Optical tools for visualizing and controlling human GLP-1 receptor activation with high spatiotemporal resolution

This paper presents two new tools for investigating GLP-1 signaling. The genetically encoded sensor GLPLight1 follows the plan for other GPCR-based fluorescent sensors, inserting a circularly permuted GFP into an intracellular loop of the GPCR. The light-uncaged agonist peptide, photo-GLP1, has no detectable agonist activity (as judged by the GLPLight1 sensor) until it is activated by light. However, based on the current characterization, it is unclear how useful either of these tools will be for investigating native GLP-1 signaling.

The GLPLight1 sensor has a strong fluorescent response to GLP-1 with an EC50 of ~10 nM, and its specificity is high, as shown by lack of response to ligands of related class B GPCRs. However, the native GLP1R enables biological responses to concentrations that are ~1000-fold lower than this (as shown, for instance, in a supplemental figure of this paper). This makes it difficult to see how the sensor will be useful for in vivo detection of GLP-1 release, as claimed; although there may be biological situations where the concentration is adequate to stimulate the sensor, this is not established. Data using a GLP-1 secreting cell line suggest that the sensor has bound some of the released GLP-1, but it is difficult to have confidence without seeing an actual fluorescence response to stimulated release.

Alternatively, the sensor might be used for drug screening, but it is unclear that this would be an improvement over existing high-throughput methods using the cAMP response to GLP1R activation (since those are much more sensitive and also allow detection of signaling through different downstream pathways).

The utility of the caged agonist PhotoGLP1 is similarly unclear. The data demonstrate a substantial antagonism of GLP-1 binding by the still-caged compound, and it is therefore unclear whether the kinetics of the response to PhotoGLP1 itself would mimic the normal activation by GLP-1 in the absence of the caged compound. A further concern is that the light-dependence of the agonist effect of PhotoGLP1 was evaluated only with the GLPLight1 sensor and not with GLP1R signaling itself, which is 1000x more sensitive and which would be the presumed target of the tool. In addition, PhotoGLP1 is based upon native GLP-1, which is rapidly truncated and inactivated by the peptidase DPPIV, expressed in most cell types, and expressed at very high levels in the plasma. The utility of PhotoGLP1 is therefore limited to acute (minutes) in vitro experiments.

The following is the authors' response to the original reviews.

We would like to thank all Reviewers for their careful evaluation of our work. Below please find our responses and comments.

Reviewer #1 (Recommendations For The Authors):

1. The detection of cell-released GLP-1 is addressed in an indirect, averaged way in Fig. 2 - Supplement 1. This question seems like a good opportunity for an antagonist experiment (Exendin-9), which presumably would require much lower concentrations than those used to antagonize a saturating dose of GLP-1. It would also be much more convincing if GLPLight1 could be used to detect stimulated release of GLP-1 from the GLUTag cells.

We tried multiple times to acutely stimulate GLUTag cells using Forskolin and IBMX, but unfortunately we did not observe any robust fluorescence increase of GLPLight1. The only observation that was consistent was the higher baseline fluorescence of GLPLight1, and the reduced maximal response to saturating GLP-1 when GLPLight1 expressing HEK cells were cultured overnight with GLUTag cells. We considered this assay to be at best qualitative and — despite the aforementioned attempts — could not determine quantitative values.

2. The excitation-ratiometric response of the sensor, shown in Fig. 1D, is usually accompanied by strong pH-dependence of sensor function. It would be valuable to characterize this pH-dependence, using permeabilized cells in which the pH is changed; the ability of small (0.2-0.5 unit) pH changes to produce changes in fluorescence, as well as to affect the dynamic range of the sensor, should be characterized. This will prevent the misidentification of agents that affect cellular pH as having (for instance) an inhibitory effect on the binding of GLP-1 to GLPLight.

The pH sensitivity of cpGFP-based sensors is a valid concern. However, considering that the cpGFP module from GLPLight1 is intracellular (and thus largely protected from potential extracellular pH changes) we assume that GLPLight1 signal should be robust in most in-vivo or cell-based assays. In fact we have previously characterized this for a similarly-built neuropeptide sensor (PMID: 35145320) and believe that this will be the case also for GLPLight1.

