Received for publication June 7, 2007.
Revised October 26, 2007.
Accepted for publication November 6, 2007.

Characterization of G-Protein Coupled Receptor Kinase Interaction with the NK-1 Receptor Using BRET

Abstract

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 to 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 while the high affinity phase of GRK2 interaction was independent of kinase activity. Surprisingly, BRET² saturation experiments indicated that before receptor activation, the full length NK-1 receptor, but not a functional C-terminal tail truncated receptor, is pre-associated with GRK5 in a relatively low affinity state. We demonstrate that GRK5 can compete for agonist induced GRK2 interaction with the NK-1 receptor while GRK2 does not compete for receptor interaction with GRK5. We suggest that GRK5 is pre-associated 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.

Key words: Tachykinin, Gs family, Gq/11 family, Desensitization/uncoupling, GRKs, barrestins