Discovery of a β-arrestin-biased CCKBR agonist that blocks CCKBR-dependent long-term potentiation

Domain: physics
Published: 2025-12-08
Editorial outcome: major_revision

Abstract
The CCKBR agonists induce neocortical long-term potentiation of excitatory synaptic transmission and enhance memory formation, while its antagonists weaken the potentiation in the amygdala and alleviate depression-like behaviors. However, the mechanism that drives CCKBR dependent long-term potentiation remains elusive. There is also no signaling pathway-biased CCKBR agonist to modulate the potentiation. Here, we discover a β-arrestin biased CCKBR agonist MF-8 with IC50 = 0.9 nM. The activation of CCKBR with MF-8 fails to induce the potentiation but efficiently induces CCKBR endocytosis. Multi-Electrode Array results demonstrate that the potentiation is dependent on Gαq/11-Ca2+ and Gαs-cAMP signaling pathways. The potentiation is entirely blocked by MF-8 through β-arrestin signaling. Furthermore, MF-8 effectively inhibits the formation of cue-to-cue associative fear memory. These results reveal the signal pathway preference of the CCKBR long-term potentiation and identify a blocker of the potentiation, which provides us with broader insights into developing drugs targeting CCKBR.

Introduction
G protein-coupled receptors (GPCRs) are the most prominent family of membrane proteins in eukaryotes, with more than 800 members in this family1. These proteins facilitate the transmission of diverse signals across the cell membrane, including gas molecules, polypeptides, photons, and small molecules2. Given their critical roles in mediating intracellular and extracellular signaling, these membrane proteins represent optimal targets for drug development. Current statistics indicate that GPCR-targeted drugs account for nearly 34% of all marketed drugs worldwide1,2.

Signal transduction upon GPCR activation was initially thought to be only G protein-dependent until arrestins were also found to play a significant role in GPCR signaling3. Besides, each GPCR was initially supposed to activate downstream signaling pathways through one unique G protein4. However, recent findings have shown that the same GPCR can activate different G proteins5,6, and arrestins can function either dependently or independently of G proteins7–9. The arrestin family contains four members, including arrestin1, arrestin2 (β-arrestin1), arrestin3 (β-arrestin2), and arrestin4. The human G protein family includes 16 Gα, 5 Gβ, and 12 Gγ4,10. There are indications that GPCR activation is biased towards different subtypes of G proteins and arrestins4,10. Therefore, even the same GPCR produces different biological effects due to the different types of G proteins and arrestins it activates. Based on this theory, drugs with high selectivity for receptor types or bias for signaling pathways may be specifically applied in treating specific diseases while avoiding numerous potential side effects during disease treatments. Hence, several signaling pathway-biased agonists and antagonists are being developed to treat specific diseases. A classic example is oliceridine, a Gαi-biased agonist of μ-opioid receptor (μ-OR) approved by the Food and Drug Administration (FDA) in 2020 for treating acute to moderate pain11. Oliceridine effectively enhances the analgesic effect while minimizing β-arrestin-mediated side effects such as tolerance, addiction, and respiratory depression11.

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the nervous system and acts in the form of sulfated CCK8s in the brain12–14. CCKAR, CCKBR, and GPR173 were discovered as receptors of CCK, and they serve important functions in the brain. For instance, CCKAR is involved in spatial memory and emotion15,16, CCKBR in associative memory, spatial learning, motor skill, and depression17–20, and GPR173 is related to inhibitory plasticity21. Studies have reported that CCKAR can activate Gαq/11, Gαi/o, Gαs, Gα12/13, and β-arrestin1&25,6,21–23. while CCKBR can also activate these signaling pathways5,6,21,23. Nevertheless, the relationship between these signaling pathways and CCK receptors’ functions in the brain has not been thoroughly understood. We recently demonstrated that a CCKBR agonist, HT-267, could be used to alleviate anterograde amnesia in CCK-deficient and aged Alzheimer’s disease mice24, and a CCKBR antagonist YM022 displayed an antidepressant-like effect in a chronic social defeat stress mouse model19. CCKBR-mediated cognitive function, emotion, and movement occur through the CCKBR-dependent long-term potentiation (CCKBR-LTP) of excitatory synaptic transmission, but the signaling mechanism of this LTP is still unclear17,19,20,24,25. Hence, revealing the signaling pathway mechanism of the CCKBR-LTP is crucial for the development of drugs targeting this receptor.

It has been more than 20 years since the cloning and purification of the CCKBR receptor in 199226, and most previous studies have focused on its Gαq/11-mediated signaling pathway27–29. CCKBR can also increase the excitability of small-sized dorsal root ganglion (DRG) neurons through Gαo-mediated inhibition of A-type K+ channels, thereby enhancing pain susceptibility30. The development of biased agonists and antagonists is beneficial not only for studying the mechanism of action of receptors but also for treating diseases more precisely and effectively2. Although there are many signaling pathways downstream of CCKBR, we found only one report about biased antagonists of CCKBR, and there is no report about biased agonists. GV150013x is a Gαq/11-biased CCKBR antagonist with no antagonistic effect on the β-arrestin signaling pathway31. However, no study has systematically evaluated the bias of this antagonist or other agonists and antagonists toward individual CCKBR signaling pathways. Therefore, finding biased agonists and antagonists of CCKBR will benefit the development of CCKBR drugs and the study of its mechanism.

In this work, we use a Multielectrode Array (MEA)-based electrophysiological method to reveal the mechanism of CCKBR-LTP in the auditory cortex (AC). Meanwhile, we identify a β-arrestin biased CCKBR agonist MF-8 through fluorescence resonance energy transfer (FRET)-based binding, bioluminescence resonance energy transfer (BRET), cyclic AMP (cAMP), calcium, receptor internalization, and PRESTO-Tango assays. This biased agonist can reduce other CCKBR signaling pathways, including Gαs-induced cAMP and Gαq/11-induced calcium signaling, thereby inhibiting the CCKBR-LTP. The blockade of MF-8 to the LTP is abolished by mutating the key site N3536.55, which determines the β-arrestin signaling bias induced by MF-8. The β-arrestin blocker barbadin and knockdown of β-arrestin expression also alleviate the inhibitory effect of MF-8 on the taburst stimulation (TBS)-induced CCKBR-LTP, suggesting that the inhibition is mediated by the β-arrestin signaling pathway. Furthermore, MF-8 effectively inhibits the formation of cue-to-cue associative fear memory. In conclusion, we uncover the mechanism of CCKBR-LTP in the AC and identify a β-arrestin-biased CCKBR agonist that blocks the LTP and CCKBR-dependent cue-to-cue associative fear memory via the β-arrestin signaling pathway.

Results
MF-8 binds to CCKBR with high affinity
CCK4 (Supplementary Table. 1) contains four amino acids at the carboxyl terminus of CCK8s, and it is considered the shortest CCK fragment that activates CCKBR32. In our previous screening campaign, we found that a CCK4-modified compound HT-178 (Supplementary Fig. 1D) exhibited a good activation effect on CCKBR downstream calcium signaling. However, we found a very interesting phenomenon. A slight change in the substituent on the amino group in the red box of HT-178 (Supplementary Fig. 1D) greatly affected the activation of calcium signals downstream of CCKBR. Since the downstream signaling pathway of CCKBR could be intricate5,6,21,23, we speculated that this slight change might not change the affinity for CCKBR, but only could change the bias for the downstream signaling pathway of CCKBR. Therefore, we selected compounds with different substituents at the amino position, as well as some other compounds based on CCK4 modification, for systematic biological evaluation (Supplementary Table. 1). They were first assayed based on established calcium mobilization and PRESTO-Tango assays to differentiate their Gαq/11-Ca2+ (Table. 1) and β-arrestin2 response (Table. 1). MF-7, MF-8, MF-9, MF-10, MF-11, MF-12, and MF-13 performed poorly in activating Gαq/11 signaling but were highly potent in inducing β-arrestin2 signaling (Table. 1). Both calcium and PRESTO-Tango assays were performed using CCK8s as a balanced agonist control. This was necessary to minimize system bias and quantitatively evaluate their bias level. According to the bias factor β33 (Table 1, Fig. 1A), we demonstrated that MF-8 is highly β-arrestin biased compared to Gαq/11-Ca2+ signaling with β > 2 (Fig. 1A–C).

To further identify these compounds, we constructed a binding system based on FRET. This system mainly consists of two components: a fluorescent probe and a modified CCKBR (Fig. 1D, E). We attached rhodamine and a HaloTag protein to the amino terminus of CCK5 and the amino terminus of CCKBR, respectively (Fig. 1D, E). After transient transfection of HaloTag-tagged CCKBR into 293T cells, we labeled CCKBR with Fluo-Halo (Fig. 1D, Supplementary Figs. 1C, 3, 4). Via this binding test, we determined that the binding affinity of MF-8 to CCKBR is close to CCK8s (IC50 (MF-8) = 0.9 nM, IC50 (CCK8s) = 1.1 nM) (Fig. 1E). The above PRESTO-Tango and binding assays prove that MF-8 is a highly efficient CCKBR agonist.

The CCKBR-LTP in the AC is not dependent on β-arrestin signaling
Several critical physiological functions, such as mood, memory, and movement within the nervous system, are dependent on LTP. We have previously revealed that CCKBR regulates LTP in the AC and facilitates the formation of associative memory17. For recording CCKBR-LTP, coronal slices (300 μm) containing AC were recovered in artificial cerebrospinal fluid (aCSF) at 28°C for 1.5 h, then positioned in a MED64 probe with continuous aCSF perfusion. Minutely field excitatory postsynaptic potentials (fEPSPs) were recorded at 30% maximal response until stable, then after 10 min of drug-treated aCSF perfusion, low-frequency electrical stimulation (LFS) was applied at 1 Hz for 60 cycles at 70% maximal response. Finally, minutely fEPSPs were re-recorded for over 60 min (Fig. 2A). Consistent with previous results, LFS along with CCK8s induced LTP in the AC (Fig. 2B, C). Since the EC50 of MF-8 equals 4.54 nM in the PRESTO-Tango assay (Fig. 1C), and its IC50 is 0.9 nM in the binding assay (Fig. 1E), therefore, 20 nM MF-8 coupled with LFS was used for LTP induction (Fig. 2B, C). However, this condition failed to induce LTP (Fig. 2B, C). We then increased MF-8’s concentration to 200 nM together with LFS to evaluate whether a higher concentration of MF-8 can induce LTP. Although a slight LTP was observed, there was no significant difference in fEPSPs before and after LFS and MF-8 treatment (Fig. 2B, C). Considering that MF-8 preferentially activates β-arrestin signaling of CCKBR, the above results demonstrate that CCKBR-LTP in AC is not dependent on β-arrestin signaling.

A β-arrestin signaling blocker, barbadin34, was also applied during LTP recording to further verify whether the CCKBR-LTP depends on the β-arrestin signaling pathway. We induced the LTP with CCK8s and LFS after the blocker was incubated with the brain slices for 1 h, but the LTP still maintained the same level as the control group (Fig. 2D, E). In addition, after β-arrestin1 and β-arrestin2 in the mouse AC were knocked down by adeno-associated virus (AAV)-mediated shRNA (Fig. 2F), the CCKBR-LTP could still be induced normally (Fig. 2G, H). Taken together, these pharmaceutical interventions and gene expression regulation studies reveal that the CCKBR-LTP is not dependent on β-arrestin signaling.

CCKBR-LTP in AC is Gαq/11 and Gαs dependent
Previous reports have indicated that CCKBR can activate a series of Gαi, Gαs, Gαq/11, and β-arrestin signaling pathways5,6,22,23,28, but the specific signaling pathway that determines CCKBR-dependent LTP remains unclear. To address this issue, we investigated the mechanism of the CCKBR-LTP formation by using a series of signaling pathway blockers. As TBS is an alternative method to induce the CCKBR-LTP17, we first induced LTP in AC using TBS (Fig. 3A, I), and confirmed that the LTP was CCKBR-dependent through treatment with the specific CCKBR antagonist YF476 (Fig. 3B, I). We then used Gαq/11 signaling pathway blockers U-73122, 2-APB, and YM254890 to confirm whether Gαq/11 is involved in the formation of the LTP (Fig. 3C–E, I). We found that all Gαq/11 inhibitors significantly and almost completely inhibited the formation of LTP (Fig. 3C–E, I). Also, the antagonist of the Gαs signaling pathway, H-89, partially inhibited LTP formation (Fig. 3G, I). However, PTX and barbadin, inhibitors of the Gαi and β-arrestin signaling pathway, respectively, could not inhibit the LTP (Fig. 3F, H, I). In addition, we used MF-8 to block TBS-induced CCKBR-LTP, and simultaneously applied chemogenetic elements hM3d(Gq)35 and rM3D(Gs)36 to activate Gq and Gs, respectively, under the induction of Clozapine N-oxide (CNO). Although MF-8 could block the LTP, the inhibitory effect of MF-8 on the LTP could be abolished by both hM3dq(Gq) and rM3D(Gs) under the activation of CNO (Supplementary Fig. 6A–F). These results indicate that the CCKBR-LTP in the AC depends on Gαq/11 and Gαs signaling pathways, especially the Gαq/11 signaling pathway.