3. The reported Kd for Exendin-9 is in the low nM range. Please explain the partial response at 1000x the concentration (including a discussion of the Kd of GLP-1 itself, as well as its off kinetics, and a comparison of this assay to the assays used previously).

The partial response is due to the presence of 1 uM GLP-1 in the imaging buffer, which is in constant competition with Exendin-9 for the binding to GLPLight1. Because GLP-1 has similar affinity as Exendin9 (see for example PMIDs: 34351033 and 21210113) and both are present at saturating concentration, we did expect to observe a partial response from GLPLight1. In this study, we did not exactly determine the on and off kinetics of both GLP-1 and Exendin9 on the GLPLight1 sensor due to technical challenges: to perform these experiments, we would need to set up a perfusion system where we could remove the unbound ligand and either wash off the bound ligand with buffer or compete it out with an antagonist. Unfortunately, we currently do not have access to such a set up.

4. Are the turn-on kinetics in Fig. 2C limited by drug application or by association? Are the on-rates much slower for the lower concentrations used for Fig. 2C? This is important for knowing how fast responses are likely to be at the lower concentrations likely to be achieved by endogenous release.

If we consider Fig 2B and 2C, we assumed the on-kinetics to be mostly driven by association since the ligand is expected to be homogeneously distributed.

The on-rate kinetics are indeed slower when lower concentrations of GLP-1 are used as shown in (Figure 2b) where we observe a TauOn of 4.7s with 10 uM GLP-1 and much slower kinetics when GLP-1 is applied a 1 uM for example (Figure 3d). As a result, we chose to incubate the ligand with GLPLight1 expressing cells for at least 30 minutes before the measurement of the dose-response to be close to equilibrium.

5. The parameters for the fitted dose-response curves in Fig 2C should be listed. The ~4x discrepancy between the dose-response in HEK-293 cells and neurons should be discussed. Are there known auxiliary subunits, dimerization, or lipid dependence that might account for this? It seems important to understand this if the sensors are to be used in an assay that may compare different systems.

We added the EC50 values to Fig 2C as requested. We did not consider a 4x discrepancy to be significant, because the measurement error in the EC50 region is relatively high and this difference seemed to be within the error range. In fact, the 95% confidence interval ranges are 7.8 to 11.1 nM in Neurons and 23.8 to 32.1 nM for HEK cells, if we consider the upper and lower boundaries of each, the difference drops to around 1-fold. We also performed a statistical test to compare the two fits (Extra sum of squares F-test) that confirmed the two fits were not significantly different (P value = 0.3736). Of course, the interaction partners and membrane composition are different in HEK cells and neurons and probably have an influence on the EC50 of GLPLight1, but their exact influence is unclear.

6. It seems surprising that removal of the endogenous N-terminal secretory sequence is actually helpful for membrane expression. Do the authors have any suggested explanation for this?

GLPLight1 contains an N-terminal hemagglutinin (HA) secretory motif. The hmGLP1R sequence that we chose also contained an endogenous secretory sequence that most likely interfered with the membrane transport mechanism and resulted in a lower sensor expression with both secretory sequences. We thus decided to keep the HA instead of endogenous to remain consistent with other sensors created in-house.

7. In Fig. 1, supplement 3, are the transient responses real? Do they occur with the control construct?

While we have not measured the G-protein recruitment on GLPLight-ctr, we have often observed this phenomenon for various receptors and ligands. The transient responses are thus most likely an artifact after manual addition of the ligand possibly due to:

• Temperature difference

• Exposure of the plate to ambient light before resuming measurement (phosphorescence)

• Re-suspension of the cells affecting the proximity to the detector

• Other unknown variables

If these responses were real, we would also expect them to be more sustained over time.

8. Please include a sentence or two explaining the luminescence complementation assay, and a reference.

We updated the results section of the manuscript with a section describing the luminescence complementation assay along with a reference:

“Next, we compared the coupling of GLPLight1 and its parent receptor (WT GLP1R) to downstream signaling. We first measured the agonist-induced membrane recruitment of cytosolic mini-G proteins and β-arrestin-2 using a split nanoluciferase complementation assay (Dixon et al., 2016). In this assay both the sensor/receptor and the mini-G proteins contains part of a functional luciferase (smBit on the sensor/receptor and LgBit for Mini-G proteins) that becomes active only when these two partners are in close proximity (Wan et al., 2018).”

Bravo to the authors for already making the sensor plasmids available at addgene.com. It would be helpful to include the plasmid IDs and/or a URL in the manuscript.

We would like to thank Reviewer #1 for noticing this. We have updated the data availability section of the manuscript and added the AddGene plasmid numbers of the constructs generated in this study.

Reviewer #2 (Recommendations For The Authors):

1. There are some parts of the introduction that need clarification. For example, GLP1 is quoted as an anorexigenic peptide, however, that is probably only true for centrally- derived GLP1. There is no evidence that enteroendocrine-derived GLP1 (the major pool) is anorexigenic- it is likely to be substantially degraded by DPPIV before reaching the brain. In any case, the discovery of GLP1 was always one of glucose-dependent insulin secretion, with the brain system being described decades later. Overall, the intro needs to be slightly reframed. While the tools presented here are more useful for assessment of central GLP1-releasing circuitry, they are ultimately based upon GLP1R signaling that is much better validated in the periphery.

We have slightly reframed the introduction accordingly.

2. "The human GLP1R (hmGLP1R) is a prime target for drug screening and drug development efforts, since GLP-1 receptor agonists (GLP1RAs) are among the most effective and widely-used weight-loss drugs available to date (Shah and Vella, 2014)." GLP1R was for two decades the breakthrough drug for treatment of type 2 diabetes mellitus and correction of glucose tolerance as assessed through HbA1c. It is only through reporting on millions of patients receiving GLP1RA that the weight loss effects were noted, leading to Phase1-3 trials and eventual approval for obesity indication. Again, some slight reframing of the introduction is required here.

Also for this point, we have slightly reframed the introduction accordingly.

3. GLP1 was applied at a maximal dose of 10 uM, which is 10-fold higher than maximal. Can the authors confirm absence of cytotoxic effects of exposing to peptide at such concentration? Ex4 (9-39) at such concentrations is usually cytotoxic at least in primary tissue.

We did not observe any obvious cytotoxic effect of GLP-1 at this concentration in HEK293T cells or Neurons.

4. "As expected, GLPLight1 responded to both GLP1RAs with almost maximal activation, on par with GLP1 (Figure 2a)." Such a claim is difficult to interpret without concentration-response curves, since the maximal concentration of liraglutide and semaglutide might not have been achieved in these experiments.

We agree with this statement is difficult to interpret without further clarification. We know from the literature that GLP-1, liraglutide and semaglutide all have very high affinity to the hmGLP1R (PMID: 31031702). We also proved that GLPLight signal saturates at concentrations above 1 uM of GLP-1 (figure 2C), we thus applied a 10x excess of all ligands and considered this signal as maximal.

5. "These results indicate that GLPLight1 can serve as a direct readout of pharmacological drug action on the hmGLP1R with higher temporal resolution than previously available approaches, such as downstream signaling assays (Zhang et al., 2020)." Many investigators use cAMP imaging to investigate GLP1R signaling, which is arguably of similar spatiotemporal resolution, also with the advantage of FRET quantification in some cases (e.g. EpacVV). Direct GLP1R signaling can also be inferred using cell lines heterologously-expressing GLP1R. Thus, the advantage of the current probes is that they can be used to readout direct GLP1R activation in native cells/tissues where promiscuous class B binding might limit signaling measures or where endogenous GLP1 release needs to be investigated.

We have edited the manuscript text accordingly.

6. "State-of-the-art techniques for detecting endogenous GLP-1 or glucagon release in vitro from cultured cells or tissues consist of costly and time-consuming antibody- based assays (Kuhre et al., 2016) or analytical chemistry procedures (Amao et al., 2015)." Agreed, but non-specificity/cross-reactivity of such assays is more prohibitive/problematic (e.g. against glicentin).

We have edited the introduction accordingly.

