Receptor determinants involved in regulatory and signalling mechanisms of orexin receptors

Werner Jaeger, Ruth Seeber, Karin Eidne, Kevin Pfleger. Western Australian Institute for Medical Research, B Block, QEII Medical Centre, Hospital Avenue, Nedlands, Western Australia, 6009, Australia

The orexin system is involved in the fundamental orchestration between sleep-wake, metabolism and the promotion of behaviour that stimulates psychochemical reward. This system mediates these activities through the action of endogenous orexin neuropeptides, orexin A and B (OxA and OxB), on two GPCRs, orexin receptors 1 and 2 (OX1R and OX2R). In addition to G protein-coupling, largely via Gq/11, these receptors interact with β-arrestins; a set of ubiquitously expressed multi-adaptor proteins that are critically involved in regulatory and signalling properties of GPCRs.

A significant orexin receptor subtype-specific difference is the temporal stability of receptor-arrestin proximity over sustained periods of stimulation as observed through the use of bioluminescence resonance energy transfer (BRET), a biophysical assay that measures protein proximity in live cells and in real-time. Specifically, OX2R displays more sustained proximity with β-arrestins than OX1R [1]. Similarly, profiles were observed that differentiated the receptor subtypes when ubiquitination of orexin receptor complexes and ERK1/2 phosphorylation were measured [1]. The molecular mechanism of this difference in stability was investigated by evaluating the contribution of the C-terminus of orexin receptors, a key binding region for β-arrestins.

Hypothesized G protein-coupled receptor kinase (GRK) phosphorylation determinants in the C-terminus of OX2R were investigated for their contribution to β-arrestin binding. Using BRET, the data indicated that unlike OX1R [2], disruption of two putative GRK phosphorylation sites at amino acid positions 406-409 (SLTT) and 427-431 (TSIST) of the OX2R C-terminal tail by mutation of S/T residues to alanine (SLTT→ALAA, (A406); TSIST→AAIAA, (A427)) was necessary to destabilize OX2R-β-arrestin interactions. BRET values between OX2R and the OX2R A406-A427 mutant were significantly different from 3-60 min post OxA (0.6 μM) stimulation, measured using two-way ANOVA and Bonferroni multiple comparison post-tests (p<0.05, n = 3). Further mutation of S/T residues in the more proximal putative GRK phosphorylation site (399-403, TSTES→AAAEA (A399)) did not have any further effect on reducing the OX2R-β-arrestin BRET signal, and the mean BRET values of this mutant (OX2R A399-A406-A427) were similarly significantly lower compared to OX2R over the same measurement period (two-way ANOVA; p<0.05, n = 3). Measurement of inositol phosphate (IP) indicated that G protein-coupling of the mutant was not compromised, but instead resulted in a significant increase in potency (pEC50 values were as follows: 8.15±0.09 (OX2R), 8.69±0.10 (OX2R A399-A406-A427 mutant); mean potency values were analyzed using a paired t-test (p<0.05, n = 5)). This increase may be due to the decreased ability of the mutant to recruit β-arrestin and desensitize G protein-mediated signalling pathways involving IP production. This study provides evidence for the molecular mechanism of this interaction that may be responsible for subtype-specific signalling and regulatory function with respect to β-arrestin, a better understanding of which may help us to develop the therapeutic potential of these receptors.

1. Dalrymple MB, Jaeger WC, Eidne KA, Pfleger KD (2011). Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes. J Biol Chem 286(19): 16726-16733.

2. Milasta S, Evans NA, Ormiston L, Wilson S, Lefkowitz RJ, Milligan G (2005). The sustainability of interactions between the orexin-1 receptor and beta-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation. Biochem. J. 387(Pt 3): 573-584.