Intramolecular fluorescence resonance energy transfer (FRET) sensors of the orexin OX1 and OX2 receptors identify slow kinetics of agonist activation

Abstract

Intramolecular fluorescence resonance energy transfer (FRET) sensors able to detect changes in distance or orientation between the 3rd intracellular loop and C-terminal tail of the human orexin OX(1) and OX(2) G protein-coupled receptors following binding of agonist ligands were produced and expressed stably. These were directed to the plasma membrane and, despite the substantial sequence alterations introduced, in each case were able to elevate [Ca(2+)](i), promote phosphorylation of the ERK1/2 MAP kinases and become internalized effectively upon addition of the native orexin peptides. Detailed characterization of the OX(1) sensor demonstrated that it was activated with rank order of potency orexin A > orexin B > orexin A 16-33, that it bound antagonist ligands with affinity similar to the wild-type receptor, and that mutation of a single residue, D203A, greatly reduced the binding and function of orexin A but not antagonist ligands. Addition of orexin A to individual cells expressing an OX(1) sensor resulted in a time- and concentration-dependent reduction in FRET signal consistent with mass-action and potency/affinity estimates for the peptide. Compared with the response kinetics of a muscarinic M(3) acetylcholine receptor sensor upon addition of agonist, response of the OX(1) and OX(2) sensors to orexin A was slow, consistent with a multistep binding and activation process. Such sensors provide means to assess the kinetics of receptor activation and how this may be altered by mutation and sequence variation of the receptors.

FIGURE 1.

Organization and expression of an OX₁ FRET sensor. A, human OX₁ receptor was modified at the N terminus by addition of a leader sequence from the metabotropic glutamate 5 receptor (mGluR5) followed by the VSV-G peptide epitope sequence while cyan fluorescent protein (CFP) was added in-frame at the C terminus. The FlAsH motif sequence FLNCCPGCCMEP was introduced into the third intracellular loop (predicted region highlighted in yellow in B) that links transmembrane domains V and VI. Energy transfer from CFP to FlAsH and subsequent output is illustrated. B, primary amino acid sequence of the human OX₁ receptor. In the sensor used for the studies reported, the FlAsH motif replaced the sequence underlined in red. This procedure shortened the predicted length of the third intracellular loop from 59 to 36 amino acids. C, following cloning into the inducible Flp-In^TM T-REx^TM locus of Flp-In^TM T-REx^TM 293 cells and induction of expression by addition of doxycycline, imaging of CFP (left hand panel) or the labeled FlAsH motif (right hand panel) demonstrated effective delivery of this sensor to the cell surface.

FIGURE 2.

Response of an OX₁ intramolecular FRET sensor to orexin peptides is both time- and concentration-dependent. Flp-In^TM T-REx^TM 293 cells induced to express mGluR5-VSV-G-OX₁-FlAsH-CFP were employed in FRET imaging studies. A and B, various concentrations of orexin A (OxA)(A) or orexin B (OxB) (B) were added at the indicated point and FRET signal monitored over the ensuing 45 s period. The orexin peptides were removed after application for 15 s (bar). C, change in normalized FRET signal from addition of the peptides to the 60 s time point was measured and plotted with the effect of 10⁻⁶ m orexin A defined as 100% and vehicle as 0%. Data represent means ± S.E., n = 5.

FIGURE 3.

Effects of orexin A at the OX₁ intramolecular FRET sensor are prevented by co-addition of an OX₁ receptor antagonist. Studies were conducted as in Fig. 2. The OX₁ receptor antagonist SB408124 (13) did not modulate the FRET signal of mGluR5-VSV-G-OX₁-FlAsH-CFP but co-addition with orexin A blocked the effect of the agonist (A). Studies are quantified in B. Data represent means ± S.E., n = 5.

FIGURE 4.

Comparisons of the responsiveness of the OX₁ and muscarinic M₃ FRET sensors. Cells induced to express mGluR5-VSV-G-OX₁-FlAsH-CFP were labeled with lumiogreen and loaded with X-rhod-1 AM. Following addition of orexin A (10⁻⁷ m) fluorescence corresponding to CFP (cyan), the FlAsH sensor (yellow) and alterations in [Ca²⁺] (red) were monitored over time (A). In B, the period of time before statistically significant changes in CFP-FlAsH FRET or [Ca²⁺]_i were recorded are displayed. The [Ca²⁺]_i response was significantly slower than the FRET response (***, p < 0.001). C, an extended time course of the [Ca²⁺]_i response (red) is compared with the change in FRET (purple). D, similar studies were performed using cells induced to express a muscarinic M₃ receptor FRET sensor (23) to which the agonist carbachol (10⁻³ m) was added at time = 0. Black, FRET signal, red, calcium response.

