The intracellular trafficking of the G protein-coupled receptor TPbeta depends on a direct interaction with Rab11.
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.
Hamelin
Emilie
E
Service de Rhumatologie, Faculté de Médecine and Centre de Recherche Clinique-CHUS, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Thériault
Caroline
C
Laroche
Geneviève
G
Parent
Jean-Luc
JL
D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000378:metabolism* / Q000502:physiology; D013997:Time Factors; D014162:Transfection; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D019204:GTP-Binding Proteins; D005982:Glutathione Transferase; D043562:Receptors, G-Protein-Coupled; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins