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Characterization of G-Protein Coupled Receptor Kinase Interaction with the Neurokinin-1 Receptor Using Bioluminescence Resonance Energy Transfer

7TM Pharma A/S, Horsholm, Denmark (R.J., A.H., T.W.S., C.E.E.); Institute of Cell Signaling, Queen's Medical Centre, Nottingham, United Kingdom (N.D.H.); Institute of Anatomy, Histology, and Embryology, Veterinary Faculty, University of Ljubljana, Slovenia (M.V); Laboratory for Molecular Pharmacology, The Panum Institute 18.6, University of Copenhagen, Copenhagen, Denmark (J.L.H., T.W.S.); and Laboratory for Molecular Cardiology, the Danish National Research Foundation Centre for Cardiac Arrhythmia, the Heart Centre, Copenhagen University Hospital, Copenhagen, Denmark (J.L.H.).

To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer² (BRET²) 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 BRET² 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.

Received for publication June 7, 2007.

Accepted for publication November 6, 2007.

Address correspondence to: Christian E. Elling, 7TM Pharma A/S, 2970 Horsholm, Denmark. E-mail: cee{at}7tm.com

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