FIGURE 5.

Modifications to generate the OX₁ intramolecular FRET sensor do not alter the ligand binding characteristics of the receptor. Saturation [³H]SB674042 binding studies were performed on intact Flp-In^TM T-REx^TM 293 cells induced to express mGluR5-VSV-G-OX₁-FlAsH-CFP (A) and compared with equivalent studies performed on cells induced to express a second OX₁ receptor construct VSV-G SNAP-OX₁ (B), previously characterized in (–24). Total binding (open circles), nonspecific binding (filled squares), and specific binding (filled circles) were assessed. Experiments shown are representative of n = 3.

FIGURE 6.

The OX₁ intramolecular FRET sensor is functional. A, Flp-In^TM T-REx^TM 293 cells harboring mGluR5-VSV-G-OX₁-FlAsH-CFP were either untreated (filled symbols) or induced to express the OX₁ sensor (open symbols) by treatment with doxycycline (100 ng·ml⁻¹, 24 h). Cells were then loaded with the ratiometric Ca²⁺ indicator dye Fura-2 AM, challenged with varying concentrations of orexin A and elevation of [Ca²⁺]_i assessed (combined results of n = 3). B, cells induced to express mGluR5-VSV-G-OX₁-FlAsH-CFP were used in ERK1/2 MAP kinase phosphorylation assays with ligand-induced phosphorylation measured using the SureFire assay kit. Orexin A, filled circles; orexin B, open circles; orexin A 16–33, filled squares (combined results of n = 3). C, although of low potency, orexin A 16–33 was also able to activate the mGluR5-VSV-G-OX₁-FlAsH-CFP sensor. (Ci) the kinetic of response and (Cii) the relative maximal effect of varying concentrations of orexin A 16–33 are shown.

FIGURE 7.

The OX₁ intramolecular FRET sensor becomes internalized in response to orexin A. A, Flp-In^TM T-REx^TM 293 cells induced to express mGluR5-VSV-G-OX₁-FlAsH-CFP were imaged to detect CFP at various times following addition of orexin A (10⁻⁷ m). B, cell surface biotinylation studies were performed on Flp-In^TM T-REx^TM 293 cells harboring mGluR5-VSV-G-OX₁-FlAsH-CFP that were either uninduced (− doxycycline) or induced to express the sensor (+ doxycycline). In the induced cells varying concentrations of orexin A were added for 40 min (left hand panel) or 1 × 10⁻⁶ m orexin A was added for varying times (right hand panel) prior to biotinylation. Cell surface biotinylated proteins were captured, resolved by SDS-PAGE, and mGluR5-VSV-G-OX₁-FlAsH-CFP detected by immunoblotting with anti-VSV-G. Orexin A induced internalization, and therefore reduced the amount of cell surface mGluR5-VSV-G-OX₁-FlAsH-CFP available to be biotinylated, in both a concentration- and time-dependent fashion. Representative examples are shown of n = 3 independent experiments.

FIGURE 8.

An D203A OX₁ sensor loses potency and responsiveness to orexin A but not affinity for antagonists. An D203A mutation was introduced into mGluR5-VSV-G-OX₁-FlAsH-CFP. Flp-In^TM T-REx^TM 293 cells able to express this variant in response to doxycycline were generated. A, saturation [³H]SB674042 binding studies were performed on membranes of these cells as in supplemental Fig. S3. Experiments shown are representative of n = 3, and specific binding of the ligand is shown. B, ability of various concentrations of orexin A to promote ERK1/2 MAP kinase phosphorylation via the D203A OX₁ sensor was quantified using the SureFire assay kit. The pEC₅₀ of orexin A at this variant was >6.0. C, responsiveness of the sensor to a high concentration of orexin A (1 × 10⁻⁶ m) was also assessed.

FIGURE 9.

Production and function of an intramolecular FRET sensor for the orexin OX₂ receptor. A similar approach to that of Fig. 1 and supplemental Fig. S1 was taken to generate potential orexin OX₂ receptor FRET sensors. See “Results” for details. Flp-In^TM T-REx^TM 293 cells harboring HA-OX₂-FlAsH-CFP were induced to express the construct and (A) the ability of orexin A (10⁻⁷ m) to modulate the basal FRET signal measured over time. B, functionality of this construct was assessed in ERK1/2 MAP kinase phosphorylation studies using the SureFire assay kit in response to varying concentration of orexin A (filled symbols) or orexin B (open symbols) (means ± S.E., n = 3).