Hamelin Emilie, Thériault Caroline, Laroche Geneviève, Parent Jean-Luc
Service de Rhumatologie, Faculté de Médecine and Centre de Recherche Clinique-CHUS, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
J Biol Chem. 2005 Oct 28;280(43):36195-205. doi: 10.1074/jbc.M503438200. Epub 2005 Aug 25.
Intracellular trafficking pathways of cell surface receptors following their internalization are the subject of intense research efforts. However, the mechanisms by which they recycle back to the cell surface are still poorly defined. We have recently demonstrated that the small Rab11 GTPase protein is a determinant factor in controlling the recycling to the cell surface of the beta-isoform of the thromboxane A2 receptor (TPbeta) following its internalization. Here, we demonstrate with co-immunoprecipitation studies in HEK293 cells that there is a Rab11-TPbeta association occurring in the absence of agonist, which is not modulated by stimulation of TPbeta. We show with purified TPbeta intracellular domains fused to GST and HIS-Rab11 proteins that Rab11 interacts directly with the first intracellular loop and the C-tail of TPbeta. Amino acids 335-344 of the TPbeta C-tail were determined to be essential for the interaction of Rab11 with this receptor domain. This identified sequence appears to be important in directing the intracellular trafficking of the receptor from the Rab5-positive intracellular compartment to the perinuclear recycling endosome. Interestingly, our data indicate that TPbeta interacts with the GDP-bound form, and not the GTP-bound form, of Rab11 which is necessary for recycling of the receptor back to the cell surface. To our knowledge, this is the first demonstration of a direct interaction between Rab11 and a transmembrane receptor.
细胞表面受体内化后的细胞内运输途径是大量研究工作的主题。然而,它们循环回到细胞表面的机制仍不清楚。我们最近证明,小GTP酶Rab11蛋白是控制血栓素A2受体(TPβ)β亚型内化后循环回到细胞表面的决定性因素。在此,我们通过在HEK293细胞中的共免疫沉淀研究证明,在没有激动剂的情况下会发生Rab11与TPβ的结合,且这种结合不受TPβ刺激的调节。我们用与GST和HIS-Rab11蛋白融合的纯化TPβ细胞内结构域表明,Rab11直接与TPβ的第一个细胞内环和C末端相互作用。TPβ C末端的氨基酸335-344被确定为Rab11与该受体结构域相互作用所必需。这一确定的序列似乎在指导受体从Rab5阳性细胞内区室向核周回收内体的细胞内运输中起重要作用。有趣的是,我们的数据表明,TPβ与Rab11的GDP结合形式而非GTP结合形式相互作用,这是受体循环回到细胞表面所必需的。据我们所知,这是Rab11与跨膜受体之间直接相互作用的首次证明。