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. 2014 Jan;171(2):364-74.
doi: 10.1111/bph.12481.

Molecular determinants of orexin receptor-arrestin-ubiquitin complex formation

Werner C Jaeger  1 , Ruth M Seeber, Karin A Eidne, Kevin D G Pfleger
Affiliations

Molecular determinants of orexin receptor-arrestin-ubiquitin complex formation

Werner C Jaeger et al. Br J Pharmacol. 2014 Jan.

Abstract

Background and purpose: The orexin system regulates a multitude of key physiological processes, particularly involving maintenance of metabolic homeostasis. Consequently, there is considerable potential for pharmaceutical development for the treatment of disorders from narcolepsy to metabolic syndrome. It acts through the hormonal activity of two endogenous peptides, orexin A binding to orexin receptors 1 and 2 (OX₁ and OX₂) with similar affinity, and orexin B binding to OX₂ with higher affinity than OX₁ receptors. We have previously revealed data differentiating orexin receptor subtypes with respect to their relative stability in forming orexin receptor-arrestin-ubiquitin complexes measured by BRET. Recycling and cellular signalling distinctions were also observed. Here, we have investigated, using BRET, the molecular determinants involved in providing OX₂ receptors with greater β-arrestin-ubiquitin complex stability.

Experimental approach: The contribution of the C-terminal tail of the OX receptors was investigated by bulk substitution and site-specific mutagenesis using BRET and inositol phosphate assays.

Key results: Replacement of the OX₁ receptor C-terminus with that of the OX₂ receptor did not result in the expected gain of function, indicating a role for intracellular domain configuration in addition to primary structure. Furthermore, two out of the three putative serine/threonine clusters in the C-terminus were found to be involved in OX₂ receptor-β-arrestin-ubiquitin complex formation.

Conclusions and implications: This study provides fundamental insights into the molecular elements that influence receptor-arrestin-ubiquitin complex formation. Understanding how and why the orexin receptors can be functionally differentiated brings us closer to exploiting these receptors as drug targets.

Keywords: BRET; GPCR; arrestin; hypocretin; orexin; ubiquitin.

