Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists

Abstract

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.

Keywords: G protein-coupled receptor; fluorescent sensor; integrator sensor; neuromodulator detection.

Fig. 1.

red-SPOTIT and SPOTcal design and testing. (A) Schematic of the original SPOTIT design (i) and the red-SPOTIT design (ii). In the original design, Nb39 inhibits cpGFP fluorophore maturation. Opioid binding recruits Nb39 to the OR, allowing the cpGFP fluorophore to mature and fluoresce. In the red-SPOTIT design, O-Geco1 replaces cpGFP from the original SPOTIT design and activation follows the same mechanism. (B) HEK293T cell testing of green or red SPOTIT. SPOTIT expressing cells were stimulated with 10 µM of fentanyl or salvinorin A for MOR or KOR, respectively. Twenty-four hours post opioid stimulation, cells were fixed, immunostained, and imaged at pH 11. GFP, cpGFP fluorescence. mApple, cpmApple fluorescence. FLAG, protein expression level. DAPI, nuclear staining. DIC, differential interference contrast. (Scale bar, 20 µm.) (C) Schematic of the opioid-activated calcium sensor, SPOTcal. Opioid binding allows the fluorophore to mature, where calcium binding leads to an increase in fluorescence in live cells. (D) Schematic of SPOTcal testing and plot of the real-time fluorescence increase from calcium stimulation. HEK293T cells expressing the opioid-activated calcium sensor were stimulated with 10 µM fentanyl 24 h post transfection. Twenty-four hours after fentanyl stimulation, cells were then imaged in real-time before and after addition of 5 mM calcium chloride and 2 µM ionomycin. Controls were also performed without opioid or without calcium and ionomycin. Mean values of replicates are indicated by the dot and error bars are the SEM. n = 30.

Fig. 2.

B2AR-SPOTall design and characterizations. (A) Schematic of B2AR-SPOTall. Nb39 inhibits cpGFP fluorophore maturation. Agonist binding recruits Nb80, sterically blocking Nb39’s interaction with cpGFP and allowing the fluorophore to mature. (B) Testing B2AR-SPOTall in HEK293T cells. B2AR-SPOTall transfected HEK293T cells were stimulated with 10 µM isoproterenol. Twenty-four hours after stimulation, cells were fixed, immunostained, and imaged with pH 11 buffer. GFP, cpGFP fluorescence. Anti-GFP, protein expression level. DAPI, nuclear staining. DIC, differential interference contrast. (Scale bar, 20 µm.) Iso, isoproterenol. (C) Quantification of the experiment described in B. The thick horizontal bar is the mean value of three technical replicates. The number above the dots is the S/N; the stars indicate statistical significance. ****P value < 0.0001. n = 3. (D) Agonist stimulation time testing of B2AR-SPOTall in HEK293T cells. Cells were stimulated with 10 µM isoproterenol for different amounts of time, washed, and then incubated for 24 h before imaging. Schematic of experiment is shown above the plot. The thick horizontal bar is the mean value of three technical replicates. The number above the dots is the S/N; the stars indicate statistical significance compared to the “no-drug” condition. 30 s: ****P < 0.0001, 5 min: ***P = 0.0005, 6 h: **P = 0.0042, 24 h: ***P = 0.0003. n = 3. (E) Maturation assay of B2AR-SPOTall in HEK293T cells. Cells were imaged 0.5, 1, 1.5, 2, 3, 4, 6, 8, or 24 h post 5-min 50 µM isoproterenol stimulation to determine the time it would take the fluorophore to mature. Schematic of experiment is shown above the plot. n = 3. Dots on the plot indicate the mean value of three technical replicates. (F) pH titration of B2AR-SPOTall expressing HEK293T cells. Twenty-four hours post 5-min 10 µM isoproterenol stimulation, cells were fixed and imaged with different pH buffers: pH 5.6, 7, 7.7, 8.1, 8.6, 9.1, 10.3, and 11. Dots on the plot indicate the mean value of three technical replicates. n = 3. (G) Screening B2AR-SPOTall expressing HEK293T cells against the catecholamines, dopamine and epinephrine. Cells were imaged 24 h post 5-min stimulation with 10 µM of drug. The thick horizontal bar is the mean value of three technical replicates. The number above the dots is the S/N and the stars indicate significance compared to the no-drug condition. Epinephrine: ****P = <0.0001, Dopamine: **P = 0.0030. n = 3. (H) Screening B2AR-SPOTall expressing HEK293T cells against different types of ligands. Cells were imaged 24 h post 5-min stimulation with 50 µM of drug. n.s., not significant. The thick horizontal bar is the mean value of three technical replicates. The number above the dots is the S/N, and the stars indicate significance compared to the no-drug condition. Isoproterenol: **P = 0.0030, indacaterol: ***P = 0.0008, arformoterol: **P = 0.0017, levalbuterol: ***P = 0.0004, iperoxo: n.s. P = 0.1580, butoxamine: n.s. P = 0.7761. n = 3. For C–H, error bars are the SEM and significance was calculated using an unpaired, two-tailed Student’s t test.

