title: Characterization of G-protein coupled receptor kinase interaction with the neurokinin-1 receptor using bioluminescence resonance energy transfer.
abstract: To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive mutants. We observed agonist induced interaction of both GRK5 and GRK2 with the activated NK-1 receptor. In saturation experiments, we observed GRK5 to interact with the activated receptor in a monophasic manner while GRK2 interacted in a biphasic manner with the low affinity phase corresponding to receptor affinity for GRK5. Agonist induced GRK5 interaction with the receptor was dependent on intact kinase-activity, whereas the high affinity phase of GRK2 interaction was independent of kinase activity. We were surprised to find that the BRET(2) saturation experiments indicated that before receptor activation, the full-length NK-1 receptor, but not a functional C-terminal tail-truncated receptor, is preassociated with GRK5 in a relatively low-affinity state. We demonstrate that GRK5 can compete for agonist induced GRK2 interaction with the NK-1 receptor, whereas GRK2 does not compete for receptor interaction with GRK5. We suggest that GRK5 is preassociated with the NK-1 receptor and that GRK5, rather than GRK2, is a key player in competitive regulation of GRK subtype specific interaction with the NK-1 receptor.
authors.affiliation: 7TM Pharma A/S, 2970 Horsholm, Denmark.
authors.forename: Rasmus
authors.initials: R
authors.lastname: Jorgensen
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Holliday
authors.forename: Jakob L
authors.initials: JL
authors.lastname: Hansen
authors.forename: Milka
authors.initials: M
authors.lastname: Vrecl
authors.forename: Anders
authors.initials: A
authors.lastname: Heding
authors.forename: Thue W
authors.initials: TW
authors.lastname: Schwartz
authors.forename: Christian E
authors.initials: CE
authors.lastname: Elling
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004735:Energy Transfer / Q000502:physiology*; D054768:G-Protein-Coupled Receptor Kinases / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D018040:Receptors, Neurokinin-1 / Q000032:analysis / Q000378:metabolism*; D048790:Renilla
chemical_list: D008164:Luminescent Proteins; D018040:Receptors, Neurokinin-1; D054768:G-Protein-Coupled Receptor Kinases