7. The studies using co-culture of GLUTag and GLP1Light1-HEK293 cells, whilst interesting, are not entirely convincing in their current form. Firstly, co-culture could influence GLP1Light expression levels (can the authors label FLAG?). Secondly, specificity of the response is not tested e.g. by adding Ex4 (9-39). Thirdly, titration with GLUTag conditioned media is not performed.

We partially addressed this issue in the answer to comment #1 from Reviewer #1. We previously performed a FLAG staining of GLPLight1 in the presence or absence of GLUTag cells and we did not notice any obvious difference. This goes in line with the fact that GLPLight1 is signaling inert, and the presence of GLP1 should not interfere with the surface expression of the sensor. We also checked that HEK293T cells did not express high levels of GLP1R according to the BioGPSCell line Gene Expression profile.

We also tried to add GLUTag media after stimulation in bolus to GLPLight1 expressing cells and observed no response. This indicated that the “sniffer” cells must be present in close proximity to GLUTag cells for an extended period of time to observe any substantial difference in response, justifying our choice of experimental setup.

8. "Given that our photocage was placed at the very N-terminus of photo-GLP1, our results show that this caging approach prevents the peptide's ability to activate GLP1R but, at the same time, preserves its ability to interact with the ECD." An alternative hypothesis is that PhotoGLP1 does activate GLP1R, but this is undetectable with the sensitivity of GLP1Light. PhotoGLP1 cAMP concentration-response assays are needed (uncaged versus cage) to properly characterize and validate the compound (as would be standard for any newly-described GLP1R peptide ligand).

While we agree that there is a chance that Photo-GLP1 could activate GLP1R at high concentrations, we think that the characterization of Photo-GLP1 has to be determined by the end user directly with the technique of choice (GLPLight1 in our case) in order to get a reliable comparison of potency and efficacy. We modified the text accordingly to more accurately reflect the direct conclusions from our data, as follows:

“our results show that this caging approach prevents the peptide's ability to activate GLPLight1”.

9. "Surprisingly, GLPLight1 shows a fluorescent response in all three uncaged areas, while its fluorescence remained unaltered throughout the rest of the FOV, indicating high spatial localization of the response to GLP-1 (Figure 3f)." Why is this surprising?

We agree that this result is, indeed, not surprising and would like to thank Reviewer #2 for spotting this mistake, which has now been corrected in the manuscript.

10. The localized PhotoGLP1 experiments are interesting and show the utility of the ligand. There is however activation outside of the region of uncaging, which would argue against a pre-bound ECD mode of action. Possibly some PhotoGLP1 is pre- bound to the ECD, and some is freely diffusing? Alternatively, the scan area might be below the diffraction limit/accuracy of the microscope?

We would like to thank Reviewer #2 for this comment and agree with their observation. There could be some free Photo-GLP1 that gets photo-activated and binds regions around the uncaging area (similar to what has been observed for Photo-OXB:,PMID: 36481097). The activation around the uncaging area could also be due to lateral diffusion of the activated receptor on the membrane. There is also most likely some light diffraction at the uncaging area that could account for this phenomenon. To increase the spatial resolution, future studies could involve uncaging during sensor imaging via two-photon microscopy.

11. What was the rationale for caging native GLP1, which is then susceptible to DPPIV-mediated degradation? Would the N-terminal cage and first 2 amino acids also not be cleaved by DPPIV, thus rendering the tool of limited in vivo application? Conversely, PhotoGLP1 provides a template for similar light-activated (stabilized) GLP1R agonists such as Ex4 or liraglutide.

Thank you for making us aware of this (in vivo) limitation. We designed photoGLP1 as a tool for neurobiological experiments in the brain, where DPPIV expression would be low compared to peripheral organs (https://www.proteinatlas.org/ENSG00000197635-DPP4/tissue). We also envisage that the presence of the photocage would be enough to hinder the binding to DPP4 that cuts the first 2 AA. This hypothesis, however, was never tested experimentally, and we, therefore, acknowledge the limitation in the manuscript. We would furthermore like to thank the reviewers for his comment on additional photo-caged GLP1 agonists, which could be developed future studies.

12. It wasn't clear how GLP1Light could be used as a HTS screen for drug discovery? Surely, conventional systems (e.g. GLP1R + BAR/Ca2+/cAMP reporting) allow signal bias, an important component of GLP1RA action, to be assessed. Or could GLP1Light1 be used as a pre-screen to exclude any ligands that do not orthosterically bind GLP1R?

