Development of a genetically encoded fluorescent indicator for facilitating deorphanization of GPR52

Reviewer #1 (Public review):

Summary:

GPR52 is an orphan receptor implicated in neuropsychiatric disorders; however, the absence of tools capable of monitoring GPR52 activity in real time has stalled both mechanistic research and ligand discovery. This study addresses this gap by reporting the development of GPR52-1.0, a genetically encoded fluorescent sensor designed to detect activation of GPR52. The sensor was systematically engineered using the established GRAB platform, yielding a construct with micromolar sensitivity and high selectivity in cell culture. The authors largely achieve their stated aims, however the biological relevance of their aims is unclear, as GPR52 is reported to be a constitutively active receptor (PMID: 32076264, PMID: 26384023). GPR52-1.0 is a validated, specific, and sensitive sensor that functions in vitro and ex vivo. The claim that electrically stimulated endogenous GPR52 ligand release occurs in the striatum is supported by the specificity of the GPR52 antagonist block using ex vivo brain slices, however, once again this aim is clouded by evidence that GPR52 is constitutively active. The sensor is presented as a tool for future deorphanization; however, this assumes that the physiological ligand is an agonist, which is unclear based on the evidence that GPR52 is constitutively active. If the authors can explain or adapt their experiments and manuscript in the context of GPR52 constitutive activity, this will be useful work to the community. The impact of this work is likely to be moderate to high within the specialized communities studying orphan GPCRs, neuronal signaling, and neuropsychiatric disease. The GRAB sensor strategy has already generated widely adopted tools for other receptors, and a validated GPR52 sensor would fill a genuine gap. The GRAB technology makes GPR52-1.0 directly applicable to in vivo studies. It is likely that GPR52-1.0 could be replicated for other orphan receptors to facilitate their deorphanization.

Strengths:

(1) Systematic and rigorous sensor optimization and characterization by screening ~800 variants with iterative linker and cpEGFP mutation step. The resulting EC50 values are characterized in HEK293T and cultured neurons.

(2) Testing GPR52-1.0 against a broad panel of neurotransmitters with no detectable off-target activation strengthens confidence in sensor specificity.

(3) The use of a selective antagonist to confirm specificity, both in cell lines and in brain slices, strengthens the conclusions significantly.

(4) Electrically stimulated GPR52-1.0 fluorescence changes in ex vivo striatal slices are blocked by a GPR52 antagonist. This is the most biologically significant result in the manuscript, as GPR52-related diseases can involve the striatum.

Weaknesses:

(1) The work, both experimentally and in its presentation, is not put into the context of what is known about GPR52 pharmacology and signaling. It is reported by multiple groups that GPR52 has high constitutive activity and does not require a ligand for high levels of signaling (PMID: 32076264, PMID: 26384023). The authors should clarify whether GPR52-1.0 senses constitutive activation and whether baseline fluorescence is stable over the timescale of their experiments. The cell and mouse work needs to be reframed and conducted in the context of the high basal activity of the receptor, or the authors need to explain the differences between their study and other studies.

(2) The electrical stimulation used in brain slice experiments is non-specific. This could be activating many cell types and neurotransmitter systems simultaneously. The pharmacological block by the GPR52 antagonist is reassuring, but the identity of the molecules driving the signal remains unknown. It could be that GPR52 is constitutively active, and that the electrical stimulation drives higher expression of GPR52 and thus constitutive signaling. This constitutive signaling can then be inhibited by the GPR52 antagonist. In this scenario, there would be no endogenous GPR52 agonist invoked by electrical stimulation.

(3) The ex vivo brain slice data rely on n=9 slices without reporting the number of animals that the slices come from. Given the importance of this result, more biological replicates and clear reporting of animal numbers would strengthen confidence.

(4) The manuscript does not benchmark GPR52-1.0 against existing approaches (e.g., HTRF, BRET, or calcium mobilization assays) to contextualize its advantages in a drug-discovery or screening workflow.

(5) The paper's title references deorphanization, but the authors have made no attempts toward this deorphanization. No candidate ligand molecules are identified or tested.

Reviewer #2 (Public review):

Summary:

This study describes the development of GPR52-1.0, a novel genetically encoded fluorescent sensor for the orphan GPCR, GPR52. The authors also utilized this sensor in vivo in brain slices and discovered that striatal neuron excititation may activate GPR52.

Strengths:

(1) The design and validation of the sensor are elegant, thorough, and rigorous. The authors conducted a systematic and impressive optimization screen of numerous variants to arrive at the top-performing GPR52-1.0 sensor. The subsequent characterization is thorough, showing excellent membrane trafficking, appropriate pharmacological profiles (EC50, IC50) by the GPR52 chemical agonist/antagonist, rapid kinetics, and high specificity against a panel of common neurotransmitters. The functional characterization was also performed in multiple experimental systems.

(2) The most exciting result is the observation that electrical stimulation may activate GPR52 in the striatum, an area where GPR52 is natively expressed. The blockade by a specific GPR52 antagonist confirms its specificity and provides the first direct evidence for activity-dependent, native GPR52 ligand in striata. This finding alone is a significant step forward and strongly justifies the sensor's development.

(3) The manuscript is well-written and logically structured. The figures are clear and effectively illustrate the key data, from the initial screening process to the final ex vivo validation. The authors did not overstate their discoveries.

Weaknesses:

(1) The sensor specificity is largely based on a single agonist/antagonist, and it might be desired for future studies to confirm this by additional agonists/antagonists or by point mutagenesis that is known to influence GPR52 activation (for example, the ones reported in (PMID: 40087539).

(2) The discovery of the existence of activity-dependent, native GPR52 ligand(s) in striata is extremely exciting. This might be further strengthened by inhibiting synaptic transmitter release with TTX, calcium channel blockers, or SNARE complex disruptors, etc.