性。通过用含有显性负性突变体Src（A430V）的逆转录病毒转导细胞来抑制Src激酶活性。使用激光扫描共聚焦显微镜监测增强型绿色荧光蛋白标记受体的亚细胞定位。两种受体变体以相同的速率脱敏；然而，CCK(2i4sv)R再敏化的速度比CCK2R快五倍。在没有激动剂的情况下，80%的CCK(2i4sv)R位于细胞内区室。相比之下，80%的CCK2R位于质膜上。用反向激动剂（YM022）处理或显性负性Src的表达阻断了CCK(2i4sv)R的组成型内化，导致其在质膜上积累。显性负性Src的表达减慢了CCK(2i4sv)R再敏化的速率。Src的抑制不影响G17诱导的任何一种受体变体的内化。CCK(2i4sv)R的组成型内化通过产生一个可以迅速循环回到质膜的细胞内受体池来增加其再敏化速率。