We would like to thank Reviewer #2 for this comment and would like to offer some clarification. We indeed thought that GLPLight1 could be used as a first line of screening to exclude ligands that do not bind in the orthosteric pocket. It is also a rather flexible method as the fluorescence increase of those sensors can be monitored using various techniques/devices that are available in most labs (e.g. microscopy, plate reader, flow cytometry).

13. Limitations of GLP1Light1 and PhotoGLP1 are not acknowledged in the discussion.

We would like to thank Reviewer #2 for pointing out the lack of description of the limitations of these tools, which have now been added to the Discussion.

14. Full characterization of PhotoGLP1 is missing, to include UV/Vis, Tr and HRMS.

PhotoGLP1 was fully characterized by UV/Vis and HRMS, and all experimental and analytical data was uploaded as supplementary data when the manuscript was initially submitted for publication in eLife.

Reviewer #3 (Recommendations For The Authors):

1. The ~1000 fold lower EC50 for GLP1 of GLPLight1 compared with native GLP1R needs to be openly acknowledged as a major limitation of the sensor, as this will substantially reduce the types of experiment for which it will be useful. Because it needs 1000 times higher GLP1 levels than wild type GLP1R to be activated, it is unlikely, for example, to be useful for monitoring the dynamics of activation of native GLP1R in vivo. The claim that the sensor could be used for in vivo imaging for fibre photometry is therefore an exaggeration.

We would like to first thank Reviewer #3 for this comment and to further provide some clarification. We recognized that the data presented in this manuscript might have been confusing when comparing the affinity of GLP1R (using cAMP) and GLPLight1 (using the fluorescence increase because there is no coupling to cAMP). We believe that the low EC50 measured in the cAMP assay cannot accurately be compared to GLPLight1 response because it is an enzymatically amplified process. In order to support this claim, we included another set of experiments where we titrated agonist- induced recruitment of miniGs protein to the GLP1R receptor and found an EC50 of 3.8 nM for native GLP-1 using this assay (added as panel l in Figure1 Supplement 3). We thus confirmed that the nature of the assay itself has a drastic influence on the EC50 measured and it is not unusual to observe 100x fold difference of EC50 for the same receptor-ligand pair.

We believe that the miniGs protein recruitment is a better comparison to GLPLight1 because it is not enzymatically amplified. This assay reveals that GLPLight1 has around 8-fold lower affinity to GLP1 compared to its parent receptor, which is in line with the EC50 loss observed previously for other GPCR-based sensors of this class. We are thus confident that GLPLight1 has to potential to be used in vivo under specific circumstances, specifically in brain tissue. We elaborated on this point in the Discussion part of the manuscript.

2. Fig2 suppl 1 is described as demonstrating a reduced response of GLPLight1 to GLP-1 when HEK cells with were cultured with GLUTag cells. However, it is speculation to conclude that this is because GLP1Light1 was partially pre-activated by endogenous GLP-1, without demonstrating the response of GLPLight1 before and after GLUTag cell stimulation. Unless additional data are generated, the presented data do not convincingly demonstrate that GLP1Light1 can detect GLP1 released from GLUTag cells.

We would like to thank Reviewer #3 for this comment which has been addressed already in the replies to Comment#1 from Reviewer #1 and Reviewer #2.

3. The authors should openly acknowledge that photo-uncaging the GLP1 probe might not be very helpful for monitoring the temporal dynamics of the GLP1-GLP1R interaction, because unless all the photocaged glp1 is released by the light stimulus, the activation of photo-released GLP1 will be slowed by the remaining caged GLP1, and the dynamics will be slower than for native GLP1. This makes it unsuitable for many temporal questions, although it might be useful to deliver GLP1 in a spatial restricted manner.