MF-8 is a β-arrestin-biased and CCKBR-selective agonist
To further explore the role of MF-8 in CCKBR-mediated LTP, we investigated the agonist’s cell signaling pathway bias and CCK receptor subtype selectivity through diverse cell pathway assays. Regarding the activation of the Gαi family (Gαi1, Gαi2, Gαi3, Gαoa, Gαob, Gαz), MF-8 showed a partial activation effect on each of the Gαi subtypes (Fig. 4A–F). For the Gα12 family, MF-8 induced weak activation on Gα12 under the high concentration condition, and the potency of MF-8 on Gα13 was about 500 times weaker than CCK8s (Fig. 4G, H). MF-8 showed partial activation effects on Gαq/11 and Gαss, but had no activity on Gαsl (Fig. 4I–L). However, MF-8 had a higher potency than CCK8s in the BRET assay for the recruitment of both β-arrestin1 and β-arrestin2 (Fig. 4M, N). The β-arrestin results were consistent with a higher affinity of MF-8 than CCK8s (Fig. 1E). We then used the results of the BRET-based test of G protein (Fig. 4A–L) and β-arrestin (Fig. 4M, N) subunits to draw a radar graph of MF-8 relative to CCK8s activity (Fig. 4O). The graph indicates that MF-8 has a clear preference for β-arrestin1 and β-arrestin2. Besides, the results of cAMP assays suggest that at a high concentration of 10 µM, the activation rate of CCKBR by MF-8 was only about 17% of CCK8s’ activation (Supplementary Fig. 7A), which is consistent with the results of BRET-based Gαss and Gαsl assays (Fig. 4I, J). We also calculated the activity of MF-8 relative to CCK8s based on the results of calcium mobilization (Fig. 1B), PRESTO-Tango (Fig. 1C), and cAMP assays (Supplementary Fig. 7A), and the results suggest that MF-8 has a higher β-arrestin preference (Supplementary Fig. 7B). Overall, compared with the endogenous agonist CCK8s, MF-8 is a potent CCKBR agonist with a strong bias for β-arrestin signaling.

Apart from CCKBR, GPR173 and CCKAR are CCK receptors. Hence, we also investigated MF-8’s activation activity on CCKAR and GPR173. CCK8s could activate the β-arrestin and Gαq/11 signaling pathways downstream of CCKAR (Fig. 5A, B). Although MF-8 weakly activated the β-arrestin signaling at 10 µM, it did not exhibit any agonistic effect on the Gαq/11 signaling of CCKAR (Fig. 5A, B). We previously found that CCK8s could trigger the release of calcium ions in monoclonal CHO cells overexpressing GPR17321, but MF-8 did not increase intracellular calcium levels in these cells (Fig. 5C). We also applied a BRET-based assay to test the activation of MF-8 on β-arrestin1/2 downstream of GPR173. CCK8s and PNX-14 were positive controls, as PNX is also a GPR173 ligand37. Although both PNX-14 and CCK8s could activate these two subtypes downstream of GPR173, MF-8 had no activating effect on them, even at 10 μM (Fig. 5D, E). The above results indicate that MF-8 is a highly selective β-arrestin-biased agonist for CCKBR.

MF-8 is able to induce CCKBR internalization
The canonical β-arrestin signaling pathway mediates endocytosis after GPCR activation38,39. Consequently, we hypothesized that MF-8 could also induce CCKBR’s endocytosis. We designed three assay systems using β-arrestin1 and β-arrestin2 to detect fluorescence distribution changes in cells after receptor activation. 1) EYFP was fused to the N-terminus of truncated β-arrestin1 (1–382aa) (Fig. 6A). 2) Fusion expression of GFP at the C-terminus of β-arrestin2 (Fig. 6D). 3) Fusion expression of EYFP at the N-terminus of β-arrestin1 (Supplementary Fig. 10A). We co-expressed these fused fluorescent protein-expressed β-arrestins with HaloTag-tagged CCKBR in 293T cells (Fig. 6A, D, Supplementary Fig. 10A). We labeled CCKBR with the fluorescent dye Rho-2CF3 by targeting HaloTag (Fig. 6A, D, Supplementary Fig. 10A) and then traced the changes in the distribution of β-arrestin and CCKBR under the stimulation of CCK8s or MF-8. We found that under control conditions, CCKBRs were mainly distributed on the cell membrane, while β-arrestin1 and β-arrestin2 were diffusely distributed within the cells (Fig. 6B, E, Supplementary Figs. 10B, 11). Within the CCK8s and MF-8 treatment groups, dense co-labeled signals of β-arrestins and CCKBR were evident in the cells (Fig. 6B, E, Supplementary Figs. 10B, 11). The results confirm that MF-8 and CCK8s can both induce receptor endocytosis. To compare the effects of endocytosis, we quantified the number of co-labeled signals in each group (Fig. 6C, F, Supplementary Fig. 10C). The number of co-labeling in CCK8s and MF-8 groups was significantly higher than that of the control group (Fig. 6C, F, Supplementary Fig. 10C). Meanwhile, the number of co-labeling in the MF-8-treated group was not significantly different from that of the CCK8s-treated group (Fig. 6C, F, Supplementary Fig. 10C), indicating that the level of endocytosis induced by MF-8 was equivalent to that of the endogenous agonist CCK8s. The above results demonstrate that MF-8 can effectively induce endocytosis of CCKBR.

CCKBR-mediated calcium, cAMP accumulation, and LTP are blocked by MF-8 with high potency
Based on the results from our previous experiments, we hypothesized that the key signaling pathways (Gαq/11 and Gαs) mediated by CCKBR would also be inhibited by MF-8. To confirm that, we applied calcium mobilization and cAMP assays to examine whether MF-8 could reduce the intracellular calcium release and cAMP accumulation induced by CCK8s. We first tested the response of CCK8s and MF-8 using 293T cells infected with a control virus. The results indicated that CCK8s and MF-8 had no agonistic effect on these cells (Supplementary Fig. 12). In the calcium mobilization assay, MF-8 was then incubated with 293T-CCKBR cells using a series of gradient concentrations for two hours, we then proceeded to add CCK8s at a final concentration of 15 nM to detect the increase in intracellular calcium (Fig. 7A). We found that MF-8, like YF476, inhibited the increase in calcium flux induced by CCK8s with IC50 = 22.7 nM (Fig. 7A). Simultaneously, we also examined cAMP accumulation after 2-h incubation of MF-8 with 293T-CCKBR cells (Fig. 7B). The results showed that MF-8, like YF476, inhibited the increase of cAMP induced by CCK8s with IC50 = 16 nM (Fig. 7B). Both calcium and cAMP assays showed that MF-8 inhibited the key signaling pathway of CCKBR-mediated LTP with high potency.

Based on the previously mentioned results, we hypothesized that MF-8 could suppress Gαq/11 and Gαs-mediated CCKBR-LTP in AC. We incubated 200 nM MF-8 with brain slices for 2 h and then examined the effect of MF-8 on TBS-induced CCKBR-LTP in AC (Fig. 7C). Compared with the control group, MF-8 significantly and completely inhibited the CCKBR-LTP (Fig. 7D). These results suggest that the β-arrestin-biased CCKBR agonist MF-8 can suppress LTP formation by blocking CCKBR-mediated cAMP and calcium signaling.

