Shi Mei, Bennett Teresa A, Cimino Daniel F, Maestas Diane C, Foutz Terry D, Gurevich Vsevolod V, Sklar Larry A, Prossnitz Eric R
Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
Biochemistry. 2003 Jun 24;42(24):7283-93. doi: 10.1021/bi0341657.
G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.
G蛋白偶联受体（GPCRs）必须不断竞争与G蛋白、激酶和阻遏蛋白的相互作用。为了更详细地评估这些蛋白质与GPCRs的相互作用，我们在N-甲酰甲硫氨酸肽受体和G(α)(i2)蛋白之间构建了一种融合蛋白。这种嵌合蛋白的功能特性在体内稳定转染的U937细胞中以及在体外使用一种新型的溶解成分重组系统进行了测定。嵌合蛋白表现出与野生型受体难以区分的细胞配体结合亲和力，并且当溶解时以一种复合物形式存在，该复合物包含βγ亚基，蔗糖密度沉降实验证明了这一点。嵌合蛋白能动员细胞内钙并在激动剂作用下正常脱敏。此外，嵌合受体以内化和再循环的速率与野生型FPR相似。共聚焦荧光显微镜显示，与野生型受体不同，用荧光配体鉴定的内化嵌合受体与阻遏蛋白以及G蛋白共定位。可溶性重组实验表明，即使在磷酸化状态下，嵌合受体在没有外源性G蛋白的情况下也以高亲和力配体G蛋白复合物的形式存在。通过添加阻遏蛋白只能部分阻止这种相互作用。此外，我们的结果表明，G蛋白α亚基的GTP结合状态对受体没有可检测到的亲和力。总之，这些结果表明，GPCRs在其未磷酸化和磷酸化状态下与G蛋白和阻遏蛋白之间存在复杂的相互作用，这导致了对GPCR功能的高度调控。