We do agree that the biggest advantage of Photo-GLP1 is its ability to be activated in a very localized manner. We also agree that the presence of caged Photo-GLP1 will influence the binding of the uncaged GLP-1. Nevertheless, there is still an advantage of using Photo-GLP1 in some assays such as pharmacological activation on brain slices. In fact, we have shown for our Photo-OXB molecule that the perfusion of OXB was much slower at eliciting neuronal depolarization compared to uncaging of Photo- OXB (see PMID: 36481097). We think that this was mainly due to the slow diffusion kinetics of the peptide into the brain tissue. We also think that uncaging can provide a more controlled activation with varying laser power and uncaging duration.

4. To claim (as currently in the discussion) that GLPLight1 has potential to be used for investigating the dynamics of endogenous GLP1, the authors would need to compare the dynamics of the GLP1Light sensor with wild type GLP1R. We do not know that its activation dynamics will reproduce native glp1r.

We would like to thank Reviewer #3 for this comment and would like to offer some clarification. Since GLPLight1 does not couple to intracellular signaling, it was impossible to compare its activation kinetics to GLP1R WT using the same assay. However, we can offer a relative comparison since we know that GLPLight1 takes around 50 seconds to be activated using 1 µM GLP-1 (figure 2B) and that it takes a similar time for GLP1R to be activated in the miniG protein recruitment assay (Fig 1 Supplement 3) using 100 nM GLP-1. Considering that GLPLight1 has a lower affinity than the GLP1R (8-10x lower), we think that the activation kinetics of both the sensor and GLP1R are comparable.

Additional comments:

1. In fig 2A,B, it is not clear whether the trace shows a partial reversal of GLP1- triggered activation by Ex9, or Ex9-independent receptor desensitization. A control trace is required to show the kinetics of GLP1-triggered activation without the addition of Ex9.

We would like to thank Reviewer #3 for this comment. We can exclude the possibility of Ex9-independent desensitization because GLPLight1 has been shown to be signaling inert to all G-proteins, Beta arrestin-2 and cAMP. Moreover, we have observed that the fluorescence signal was stable for more than 30 minutes for the GLP-1 titrations, even at high concentrations of ligand.

2. It would be helpful if the pEC50 for WT GLP1 were also shown in table 1, for comparison with the GLP1 mutants.

We would like to thank Reviewer #3 for this comment, and we have now added the respective pEC50 for WT GLP1 to Table 1.

3. Fig2 suppl 1. The methods and analysis for this figure are inadequately explained. To show that the HEK-GLPLight1 cells are responding to GLP1 released from GLUTag cells, the GLPLight1 response needs to be shown before and after GLUTag cell stimulation with an agent that should trigger GLP-1 release.

We would like to thank Reviewer #3 for this comment which has been partially addressed already in the replies to Comment#1 from Reviewer #1 and Reviewer #2.

Since we did not observe any response to acute stimulation of GLUTag cells we considered the high glucose concentration present in the culture media being a stimulation agent for GLUTag cells, which has been previously reported (PMID: 17643200).

4. Fig 3g and others: The end of the photo activation period needs to be represented correctly on the timeline. In 3g, the bar that should indicate when photoactivation was applied does not end at the zero time point (which is labelled as the time relative to photoactivation).

We would like to thank Reviewer #3 for pointing this out. The shaded area representing the photo-activation has been matched accordingly.

5. Discussion para 1: the authors claim their data show that ligand induced activation of human GLP1R occurs more slowly than others similar GPCR sensors - they should give actual data to substantiate this claim, since the time course of glp1r activation has not been analysed and compared with other sensors in the manuscript.

We added data to support this claim to the discussion: “As a reference, other previously-characterized class-A GPCR-based neuropeptide biosensors showed sub- second activation kinetics (Duffet et al., 2022a; Ino et al., 2022).”

6. Methods: what wavelength was used for recording emission from GLP1Light1? The excitation wavelength is given, but I can't see the emission wavelength(s). In fig 1d, the excitation and emission spectra should be depicted in different colours/line properties, otherwise this figure is very confusing.

We updated figure1d and changed the colors to improve data visualization. Regarding the missing wavelength, we would like to clarify that both wavelengths were already described in the methods section as: “The excitation and emission spectra were measured at λem = 560nm and λex = 470nm, respectively, on a TECAN M200 Pro plate reader at 37 °C. “. We would be happy to rewrite this paragraph, if necessary, shall it remain unclear to the reader.