MF-8 blocks CCKBR-LTP in a β-arrestin dependent manner
To further verify whether MF-8 inhibits CCKBR-LTP through β-arrestin signaling, we attempted to discover the key amino acid sites in CCKBR that are associated with this pathway. Simply relying on molecular docking, such as autodock, cannot obtain accurate docking postures, especially for highly flexible polypeptide molecules40. Here, we used autodock vina to perform molecular docking of CCKBR and MF-8 (Supplementary Data 1), and then we selected the docking results based on the conformation and orientation of gastrin-17 in the Cyro-EM gastrin-17-CCKBR complex41. Molecular dynamics simulations with GROMACS were used to evaluate the binding mode of CCKBR and MF-8 (Supplementary Data 2). We monitored interactions formed between the receptor and ligand during the three simulations using multiple bioinformatics tools (Supplementary Figs. 13–15). Then point mutations in CCKBR combined with a BRET-based β-arrestin assay and PRESTO-Tango assay were applied to verify hydrogen bond interactions of MF-8 and CCKBR (Fig. 8A, Supplementary Fig. 16). As shown in Fig. 8A, CCKBR could form stable hydrogen bond interactions with MF-8 through residues N1152.65, T1112.61, H3767.39, Y3807.43, N3536.55, Y1894.60, H207ECL2, R3566.58, and H3647.27 (Fig. 8A, B, Supplementary Figs. 13–16), which was similar to the bonds formed by gastrin-17-CCKBR41. Among them, N1152.65 and H3647.27 could not form stable hydrogen bond interactions with the CCK4 part of gastrin-1741, which might explain why MF-8 could act as a potent agonist of CCKBR.

Considering the drastic effect of N3536.55 on MF-8-mediated β-arrestin signaling (Fig. 8B, Supplementary Fig. 16) and the weak effect of this site on the Gαq signaling pathway41, we speculated that mutation of this site would not affect TBS-induced CCKBR-LTP, but would eliminate the blockade of MF-8 on the TBS-induced CCKBR-LTP since the LTP mainly depends on the CCKBR-Gαq/11-mediated signaling pathway. We then adopted CCKBR knockout mice to verify the integrity of TBS-induced CCKBR-LTP in the AC (Fig. 8C). The TBS-induced LTP was completely abolished in CCKBR knockout mice (Fig. 8C, F, G). We also overexpressed CCKBR in AC via AAV injection (Supplementary Fig. 17), and TBS induced CCKBR-LTP formation regardless of whether we overexpressed the human or mouse CCKBR (Fig. 8C, F, G, D, H, I). Meanwhile, 200 nM of MF-8 completely abolished TBS-induced CCKBR-LTP in the AC of human CCKBR-overexpressing CCKBR-KO mice (Fig. 8D, H, I). However, when we overexpressed the human CCKBR with the N3536.55A mutation, we found that although TBS induced CCKBR-LTP formation, this LTP was not blocked by MF-8 (Fig. 8E, J, K). We also used barbadin to inhibit β-arrestin signaling and then determined whether MF-8 could inhibit TBS-induced LTP in the presence of barbadin. The results indicated that barbadin could indeed eliminate the inhibitory effect of MF-8 on this LTP (Fig. 8L, N, O). Meanwhile, AAV-mediated shRNA was applied to knock down β-arrestin1 and β-arrestin2 (Fig. 2F). The inhibitory effect of MF-8 on the LTP was significantly reduced after the two β-arrestins were knocked down (Fig. 8M, P, Q). These results suggest that MF-8 reduces CCKBR-LTP via a β-arrestin-dependent signaling pathway.

MF-8 treatment inhibits the CCKBR-LTP mediated cue-to-cue fear memory
Enhanced fear memories are thought to be closely linked to anxiety, depression, and post-traumatic stress disorder42–44. Our previous study found that the CCKBR-LTP in AC can enhance cue-to-cue fear memory, while CCKBR antagonists could inhibit the formation of fear memory17. This study found that the CCKBR-LTP in AC is mainly mediated by the Gαq/11 and Gαs signaling pathways. MF-8, as a β-arrestin biased CCKBR agonist, can inhibit the CCKBR-LTP. Therefore, MF-8 may also be able to inhibit the CCKBR-LTP mediated cue-to-cue fear memory. Here, we used the previous behavioral paradigm and found that MF-8 can indeed inhibit cue-to-cue fear memory (Fig. 9), which highlights the translational value of MF-8 in fear memory-related disorders.

Methods
Ethics statement
All animal experimental procedures were reviewed and approved by the Animal Subjects Ethics Sub-committees of the City University of Hong Kong. The following mice were used for electrophysiology: C57BL/6J (C57) and CCKBR-KO mice with a 129S1 background (Stock# 006369, Jackson Laboratory). All mice were randomly allocated into the experimental and control groups. Male C57 and CCKBR-KO mice, with 8–24 weeks, were used for MEA recording. Male C57 with 6–8 weeks were used for animal behavior analysis. Mice were housed at 20–24°C with 40–60% humidity under a 12-h light/12-h dark cycle (dark from 9:00 to 21:00) and given food and water ad libitum.

Plasmid construction
Tango-CCKBR was constructed in our previous work21. To examine the effects of mutations to alanine (A) at Asn115, Gln204, His207, and Asn353 on β-arrestin signaling, Tango-CCKBR (N115A), Tango-CCKBR (Q204A), Tango-CCKBR (H207A), and Tango-CCKBR (N353A) were generated by replacing the CCKBR sequences with point mutations into Tango-CCKBR. To construct pcDNA3.1(+)-HaloTag-CCKBR, the full length of Homo sapiens CCKBR (NM_176875.4) complementary DNA was cloned into the pcDNA3.1(+) vector (Invitrogen) containing a hGH signal sequence followed by a HaloTag sequence before the receptor sequence. Full length of Homo sapiens GPR173 (NM_018969.5), CCKAR (NM_000730), and CCKBR ((NM_176875.4)) were cloned into the multiple cloning site (MCS) of pLVX-puro vector to generate pLVX-puro-GPR173, pLVX-puro-CCKAR, and pLVX-puro-CCKBR. The coding sequence of β-arrestin2 (NM_004313) with GFP conjugated to its carboxyl terminus was cloned into pcDNA3.1(+) vector to generate pcDNA3.1(+)-βarr2-GFP. To construct pcDNA3.1(+)-YFP-βarr1, the coding sequence of β-arrestin1 (NM_004041.5) with YFP conjugated to its amino terminus was cloned into pcDNA3.1(+) vector. pcDNA3.1(+)-YFP-βarr1_1-382aa was constructed by replacing nucleotides GAT encoding Asp-383 with nucleotides TAG encoding a stop codon of YFP-βarr1 in pcDNA3.1(+)-YFP-βarr1. pcDNA3.1(+)-Flag-CCKBR, pcDNA3.1(+)-Flag-CCKBR mutants, pcDNA3.1(+)-rGFP-CAAX, pcDNA3.1(+)-β-arrestin1-RlucII, and pcDNA3.1(+)-β-arrestin2 -RlucII were obtained from PPL (Public Protein/Plasmid Library, China). All final recombinant vectors were confirmed by sequencing analysis.