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Figures

Figure 1
Figure 1
Diagrammatic representation of the primary amino acid structures of the C-termini of OX1, OX2 and the OX1ctOX2 mutant receptors. Dots above residues indicate identical amino acids in OX1 and OX2 receptors when aligned from the NPIIY motif at the end of transmembrane domain 7. The OX1ctOX2 mutant contains amino acids 1–367 of the OX1 receptor and amino acids 374–444 of the OX2 receptor, as indicated. Underlined, bold residues are putative GRK phosphorylation cluster sites in OX1 and OX2 receptors.
Figure 2
Figure 2
BRET proximity data between Rluc8-Kras and Venus-tagged OX1, OX2, OX1ctOX2 receptors (A), or Venus-Kras and Rluc8-tagged OX1, OX2 and OX1ctOX2 receptors (B). Concentration-response data of inositol phosphate production for OX1, OX2 and OX1ctOX2 receptors. HEK293FT cells were transiently transfected with C-terminally Venus-tagged OX1, OX2 or OX1ctOX2 receptors and treated with orexin A at concentrations shown (C). pEC50 values were as follows: 8.07 ± 0.15 (OX1), 8.15 ± 0.09 (OX2) and 7.81 ± 0.17 (OX1ctOX2). These values were not significantly different from each other (anova; P = 0.28). Significant differences in maximal efficacy were also not observed (anova; P = 0.052). Values for maximal efficacy of OX1 and OX1ctOX2 receptors were 95.4 ± 4.4% and 86.1 ± 3.3% of OX2 receptor respectively. ‘UNT’ refers to untreated cells transfected with each OX receptor construct (C). Data are expressed as mean ± SEM of at least three independent experiments. * P < 0.05, significantly different, as indicated.
Figure 3
Figure 3
eBRET kinetic data for OX1, OX2 and OX1ctOX2 receptors. HEK293FT cells transiently transfected with C-terminally Venus-tagged receptors and Rluc8-tagged β-arrestin1 (A) or β-arrestin2 (B), or C-terminally Rluc8-tagged receptors and Venus-tagged β-arrestin1 (C) or β-arrestin2 (D) were treated with 0.6 μM orexin A. Data are presented as mean ± SEM of three independent experiments.
Figure 4
Figure 4
Diagrammatic representation of OX2 and each of the OX2 C-terminal mutant receptors used in this study in terms of primary amino acid structure. Amino acids 360–444 corresponding to the C-terminal tail region of the OX2 receptor are shown. Residues indicated in bold are in the serine/threonine (S/T) clusters that were assessed as putative GRK phosphorylation sites (Oakley et al., 2001). Underlined bold residues (in red) indicate amino acids within each of the clusters that were mutated to alanine. Additionally, glutamate 402 was mutated to glutamine as indicated (in green).
Figure 5
Figure 5
eBRET data indicating proximity between OX2 or OX2 C-terminal tail mutant receptors with β-arrestin1 or 2. HEK293FT cells were transiently transfected with either C-terminally Venus-tagged (A-D), or Rluc8-tagged (E-H) OX2 or each of the single (A, C, E, G) or double/triple (B, D, F, H) C-terminal OX2 mutant receptors in the presence of either Rluc8-tagged β-arrestin1 (A, B) or β-arrestin2 (C, D), or Venus-tagged β-arrestin1 (E, F) or β-arrestin2 (G, H). The zero time point indicates the point at which 0.6 μM orexin A was added. Data are presented as mean ± SEM of three independent experiments.
Figure 6
Figure 6
eBRET data indicating proximity between Rluc8-Kras and Venus-tagged OX2 wild-type and mutant receptors (A), or Venus-Kras and Rluc8-tagged OX2 wild-type and mutant receptors (B). Inositol phosphate concentration-response data for OX2 wild-type and mutant receptors. Transiently transfected HEK293FT cells with C-terminally Venus-tagged wild-type OX2 or OX2 mutant receptors (Δ406, Δ406-Δ427 or Δ399-Δ406-Δ427) were treated with doses of orexin A as shown (C). pEC50 values were: 8.15 ± 0.09 (OX2); 8.51 ± 0.08 (Δ406); 8.62 ± 0.09 (Δ406-Δ427) and 8.69 ± 0.10 (Δ399-Δ406-Δ427). Values for maximal efficacy as a percentage of OX2 receptors are as follows: 101.2 ± 0.2 (Δ406), 102.1 ± 1.7 (Δ406-Δ427), 101.1 ± 3.3 (Δ399-Δ406-Δ427). ‘UNT’ refers to untreated cells transfected with each OX receptor construct (C). Data are presented as mean ± SEM of at least three independent experiments. * P < 0.05, significantly different from wild-type OX2 receptor.
Figure 7
Figure 7
eBRET dose-response data indicating proximity between β-arrestin2 and OX2, or OX2 mutants Δ406 or Δ406-Δ427, at 20 and 120 min post-agonist stimulation. pEC50 values were as follows: 7.34 ± 0.11 (OX2), 6.91 ± 0.05 (Δ406), 6.64 ± 0.14 (Δ406-Δ427) at 20 min; 7.11 ± 0.10 (OX2), 6.79 ± 0.08 (Δ406), 6.42 ± 0.43 (Δ406-Δ427) at 120 min. Maximal BRET efficacy values are as follows: 0.91 ± 0.07 (OX2), 0.85 ± 0.04 (Δ406), 0.40 ± 0.02 (Δ406-Δ427) at 20 min; 0.85 ± 0.06 (OX2), 0.61 ± 0.01 (Δ406), 0.21 ± 0.03 (Δ406-Δ427) at 120 min. Data are presented as mean ± SEM of three independent experiments. * P < 0.05, significantly different from OX2 receptors.
Figure 8
Figure 8
eBRET data comparing proximity between β-arrestin1 or 2 and OX2, OX2 Δ399 or OX2 Δ399-Δ406-Δ427 receptors, with or without the E402Q mutation. HEK293FT cells were transiently transfected with Venus-tagged OX2 or mutant receptors and either Rluc8-tagged β-arrestin1 (A) or β-arrestin2 (B). The zero time point indicates when 0.6 μM orexin A was added. Data are presented as mean ± SEM of three independent experiments.
Figure 9
Figure 9
eBRET data indicating proximity between ubiquitin and β-arrestin2 in the presence of wild-type OX1, OX2 or OX2 mutant receptors. HEK293FT cells were transiently transfected with N-terminally Venus-tagged ubiquitin, C-terminally Rluc8-tagged β-arrestin2 and non-BRET-tagged OX1, OX2, OX2 Δ406 or OX2 Δ406-Δ427 receptors. The zero time point indicates when 0.6 μM orexin A was added. Data are presented as mean ± SEM of three independent experiments.
Figure 10
Figure 10
Diagram summarizing the apparently critical putative GRK phosphorylation sites in the C-terminal tail of OX1 and OX2 receptors. Underlined residues indicate the clusters that were examined for OX1 receptors previously (Milasta et al., 2005) and for OX2 receptors in this study. The boxed residues indicate clusters that had a notable effect on β-arrestin-mediated recruitment/colocalization with OX1 in the work by Milasta et al. (2005), and with OX2 receptors in the current study. Note that from our data, mutation of the 406 cluster in OX2 receptors had the most influence on the receptor-arrestin-ubiquitin complex over time, but mutation of both 406 and 427 clusters was required to substantially reduce the initial strength of complex formation.
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