Time-resolved FRET between GPCR ligands reveals oligomers in native tissues.
G protein-coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these transfected cells. More notably, the comparison between FRET signals measured with sets of fluorescent agonists and antagonists was consistent with an asymmetric relationship of the two protomers in an activated GPCR dimer. Finally, we applied the strategy to native tissues and succeeded in demonstrating the presence of oxytocin receptor dimers and/or oligomers in mammary gland.
Albizu
Laura
L
Institut de Génomique Fonctionnelle, Centre National de la Recherche Scientifique, Montpellier, France.
Cottet
Martin
M
Kralikova
Michaela
M
Stoev
Stoytcho
S
Seyer
René
R
Brabet
Isabelle
I
Roux
Thomas
T
Bazin
Hervé
H
Bourrier
Emmanuel
E
Lamarque
Laurent
L
Breton
Christophe
C
Rives
Marie-Laure
ML
Newman
Amy
A
Javitch
Jonathan
J
Trinquet
Eric
E
Manning
Maurice
M
Pin
Jean-Philippe
JP
Mouillac
Bernard
B
Durroux
Thierry
T
D000465:Algorithms; D000818:Animals; D065092:Antidiuretic Hormone Receptor Antagonists; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D019281:Dimerization; D065127:Dopamine D2 Receptor Antagonists; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D008024:Ligands; D008321:Mammary Glands, Animal / Q000378:metabolism; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000819:agonists / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018045:Receptors, Oxytocin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D017483:Receptors, Vasopressin / Q000819:agonists / Q000378:metabolism
D065092:Antidiuretic Hormone Receptor Antagonists; D065127:Dopamine D2 Receptor Antagonists; D005456:Fluorescent Dyes; D008024:Ligands; D009842:Oligopeptides; D017448:Receptors, Dopamine D2; D043562:Receptors, G-Protein-Coupled; D018045:Receptors, Oxytocin; D017483:Receptors, Vasopressin