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.Seven transmembrane (7TM) G-protein-coupled receptors interact with heterotrimeric G-proteins upon activation. Receptor activation results in initiation of G-protein mediated signal transduction cascades leading to up-or down-regulation of intracellular levels of secondary messengers such as cAMP, inositol trisphosphate, or Ca 2ϩ . Termination of the receptor-mediated signaling is regulated by second messengerϩregulated kinases and Gprotein-coupled receptor kinases (GRKs). GRK-mediated phosphorylation of the receptor promotes receptor recruitment of the intracellular scaffolding proteins ␤arrestins (␤arrs) from the cytosol, thereby excluding the receptor from further G-protein interaction (Luttrell and Lefkowitz, 2002). In addition to uncoupling 7TM receptors from G-proteins, ␤arrs recruit the receptor to clathrin-coated pits (Goodman et al., 1996) and initiates signaling pathways such as mitogenactivated protein kinase cascades (Luttrell and Lefkowitz, 2002). Seven members of the mammalian GRK family have so far been described. These GRKs are classified in three subgroups based on sequence homology and function: the visual GRK1 and -7, nonvisual cytosolic GRK2 and -3, and nonvisual membrane-associated GRKs 4, 5, and 6. With GRK4 as the exception, nonvisual GRKs are rather ubiquitously expressed in mammalian tissues. Although very little is known about GRK subtype-specific phosphorylation patterns of 7TM receptors, recent publications report GRK subtype-specific initiation of cellular events J.L.H.

Beta-Arrestin2 as a Competitor for GRK2 Interaction with the GLP-1 Receptor upon Receptor Activation

“…We used GFP 2 which is a blue shifted variant of GFP developed for the BRET 2 variant of the BRET assay [13] . In previous reports, we have demonstrated the functionality of the tagged constructs and the suitability of the BRET 2 assay for measuring ␤ arr2 and GRK2 interaction with the GLP-1R [12,14,15] . Using a BRET 2 assay, we have measured agonist-induced ␤ arr2 recruitment to the GLP-1R with and without co-expression of GRK2, and found that co-expression of GRK2 enhances ␤ arr2 recruitment to the activated GLP-1R (data not shown).…”

Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

“…In similar BRET 2 studies on the neurokinin NK1 receptor, Jorgensen et al (2008) investigated the interaction of the receptor with GRK2 and GRK5. Both showed a rapid signal that peaked after 10 to 20 s, but the GRK2 signal started from much lower basal values and was therefore of a larger amplitude; the authors interpreted this as a preassociation of receptors and (membrane-localized) GRK5, whereas GRK2 needs to be recruited from the cytosol.…”

Identification of Serine 348 on the Apelin Receptor as a Novel Regulatory Phosphorylation Site in Apelin-13-induced G Protein-independent Biased Signaling

“…In recent years, a number of studies have now utilized BRET to monitor the interactions between GPCRs and GRKs or ␤-arrestins (27)(28)(29)(30). High-throughput screening of GPCR-specific drugs was also performed by a BRET assay (31).…”