Generation of GPCRs overexpression-cell lines
HEK293T, 293T-puro (HEK293T cells infected with empty lentivirus), CCKAR-overexpressing HEK293T (293T-CCKAR), CCKBR-overexpressing HEK293T (293T-CCKBR), and GPR173-overexpressing HEK293T (293T-GPR173) cells were maintained in DMEM medium (Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (Gibco) and 1% Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture (Thermo Fisher Scientific) at 37°C in a humidified atmosphere of 5% CO2.

The procedure for generating 293T-puro, 293T-GPR173, 293T-CCKAR and 293T-CCKBR is outlined as follows. To produce the lentivirus required for GPCRs overexpression, HEK293T cells were seeded for 70%–90% confluency in 6-well tissue culture plates and incubated for 10–12 h in the mixture of calcium phosphate transfection reagent (31.25 μl 2M CaCl2 solution, 250 μl 2×HBS (0.05 mol/L HEPES, 0.012 mol/L D-(+)-Glucose, 0.28 mol/L NaCl, 0.023 mol/L KCl, 0.0015 mol/L Na2HPO4), 218.75 μl H2O) and 1 ml fresh medium, with 4 μg pLVX-puro-GPCR plasmids (Public Protein/Plasmid Library, Nanjing, China) or pLVX-puro, 3 μg pSPAX2 plasmid (Addgene, plasmid #12260) and 1.2 μg pMD2.G plasmid (Addgene, plasmid #12259). The cells were cultured for an additional 36 h in their normal medium before collecting the virus supernatant and infecting HEK293T cells. Meanwhile, around 1 × 106 HEK293T cells were seeded in 6-well plates, each well for 12 h, 0.5 mL virus supernatant, and 0.1% polybrene (Sigma) in 1 mL fresh normal medium were mixed and added. The second infection was performed at the interval of 24 h, and the virus supernatant mix was replaced by the fresh normal medium of HEK293T after 6–8 h of each time infection. After 2 days of the second infection, puromycin (Invitrogen) was added at the final concentration of 10 μg/mL for one week. The screened cells were going to be used for subsequent assays. Signaling pathway-based cell assays were used to validate the 293T-CCKAR (Supplementary Fig. 9) and 293T-CCKBR cell lines (Supplementary Fig. 8).

Calcium mobilization assays
HEK293T (ATCC, CRL-3216) cells stably expressing GPCRs were plated at 6 × 104 (1 × 104) cells per well in black and optical-bottom 96/384-well plates (Corning). The next day, the Ca2+ release response of cells was measured by a Fluo-8 No Wash Calcium Assay Kit (AAT-Bioquest) according to the manufacturer’s instructions. All measurements of calcium mobilization assay were performed by EnVision 2104 Multilabel Reader (Perkin Elmer) or CLARIOstar® Plus Multi-Mode Plate Reader (BMG LABTECH). The relative luminescence was exported into an Excel sheet and processed with Excel and GraphPad Prism. The cells were cultured at 37°C, 5% CO2 in DMEM medium (Thermo Fisher Scientific) containing 10% FBS (Natocor-Industria Biológica) and washed with HHBS once before incubating with Fluo-8.

The procedure for calcium mobilization assay of CHO-GPR17321 was identical to the other GPCR-overexpressing HEK293T cells apart from the culture medium. CHO-GPR173 were cultured at 37°C and 5% CO2 in DMEM/F12 medium (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific).

PRESTO-Tango assays
PRESTO-Tango assays were performed as described in59 with some modifications. HTLA cells (A gift from the R. Axel’s Laboratory) grown in 6-well plate were transfected with 1 μg Tango-CCKAR21 or Tango-CCKBR21 per well according to the instructions of Lipo8000 (Beyotime). The transfected cells were seeded in poly-L-lysine-coated 384-well plates (JingAn Biological) at 10000 to 15000 cells per well in 50 μl of medium after 24-h culture. The medium was replaced with fresh medium containing a series concentration of compounds after 24-h culture in 384-well plate. Medium and drug solutions were removed from each well after ~18-h incubation with cells, and 20 μl per well of Bright-Glo solution (Promega) diluted 20-fold in assay buffer (1x HBSS, 2.5 mM probenecid, 20 mM HEPES, pH = 7.4) was added into each well. After ~15 min incubation at room temperature, luminescence was measured in FlexStation 3 (Molecular Devices) or CLARIOstar® Plus Multi-Mode Plate Reader (BMG LABTECH). The relative luminescence was exported into an Excel sheet and processed with Excel and GraphPad Prism.

BRET-based β-arrestin assays
The procedure of BRET-based β-arrestin assays was modified based on a previously reported paper60. HEK293T cells were seeded into 6-well culture plates and maintained in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) Penicillin-Streptomycin-Neomycin Antibiotic Mixture (Gibco) at 37°C in a humidified incubator with 5% CO2. Plasmids encoding CCKBR (pcDNA3.1(+)-ssflag-CCKBR), an effector-RlucII fusion protein (pCDNA3.1(+)-β-arrestin1/2 -RlucII), and pCDNA3.1(+)-rGFP-CAAX were transiently transfected into cells using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. For BRET-based β-arrestin assays of GPR173, 293T-GPR173 cells were used and only plasmids containing β-arrestin1/2-RlucII and rGFP-CAAX were transfected. Twenty-four hours after transfection, cells were trypsinized, resuspended in DMEM, and seeded into white clear-bottom 96-well microplates (Greiner Bio-One) at a density of 5 × 104 cells per well. The BRET substrate Coelenterazine 400a (Nanolight Technologies) was added to each well to a final concentration of 5 μM. BRET signals were acquired using a CLARIOstar microplate reader (BMG Labtech) equipped with dual emission filters (480 nm for RlucII and 530 nm for rGFP). Background luminescence was measured using untransfected cells, and background-subtracted BRET ratios were calculated as the 530 nm/480 nm emission intensity ratio.