Fig. 3.

Design and testing of DRD1- and CHRM2-SPOTall. (A) Schematic of DRD1- and CHRM2-SPOTall. (B) HEK293T cell testing of DRD1- and CHRM2-SPOTall. HEK293T cells expressing DRD1- and CHRM2-SPOTall were stimulated with 100 µM dopamine and iperoxo, respectively. Cells were fixed, immunostained, and imaged 24 h post stimulation. GFP, cpGFP fluorescence. Anti-GFP, protein expression level. DAPI, nuclear staining. DIC, differential interference contrast. (Scale bar, 20 µm.) (C) Quantification of B. Error bars are the SEM. The thick horizontal bar is the mean value of three technical replicates. The number above the dots is S/N and the stars indicate significance compared to the “−drug” condition. Significance was calculated using an unpaired, two-tailed Student’s t test. DRD1: ***P value = 0.0002, CHRM2: ***P value = 0.0005. n = 3.

Fig. 4.

Mouse testing of M-SPOTIT2 with AAV1/2 viral serotype. (A) Experimental protocol for M-SPOTIT2 mouse testing. (B) Representative images of M-SPOTIT2 mouse testing. About 100 mg/kg of morphine or saline was administered through IP injection 6 d after viral delivery to the preBötC. GFP, cpGFP fluorescence. Anti-GFP, protein expression levels. (Scale bar, 300 µm.) Images are cropped to show the injection site. Uncropped images are found in SI Appendix, Fig. S7. (C) Statistics of experiment described in B, as well as 60 and 30 mg/kg morphine testing. Mean is represented by the horizontal bar. Each dot is one animal. Error bars, SEM. Stars indicate significance after performing an unpaired, two-tailed Student’s t test compared to the Saline condition. 100 mg/kg: *P = 0.0303, 60 mg/kg: *P = 0.0201, 30 mg/kg: n.s.= 0.3483. n = 3 to 5.

Fig. 5.

Mouse testing of B2AR-SPOTall with AAV1/2 viral serotype. (A) Experimental protocol for B2AR-SPOTall mouse testing. (B) Representative images of mouse testing. Here, 1 µL of 10 mM isoproterenol, 10 mM epinephrine, or saline was locally injected to the lateral hypothalamus area 7 d after viral delivery to the same area. GFP, cpGFP fluorescence. Anti-GFP, protein expression levels. (Scale bar, 300 µm.) (C) Statistics of experiment described in A. Mean is represented by the horizontal bar. Each dot is one animal. Error bars, SEM. Stars indicate significance after performing an unpaired, two-tailed Student’s t test. Iso: *P = 0.0233. Epi: **P = 0.0059. n = 4. Iso, isoproterenol. Epi, epinephrine.