Arrestin-independent internalization and recycling of the urotensin receptor contribute to long-lasting urotensin II-mediated vasoconstriction.
Urotensin II (UII), which acts on the G protein-coupled urotensin (UT) receptor, elicits long-lasting vasoconstriction. The role of UT receptor internalization and intracellular trafficking in vasoconstriction has yet not been analyzed. Therefore, UII-mediated contractile responses of aortic ring preparations in wire myography and rat UT (rUT) receptor internalization and intracellular trafficking in binding and imaging analyses were compared. UII elicited a concentration-dependent vasoconstriction of rat aorta (-log EC50, mol/L:9.0+/-0.1). A second application of UII after 30 minutes elicited a reduced contraction (36+/-4% of the initial response), but when applied after 60 minutes elicited a full contraction. In internalization experiments with radioactive labeled VII ((125)I-UII), approximately 70% of rUT receptors expressed on the cell surface of human embryonic kidney 293 cells were sequestered within 30 minutes (half life [t(h)]: 5.6+/-0.2 minutes), but recycled quantitatively within 60 minutes (t(h) 31.9+/-2.6 minutes). UII-bound rUT receptors were sorted to early and recycling endosomes, as evidenced by colocalization of rUT receptors with the early endosomal antigen and the transferrin receptor. Real-time imaging with a newly developed fluorescent UII (Cy3-UII) revealed that rUT receptors recruited arrestin3 green fluorescent protein to the plasma membrane. Arrestin3 was not required for the endocytosis of the rUT receptor, however, as internalization of Cy3-UII was not altered in mouse embryonic fibroblasts lacking endogenous arrestin2/arrestin3 expression. The data demonstrate that the rUT receptor internalizes arrestin independently and recycles quantitatively. The continuous externalization of rUT receptors provides the basis for repetitive and lasting UII-mediated vasoconstriction.
Giebing
Günter
G
Med. Klinik IV-Nephrologie, Charité, Campus Benjamin Franklin, Berlin, Germany.
Tölle
Markus
M
Jürgensen
Jana
J
Eichhorst
Jenny
J
Furkert
Jens
J
Beyermann
Michael
M
Neuschäfer-Rube
Frank
F
Rosenthal
Walter
W
Zidek
Walter
W
van der Giet
Markus
M
Oksche
Alexander
A
D000818:Animals; D019390:Arrestins / Q000502:physiology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D014579:Urotensins / Q000494:pharmacology*; D014661:Vasoconstriction / Q000187:drug effects*
D019390:Arrestins; D043562:Receptors, G-Protein-Coupled; C121697:UTS2R protein, human; D014579:Urotensins; C082333:arrestin3; C029677:urotensin II