Immunofluorescence
For brain slice immunofluorescence in Fig. 2D1, Supplementary Fig. 6A, and 17, mice were anesthetized with pentobarbital sodium and transcranial-perfused with 30 ml cold PBS and 30 ml cold 4% (w/v) PFA in PBS. Brain tissue was removed, postfixed with 4% PFA, and treated with 30% (w/v) sucrose in PBS at 4°C for two days. Brain tissue was sectioned on a cryostat (Leica CM3500) with 30 μm thickness and preserved with antifreeze buffer (20% v/v glycerin and 30% v/v ethyleneglycol diluted in PBS) at −20°C. Brain sections in Supplementary Figs. 6A and 17 were washed three times with PBS and then incubated with DAPI for 30 min. Next, the sections were washed three times with PBS and mounted with 70% (v/v) glycerol in PBS on a slide. Brain sections in Fig. 2D1 were rinsed three times with PBS and blocked with blocking buffer (10% v/v goat serum in PBS with 0.25% v/v Triton X-100) for 1 h at room temperature. Sections were incubated with primary antibodies (1:50 dilution, β-arrestin1/2 antibody, SANTA, Catlog No: SC-74591) at 4°C for 36~48 h. After washing thrice (15 min for each) in PBS, sections were incubated with the corresponding secondary antibodies (1:200 dilution, Goat anti-mouse 594, Jackson ImmunoResearch, Catlog No: 115-585-003) and DAPI for two hours at room temperature. We mounted all sections with 70% (v/v) glycerol in PBS on a slide after rinsing them with PBS three times. Images of the sections were acquired using Nikon Confocal Imaging System AXR and processed with Nikon NIS element and ImageJ (NIH).

FRET-based binding assays
For FRET-based binding assays using CCKBR N-terminal labeled with HaloTag, cultivation of 293T cells was performed in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin-Neomycin Antibiotic Mixture at 37°C in a humidified 5% CO2 atmosphere. The 293T cells were transiently transfected with pcDNA3.1(+)-HaloTag-CCKBR using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher, Catalog Number: L3000015) according to the manufacturer’s instructions. The transfected cells were seeded at 15,000-20,000 cells per well in a black transparent-bottomed 384-well microplate and allowed to adhere overnight. For fluorescence resonance energy transfer (FRET) measurements, serial dilutions of tested compounds were prepared. A dual-labeled detection system was established using 500 nM SRB-CCK5 and 1000 nM Fluo-Halo. Prior to assay initiation, growth medium was aspirated from designated wells. Aliquots of 20 μL of the prepared solution with compounds were dispensed into each well, followed by the addition of 40 μL combined probe solution to achieve final concentrations of 500 nM SRB-CCK5 and 1000 nM Fluo-Halo in a total reaction volume of 60 μL. After 5-min incubation at room temperature, FRET signals were measured simultaneously through dual excitation/emission channels (475 nm/520 nm, 475 nm/586 nm). The final concentration-dependent response curve was plotted using GraphPad Prism after subtracting the FRET ratio (F586 nm/F520 nm) values of the control group.

TRUPATH assays (BRET2 assays)
The TRUPATH suite (Kit #1000000163)61, which includes 14 optimized BRET Gαβγ biosensors, was obtained from Addgene. We used Lipo8000TM Transfection Reagent (Beyotime Biotechnology) for plasmid transfection according to the manufacturer’s instructions. The Gαβγ biosensor’s plasmids with the best single pathway resolution were transfected into HEK293T cells for 24 h, using a 2:1:1:1 DNA ratio of receptor: Gα-RLuc8: Gβ: Gγ-GFP2 (1 μg per construct for 6-well dishes). After 24 h transfection, cells were harvested and incubated in poly-L-lysine-coated white, clear-bottom 96-well assay plates (Greiner Bio-One) at a density of 4 × 104–6 × 104 cells per well. The next day, we inspected the cell density and plastered the white backing (Perkin Elmer) to the bottom of the plate. After cautiously aspirating the growth medium, 60 μl BRET assay buffer (1× Hank’s balanced salt solution (HBSS) + 20 mM HEPES, pH 7.4) was added to each well, followed by the addition of 10 μl freshly prepared 50 μM coelenterazine 400a (Nanolight Technologies). After 5 min, cells were treated with 30 ul of gradient concentration drug solution. The plate was read using a CLARIOstar plate reader (BMG LABTECH Ltd) with 395 nm (RLuc8-coelenterazine 400a) and 510 nm (GFP2) emission filters, with an integration time of 1 s per well. Plates were read serially 4 times, and the measurements from the last read were used for all analyses. BRET2 ratios were calculated as the ratio of the 510 nm emission to the 395 nm emission.

CCKBR internalization assays
HEK293T cells were transfected at a 1:1 ratio with pcDNA3.1(+)-Halotag-CCKBR and β-arrestins subunit (pcDNA3.1(+)-YFP-βarr1, pcDNA3.1(+)-YFP-βarr1_1-382aa, or pcDNA3.1(+)-βarr2-GFP respectively) by Lipo8000TM (Beyotime Biotechnology). The next day, the cells were reseeded onto poly-L-lysine-coated coverslips. After 24 h, the cells were incubated with drugs at specific concentrations and 500 nM Rho-2CF3 for 1~2 h. The cells were then fixed with 4% PFA in PBS for 15 min at room temperature. Afterward, the cells were washed thrice for five minutes in PBS. Thereafter, the cells’ attached coverslips were mounted on glass slides with Anti-Fade Fluorescence Mounting Medium (Abcam, ab104135) for imaging with Super Resolution Confocal-Leica Stellaris STED. For culture cell immunofluorescence in Fig. S11, the fixed cells were co-stained with DAPI (ChemCruz) for 10 min at room temperature. After three washings with PBS, we mounted them on a microscope slide for imaging. For the analysis, all detection, colocalization, and quantification were performed using ComDet v.0.5.5 plugin for ImageJ (https://github.com/UU-cellbiology/ComDet). The colocalization between β-arrestin-fused fluorescent proteins and Rho-2CF3-labeled CCKBR was assessed for statistical differences. A minimum of 30 cells was utilized in each group for analysis.

GloSensor™ cAMP accumulation assays
The 293T-CCKBR cells were cultured in DMEM (Gibco) with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin (P/S) in a humidified 37°C incubator with 5% CO2. The cells were transfected by the transfection mixture, which was diluted with Opti-MEM medium with a ratio of 2 μl Lipo8000TM (Beyotime Biotechnology) per 1 μg pGloSensor™-22F. On the day after transfection, transfected cells were transferred and incubated overnight in white, clear-bottom Poly-L-Lysine-coated 96-well microplates with approximately 40000 cells per well. On the following day, the white backing (Perkin Elmer) was sealed to the bottom of the plate before measurement. The medium was replaced by 50 μl D-luciferin drug buffer, which was made with the Equilibration medium (20 mM HEPES pH 7.5, 1x Hank’s Balanced Salt Solution (HBSS, Gibco), 0.1% w/v Bovine Serum Albumin (BSA)) with 2% GloSensor™ cAMP Reagent stock solution. Cells were incubated for 2 h at room temperature with D-luciferin buffer. After that, the tested compounds were gradient diluted in HHBS buffer (20 mM HEPES, pH 7.5, 1x HBSS) at 2x final concentration. 50 μL gradient diluted drugs were added to each well of the assay plate. The Plate was read using a CLARIOstar plate reader after 30 min. All drugs or compounds used here were dissolved in dimethyl sulfoxide (DMSO) and diluted to 10 mM stocks stored at −20°C before use.

