Recycling of the human prostacyclin receptor is regulated through a direct interaction with Rab11a GTPase.
The human prostacyclin receptor (hIP) undergoes agonist-induced internalization but the mechanisms regulating its intracellular trafficking and/or recycling to the plasma membrane are poorly understood. Herein, we conducted a yeast-two-hybrid screen to identify proteins interacting with the carboxyl-terminal (C)-tail domain of the hIP and discovered a novel interaction with Rab11a. This interaction was confirmed by co-immunoprecipitations in mammalian HEK293 and was augmented by cicaprost stimulation. The hIP co-localized to Rab11-containing recycling endosomes in both HEK293 and endothelial EA.hy 926 cells in a time-dependent manner following cicaprost stimulation. Moreover, over-expression of Rab11a significantly increased recycling of the hIP, while the dominant negative Rab11(S25N) impaired that recycling. Conversely, while the hIP co-localized to Rab4-positive endosomes in response to cicaprost, ectopic expression of Rab4a did not substantially affect overall recycling nor did Rab4a directly interact with the hIP. The specific interaction between the hIP and Rab11a was dependent on a 22 amino acid (Val(299)-Gln(320)) sequence within its C-tail domain and was independent of isoprenylation of the hIP. This study elucidates a critical role for Rab11a in regulating trafficking of the hIP and has identified a novel Rab11 binding domain (RBD) within its C-tail domain that is both necessary and sufficient to mediate interaction with Rab11a.
Wikström
Katarina
K
UCD School of Biomolecular and Biomedical Sciences, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
Reid
Helen M
HM
Hill
Maria
M
English
Karol A
KA
O'Keeffe
Martina B
MB
Kimbembe
Cisca C
CC
Kinsella
B Therese
BT
D011464:Epoprostenol / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D016297:Mutagenesis, Site-Directed; D044006:Receptors, Epoprostenol / Q000819:agonists / Q000378:metabolism*; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000819:agonists / Q000378:metabolism*; D020695:rab4 GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D011464:Epoprostenol; D044006:Receptors, Epoprostenol; D020691:rab GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C043867:cicaprost