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

Abstract

The orexin-1 receptor interacts with beta-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with beta-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of beta-arrestin-2-GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with beta-arrestin-2, studies in wild-type and beta-arrestin-deficient mouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a beta-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca2+ levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with beta-arrestin-2, and indicate an important role of beta-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation.

Figure 1. Orexin-1 receptor co-internalizes with β-arrestin-2

HEK-293T cells were transfected with various forms of the human orexin-1 receptor and β-arrestin-2. Post-transfection (24 h) cells were challenged with orexin-A (0.5 μM) for 30 min. Cells were then fixed and visualized. The distribution of the orexin-1 receptor (i), β-arrestin-2 (ii) and a composite of these images (iii) are shown. (A) Wild-type orexin-1 receptor+β-arrestin-2–GFP, stimulated with TAMRA-orexin-A. (B) N-terminally HA-tagged orexin-1 receptor+β-arrestin-2–GFP, stimulated with orexin-A in the presence of anti-HA antibody. (C) Orexin-1 receptor–eYFP+β-arrestin-2–RFP, stimulated with orexin-A. (D) N-terminally VSV-G-tagged orexin-1 receptor+β-arrestin-2–GFP, stimulated with orexin-A in the presence of CypHer-5-labelled anti-VSV-G antibody.

Figure 2. C-terminal truncation of the orexin-1 receptor prevents sustained interactions with β-arrestin-2 but not agonist-induced internalization

A series of C-terminal truncation mutants of the HA-tagged orexin-1 receptor was generated (A). These were transfected into HEK-293T cells along with β-arrestin-2–GFP. The cells were then stimulated with orexin A (0.5 μM, 30 min) in the presence of anti-HA antibody and visualized following permeabilization and addition of secondary antibody. Representative data are shown for the 394-Stop (B) and 374-Stop (C) mutants. The distribution of the orexin-1 receptor (i), β-arrestin-2 (ii) and a composite of these images (iii) are shown.

Figure 3. A single cluster of hydroxy amino acids is responsible for sustained interactions between the orexin-1 receptor and β-arrestin-2

The cluster of four hydroxy amino acids between residues 418–422 (C1) and a second cluster of three hydroxy amino acids between residues 393–396 (C2) of the orexin-1 receptor were mutated to alanine residues. These two sets of mutations were also combined (C1,C2) (A). These were transfected into HEK-293T cells along with β-arrestin-2–GFP and treated as described in Figure 2. Representative data are shown for the C2 (B), C1 (C) and C1,C2 (D) mutants. The distribution of the orexin-1 receptor (i), β-arrestin-2 (ii) and a composite of these images (iii) are shown.

Figure 4. Double, but not single, point mutations in C1 of the orexin-1 receptor disrupt sustained interactions with β-arrestin-2

The cluster of four hydroxy amino acids between residues 418–422 of the orexin-1 receptor were mutated either singly (A, B) or in pairs (C, D). These were transfected into HEK-293T cells along with β-arrestin-2–GFP and treated as described in Figure 3. Representative data are shown for T418A (B, a), S419A (B, b), T421A (B, c), T422A (B, d), T418A/S419A (D, a), T418A/T421A (D, b), T418A/T422A (D, c), S419A/T421A (D, d), S419A/T422A (D, e) and T421A/T422A (D, f). The distribution of the orexin-1 receptor (i), β-arrestin-2 (ii) and a composite of these images (iii) are shown.

Figure 5. C1 mutant binds β-arrestin-2 less well than the wild-type orexin 1 receptor

VSV-G-tagged forms of the wild-type (WT) and C1 orexin-1 receptor were transiently co-expressed with β-arrestin-2–GFP (β-arr 2 GFP) in HEK-293T cells. Post-transfection (24 h) the cells were stimulated with vehicle or 0.5 μM orexin A for 15 min. Following addition of 2 mM dithiobis(succinimidyl propionate), β-arrestin-2–GFP was immunoprecipitated (IP) using an anti-GFP serum and the samples resolved on SDS/PAGE. β-Arrestin-2–GFP and forms of the receptor bound were monitored by immunoblotting (IB) with the anti-GFP and anti-VSV-G sera respectively. Two further experiments produced similar results.

Figure 6. Disruption of sustained interactions with β-arrestin-2 does not alter the pathways of agonist-induced internalization of the orexin-1 receptor

N-terminally VSV-G-tagged forms of the wild-type (a) or C1 mutant (b) of the orexin-1 receptor were transfected into HEK-293T cells along with β-arrestin-2–GFP. Cells were untreated (A) or treated with sucrose (0.4 M) (B). Subsequent to addition of anti-VSV-G antibody labelled with CypHer-5, orexin A was added (0.5 μM, 30 min) and the cells visualized. The distribution of the orexin-1 receptor (i), β-arrestin-2 (ii) and a composite of these images (iii) are shown.

Figure 7. Internalization of both the wild-type orexin-1 receptor and the C1 mutant is β-arrestin-dependent

(A) Wild-type (a) or β-arrestin-1 and -2 double knock-out (b) MEF cells were transiently transfected with C-terminally eYFP-tagged forms of the wild-type (WT) orexin-1 receptor or the C1 mutant (C1). Post-transfection (24 h) the cells were treated with vehicle (i) or agonist (0.5 μM orexin A, 30 min; ii), fixed and visualized. (B) β-Arrestin-2–RFP was co-transfected with eYFP-tagged forms of the wild-type or the C1 mutant of orexin-1 receptor mutant. The cells were challenged with vehicle (a) or 0.5 μM orexin A for 30 min (b). The distribution of the orexin-1 receptor (i), β-arrestin-2 (ii) and a composite of these images (iii) are shown.

Figure 8. Sustained interactions between β-arrestin-2 and the orexin-1 receptor prolong ERK1/2 activation

HEK-293T cells transiently expressing the wild-type orexin-1 receptor or the C1 mutant for 24 h were serum starved for 2 h and then stimulated with 0.5 μM orexin A for the times indicated. (A) The activation of ERK1 and 2 was assessed by immunoblotting with a anti-phospho-ERK1/2 antibody (P-ERK1/2). Expression levels of ERK1 and ERK2 were monitored using antibodies directed against total population of ERK1 and ERK2. (B) Quantitation of densitometric analysis of four similar experiments. The results are represented as the means±S.E.M. Orexin-1 receptor (■) or the C1 mutant (◆).

Figure 9. Wild-type and C1 mutant forms of the orexin-1 receptor have similar effects on intracellular [Ca²⁺]

(A) N-terminally VSV-G-tagged forms of the wild-type (WT) and C1 forms of the orexin-1 receptor (Orexin1R) were expressed in HEK-293T cells. (B) C-terminally eYFP-tagged forms of the wild-type, C1 and C2 mutants of the orexin-1 receptor were transfected along with Gα₁₁ into Gα_q/Gα₁₁ knock-out MEF cells. The effect of 0.5 μM orexin A on intracellular [Ca²⁺] was then recorded in individual cells. The results are pooled from 6 cells expressing each construct.