Chemicals
YF476 (Cat. No. HY-14850) and A-71623 (Cat. No. 2411/1) were purchased from Sigma-Aldrich and Tocris Bioscience, respectively. Barbadin (Cat. No. HY-119706) and YM254890 (Cat. No. HY-111557) were purchased from MCE. CCK4, CCK8, and CCK8s were chemically synthesized by Bankpeptide Biotechnology. PNX-14 was chemically synthesized by Hefei Scier Bio-Tech Co., Ltd. MF-1, MF-2, MF-3, MF-4, MF-5, MF-6, MF-7, MF-8 (Supplementary Fig. 1), MF-9, MF-10, 5r11, MF-12, and MF-13 were synthesized by Shanghai Top-peptide Bio Co., ltd and NovoPro Bioscience Inc. Rho-2CF3 was synthesized according to our previous work62. HPLC analysis results ensured that the purity of chemicals was above 95%. Mass spectrometry further confirmed the molecular weight. We listed the chemical information in Supplementary Table. 1.

4(5)-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)−2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid (Fluo-Halo).
The route (Fig. 10) describes the synthetic scheme of Fluo-Halo. To a solution of 5(6)-Carboxyfluorescein (50 mg, 0.13 mmol) in dry DMF (5 ml) was added HBTU (65.62 mg, 0.17 mmol). After being stirred at room temperature for 20 min, the mixture was added HaloTag(O)2 amine (59 mg, 0.27 mmol) and DIEA (46 μL, 0.27 mmol). After being stirred at room temperature for 3 h, the reaction was quenched by NH4Cl (0.5 ml). The mixture was extracted by DCM (3 × 20 ml), and the combined organic phase was washed by H2O (6 × 20 ml), dried over Na2SO4, filtered and concentrated in vacuo. The solvent was evaporated and the residue was purified by silica gel flash column chromatography using dichloromethane: methanol (30:1 to 10:1) to afford Fluo-Halo (37.2 mg, 48.1% yield) as a yellow solid. 1H NMR (400 MHz, Chloroform-d) δ 8.40 (s, 1H), 8.14 (d, J = 4.0 Hz, 1H), 8.03 (s, 1H), 7.96 (s, 0.5H), 7.81 (s, 0.5H), 7.81 (s, 0.5H), 7.19 (d, J = 8.0 Hz, 0.5H), 6.62 (d, J = 4.0 Hz, 1H), 6.58 (s, 1H), 6.52 (dd, J = 16.0, 12.0 Hz, 2H), 6.44 (d, J = 8.0 Hz, 2H), 3.67–3.60 (m, 4H), 3.55-3.46 (m, 7H), 3.38 (t, J = 8.0 Hz, 1H), 1.72–1.67 (m, 2H), 1.60-1.48 (m, 2H), 1.38-1.25 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 168.24, 168.11, 164.71, 164.60, 159.68, 159.68, 151.87, 140.61, 136.19, 134.75, 129.30, 128.23, 126.52, 124.31, 123.36, 122.35, 112.77, 112.77, 109.16, 109.16, 109.11, 109.11, 102.29, 102.29, 70.24, 69.67, 69.58, 69.50, 69.37, 68.79, 68.65, 45.40, 32.05, 29.11, 26.16, 24.97. MS (ESI) (m/z): [M+H]+ calcd. for C31H33Cl2NO8+, 582.2; found, 582.2.

N-(9-(4-(N-(4-((1H-indol-3-yl)methyl)−14-amino-13-benzyl-7-butyl-10-(carboxymethyl)−2,5,8,11,14-pentaoxo-3,6,9,12-tetraazatetradecyl)sulfamoyl)−2-sulfophenyl)−6-(diethylamino)−3H-xanthen-3-ylidene)-N-ethylethanaminium (SRB-CCK5).
Peptide GW(Nle)DF-NH2 (NovoPro, 15 mg, 0.025 mmol) and Lissamine rhodamine B sulfonyl chloride (17.5 mg, 0.03 mmol) were dissolved in 2 mL DMF, then the DIEA (9 μL, 0.052 mmol) was added. The mixture was stirred at room temperature for 3 h. The product SRB-CCK5 (12 mg, 41%) was purified by HPLC (Fig. S5). MS (ESI) (m/z): [M]+ calcd. for C59H70N9O13S2+, 1176.5; found, 1176.9 (Supplementary Fig. 5).

Molecular docking and molecular dynamics simulations
The Cryo-EM structure of the CCKBR in complex with gastrin-17 (PDB ID: 7F8V)41 was obtained from the RCSB Protein Data Bank. Non-protein components were removed, and the missing intracellular loop 3 (ICL3, residues 250–325) was reconstructed using MODELER (version 10.6)63, and the model with the lowest zDOPE (normalized Discrete Optimized Protein Energy) score and without traversing the intracellular gap between TM5 and TM6 was selected for subsequent molecular docking. The protonation state of the protein was assigned by adding hydrogens in UCSF ChimeraX (version 1.9)64 at pH 7.0. MF-8 was protonated at pH 7.0 using Marvin Sketch (version 23.4.0, http://www.chemaxon.com/products/marvin/marvinsketch/), and no automers were identified. Three methods were then used to generate diverse 3D conformers. Marvin Sketch provided one low-energy conformer. Open Babel (version 3.1.1)65 and RDKit (version 2024.09.2) (https://www.rdkit.org) generated 10 conformers separately, and the lowest-energy structure from each was selected as the input conformation for docking (Supplementary Data 1). The receptor was prepared using ADFR Suite (version 1.9)66, and ligands were processed with Meeko (version 0.6.1) (https://github.com/forlilab/Meeko). Molecular docking was performed using AutoDock Vina (version 1.2.5)67, generating 20 binding poses for each input conformer. Docked poses were visually inspected and those that preserved a backbone orientation consistent with gastrin-17 and formed multiple similar conformations across docking runs were considered, the one with the highest docking score was selected for molecular dynamics (MD) simulations.

For MD simulations (Supplementary Data 2), the selected receptor-ligand complex was embedded into a heterogeneous 100×100 Å lipid bilayer composed of POPC and cholesterol in a 55:45 ratio at pH 7, with the receptor orientation determined using PPM 2.068. Protonation states of the receptor and ligand were assigned according to pH 7. The system was solvated with 22.5 Å of water on both sides of the membrane, neutralized with 0.15 M KCl, and assembled using Amber force fields (FF19SB69 for protein, Lipid 2170 for lipid, GAFF271 for ligand parameters, and OPC model72 for water). The steps were carried out using CHARMM-GUI (version 3.7)73. Simulations were performed in triplicate using GROMACS (version 2023.3)74, including energy minimization, equilibration under the NPT ensemble at 303.15 K and 1 atm, and a 200 ns production run.

