Yang W, Schraw W P, Mueller S G, Richmond A
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2175, USA.
Biochemistry. 1997 Dec 9;36(49):15193-200. doi: 10.1021/bi971594u.
CXCR2 is a seven-transmembrane receptor that transduces intracellular signals in response to the chemokines IL-8, MGSA/GRO, and other ELR motif-containing CXC chemokines by coupling to heterotrimeric GTP-binding proteins. In this study, we have mutated two putative G protein-coupling regions of CXCR2 and characterized the effects of these mutations on ligand-activated signal transductions: aspartic acid 89 in the second transmembrane domain and the HRAMR sequence (BBXXB motif, found in the third intracellular loop where B indicates a basic amino acid and X represents any amino acid). The Asp89 was replaced by either asparagine (D89N) or glutamic acid (D89E). For the BBXXB motif, the first two basic amino acids were mutated to two neutral isoleucines (HR-II), or alternatively, two isoleucines were inserted between alanine and methionine (II-insert). When expressed in human embryonic kidney 293 cells, the D89E mutant was localized intracellularly with no detectable cell surface expression. In contrast, D89N, HR-II, and II-insert mutants displayed cell surface expression, with Kd values and expression levels similar to that of the wild-type transfectant. The ability of the mutants to transduce signal was assessed by ligand-stimulated GTPgamma35S binding, mobilization of intracellular free Ca2+, and chemotaxis assays. Both D89N and HR-II mutants signaled similarly to a wild-type receptor in all three assays. However, the II-insert mutant exhibited a loss of ligand-stimulated GTPgamma35S binding, calcium mobilization, and chemotaxis. Unexpectedly, this receptor underwent ligand-induced sequestration comparable to wild-type CXCR2. These data indicate that Asp89 and the basic amino acids in the third intracellular domain do not play essential roles in ligand-induced signal transduction through CXCR2. However, proper secondary structure and orientation of the third intracellular loop of CXCR2 are essential for ligand-mediated signal transduction but not for receptor sequestration.
CXCR2是一种七跨膜受体，通过与异源三聚体GTP结合蛋白偶联，响应趋化因子IL-8、MGSA/GRO和其他含ELR基序的CXC趋化因子转导细胞内信号。在本研究中，我们对CXCR2的两个假定G蛋白偶联区域进行了突变，并表征了这些突变对配体激活信号转导的影响：第二个跨膜结构域中的天冬氨酸89以及HRAMR序列（BBXXB基序，位于第三个细胞内环中，其中B表示碱性氨基酸，X代表任何氨基酸）。天冬氨酸89被天冬酰胺（D89N）或谷氨酸（D89E）取代。对于BBXXB基序，前两个碱性氨基酸被突变为两个中性异亮氨酸（HR-II），或者在丙氨酸和甲硫氨酸之间插入两个异亮氨酸（II-插入）。当在人胚肾293细胞中表达时，D89E突变体定位于细胞内，未检测到细胞表面表达。相反，D89N、HR-II和II-插入突变体显示出细胞表面表达，其解离常数（Kd）值和表达水平与野生型转染子相似。通过配体刺激的GTPγ35S结合、细胞内游离Ca2+的动员和趋化性测定来评估突变体转导信号的能力。在所有这三种测定中，D89N和HR-II突变体的信号传导与野生型受体相似。然而，II-插入突变体表现出配体刺激的GTPγ35S结合、钙动员和趋化性丧失。出乎意料的是，该受体经历了与野生型CXCR2相当的配体诱导的隔离。这些数据表明，天冬氨酸89和第三个细胞内结构域中的碱性氨基酸在通过CXCR2的配体诱导信号转导中不发挥重要作用。然而，CXCR2第三个细胞内环的适当二级结构和取向对于配体介导的信号转导是必不可少的，但对于受体隔离则不是。
