QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: mol.107.038877v1 73/2/349    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jorgensen, R. Articles by Elling, C. E. Search for Related Content PubMed PubMed Citation Articles by Jorgensen, R. Articles by Elling, C. E.

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.

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