Dissociation of beta-arrestin from internalized bradykinin B2 receptor is necessary for receptor recycling and resensitization.
Beta-arrestins are multifunctional adaptors that bind agonist-activated G protein-coupled receptors (GPCRs), mediate their desensitization and internalization, and control the rate at which receptors recycle back at the plasma membrane ready for subsequent stimulation. The activation of the bradykinin (BK) type 2 receptor (B2R) results in the rapid desensitization and internalization of the receptor. Little is known, however, about the role of beta-arrestin in regulating the intracellular trafficking and the resensitization of the B2R. Using confocal microscopy, we show that BK stimulation of COS-7 cells expressing B2R induces the colocalization of the agonist-activated receptor with beta-arrestin into endosomes. Fluorescent imaging and ligand binding experiments also reveal that upon agonist removal, beta-arrestin rapidly dissociates from B2R into endosomes, and that receptors return back to the plasma membrane, fully competent for reactivating B2R signaling as measured by NO production upon a second BK challenge. However, when the receptor is mutated in its C-terminal domain to increase its avidity for beta-arrestin, B2R remains associated with beta-arrestin into endosomes, and receptors fail to recycle to the plasma membrane postagonist wash. Similarly, the recycling of receptors is prevented when a beta-arrestin mutant exhibiting increased avidity for agonist-bound GPCRs is expressed with B2R. Stabilizing receptor/beta-arrestin complexes into endosomes results in the dampening of the BK-mediated NO production. These results provide evidence for the involvement of beta-arrestin in the intracellular trafficking of B2R, and highlight the importance of receptor recycling in reestablishing B2R signaling.
Simaan
May
M
Hormones and Cancer Research Unit, Department of Medicine, McGill University, Royal Victoria Hospital, 687 Pine Avenue West, Room H7-67, Montreal, Quebec, Canada.
Bédard-Goulet
Sara
S
Fessart
Delphine
D
Gratton
Jean-Philippe
JP
Laporte
Stéphane A
SA
D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D021381:Protein Transport; D043782:Receptor, Bradykinin B2 / Q000819:agonists / Q000378:metabolism*; D000071557:beta-Arrestins
D019390:Arrestins; D043782:Receptor, Bradykinin B2; D000071557:beta-Arrestins; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III