Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization, efficient signaling, and interaction with beta-arrestin 2.
This study was focused on the relationship between the plasma-membrane localization of neurokinin-1 receptor (NK1-R) and its endocytic and signaling properties. First, we employed electron paramagnetic resonance (EPR) to study the domain structure of HEK-293 cells and NK1-R microlocalization. EPR spectra and the GHOST condensation routine demonstrated that NK1-R was distributed in a well-ordered domain of HEK-293 cells possibly representing lipid raft/caveolae microdomains, whereas the impairment of caveolae changed the NK1-R plasma-membrane distribution. Internalization and second messenger assays combined with bioluminescence resonance energy transfer were employed subsequently to evaluate the functional importance of the NK1-R microlocalization in lipid raft/caveolae microdomains. The internalization pattern was delineated through the use of dominant-negative mutants (DNM) of caveolin-1 S80E (Cav1 S80E), dynamin-1 K44A (Dyn K44A), and beta-arrestin (beta-arr 319-418) and by means of cell lines that expressed various endogenous levels of beta-arrestins. NK1-R displayed rapid internalization that was substantially reduced by DNMs of dynamin-1 and beta-arrestin and even more profoundly in cells lacking both beta-arrestin1 and beta-arrestin2. These internalization data were highly suggestive of the predominant use of the clathrin-mediated pathway by NK1-R, even though NK1-R tended to reside constitutively in lipid raft/caveolae microdomains. Evidence was also obtained that the proper clustering of the receptor in these microdomains was important for effective agonist-induced NK1-R signaling and for its interaction with beta-arrestin2.
Kubale
Valentina
V
Institute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Gerbiceva 60, SI-1000, Ljubljana, Slovenia.
Abramović
Zrinka
Z
Pogacnik
Azra
A
Heding
Anders
A
Sentjurc
Marjeta
M
Vrecl
Milka
M
D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism; D034284:Dynamin I / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004705:Endocytosis / Q000502:physiology; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D011940:Receptor Aggregation / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; C000605258:Arrb2 protein, mouse; D019390:Arrestins; C495540:CAV1 protein, human; D051242:Caveolin 1; D002966:Clathrin; D011956:Receptors, Cell Surface; D018040:Receptors, Neurokinin-1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D034284:Dynamin I