Binding interactions, including hydrogen bonds, salt bridges, and hydrophobic interactions, were tracked using GROMACS (version 2023.3), PLIP (version 2.4.0)75, and ProLIF (version 2.0.3)76. The key binding pose was visualized in ChimeraX.

Local virus injection in the AC
Animals were anesthetized with pentobarbital (100 mg/kg) supplemented with atropine (0.05 mg/kg, Sigma). The anesthetized mice’s skull was fixed to the stereotaxic device (RWD Life Science). The auditory cortex (AP: −2.5 to −3.0 mm posterior to the bregma, ML: 4.1 to 4.3 mm to the middle, DV: −0.600 mm from the endocranium) was labeled. Then, craniotomy was performed on the labeled site. Adeno-associated viral type 9 (AAV9) vector expressing mouse CCKBR (rAAV-CaMkIIa-mCCKBR-P2A-EGFP-WPRE-hGH, 5.0E+12 vg/ml, BrainVTA) or human CCKBR (rAAV-hsyn-hCCKBR-P2A-EGFP, 5.00E+12 μg/mL, BrainVTA) or human CCKBR with the 353 site mutation fused with an enhanced GFP (rAAV-hsyn-hCCKBR(Asn353AIa) -P2A-EGFP, 5.00E+12 μg/mL, BrainVTA) or AAV2/9-mCaMKIIa-MasterRNAi30e(NC)-eGFP-WPER (Taitool, Shanghai, China, 2E+12 VG/ml, Cat. No. S1273-9, targeting sequence for: 5’-GCTGAGTACTTCGAAATGTCA-3’) or AAV2/9-mCaMKIIa-MasterRNAi30e(mArrb1) (Taitool, Shanghai, China, 2E+12 VG/ml, targeting sequence for mouse β-arrestin1: 5’-TCCTGGTGGATCCTGAGTATC-3’)-eGFP-WPER or AAV2/9-mCaMKIIa-MasterRNAi30e -(mArrb2)#1-eGFP-WPER (Taitool, Shanghai, China, 2E+12 VG/ml, targeting sequence for mouse β-arrestin2: 5’-GGCTTGTGGAGTAGACTTTGA-3’) or AAV2/9-mCaMKIIa -MasterRNAi30e(mArrb2)#2-eGFP-WPER (Taitool, Shanghai, China, 2E+12 VG/ml, targeting sequence for mouse β-arrestin2: 5’-GAGGAACTCTGTGCGGCTTAT-3’) or rAAV-CaMKIIa -hM3D(Gq)-EGFP-WPRE (Taitool, Shanghai, China, 1.79E+13 VG/ml, Cat. No. S0140-9-H20) or AAV2/9-mCaMKIIa-rM3D(Gs)-TS-EGFP-ER2-WPRE (Taitool, Shanghai, China, 1E+13 VG/ml, Cat. No. S0571-9) or AAV2/9-mCaMKIIa-EGFP-ER2-WPRE (Taitool, Shanghai, China, 1E+13 VG/ml, Cat. No. S0241-9) was injected into the two sides of AC of CCK-BR-KO mice. To knockdown β-arrestin2, #1 and #2 viruses were mixed and injected at a 1:1 ratio. rAAV-hsyn-EGFP (5.0E+12 vg/ml, BrainVTA) was injected into the AC of CCK-BR-KO mice as a negative control. The diluted AAV vectors with aCSF were injected into 4 selected locations at a speed of 10 nL/min (Nanoliter Injector, Shanghai-GAOGE).

Multielectrode array (MEA) assays
Fresh aCSF was prepared in advance from 119 mM NaCl, 2.5 mM KCl, 1.25 mM NaHPO4, 24 mM NaHCO3, 12.5 mM glucose, 2 mM CaCl2·4H2O, and 2 mM MgSO4·7H2O, ~25°C). The mice were euthanized with gaseous isoflurane, after which their brain was extracted and immersed in 95% O2 and 5% CO2 bubbled cold aCSF for approximately 1 min. 300 μm coronal brain slices with auditory cortex were obtained using a vibratome (Leica VT1000S) and were incubated in a 28°C, 95% O2 and 5% CO2 bubbled aCSF for at least 1.5 h.

The target area of the incubated slice was placed on a probe (MED-PG515A), and an anchor (Warner Instruments, Harvard) overlayed the slice to stabilize it. We adjusted the position of the slice through the camera (DP70, Olympus) connected to an inverted microscope until the region of interest could be recorded. The fresh gas bubbled aCSF was perfused into the probe, and the temperature was kept at 32-34°C throughout the recording period. All the recordings were carried out using the MED64 system (MED, Panasonic Alpha-Med Sciences). Firstly, the I/O curve was recorded to obtain the recording voltage (the voltage value that can induce 30% of maximal response) and stimulation voltage (the voltage value that can induce 75% of maximal response). Secondly, we recorded the basal signal of synaptic response with recording voltage until more than 30 min of stable signal was obtained. Afterward, a group of specific high/low-frequency stimulation with/without drug treatments was applied before re-recording signals under recording voltage for more than an hour. Mean fEPSP amplitude/slope before and after the stimulation were analyzed using two-way ANOVA. For comparison of the LTP magnitude between different treatments, the averaged value of the last 20 min of recordings was compared statistically. The data was exported into an Excel sheet and processed with Excel and GraphPad Prism.

Cue-to-cue fear memory
This method is similar to the one we reported previously17. A brief description is given below. Two tones were selected at a frequency of 1 kHz (f1) and 4 kHz (f2) with a duration of 3 s. The tones were set at 70 dB SPL, which could evoke neuronal responses in the auditory cortex. During the first three days of training (days 1–3), f1 and f2 were presented in pairs with 0.5 s interval to C57 mice for 25 trials in each session. We conducted 4 sessions of training for each day, with 5 min interval between each session. On day 4, a baseline test was performed to calculate the percentage of freezing over a period of 10 s after the f1 and f2 tones were presented separately. The f2 tone was then conditioned for 3 trials with a footshock. A delayed conditioning was adopted, in which the f2 tone was presented firstly and the footshock and f2 tone were terminated simultaneously. On day 5, we performed a post-conditioning test to f1 and f2 tone, and the percentage of freezing was calculated. MF-8 was intraperitoneally injected at 100 μg/kg on the day before each training session, and the same volume of saline was injected into the control group mice. Tones were digitally generated using a Tucker-Davis Technologies (TDT, Alachua, FL) workstation and delivered by a coupled electrostatic speaker. The electrical stimuli of the footshock were generated using a TDT workstation and delivered by an isolator (Isoflex, AMPI, Israel). The current of the electrical stimuli of the footshock was set at 50~200 μA with a duration of 0.5 ms. The freezing percentage of freezing was analyzed with Smart 3.0 video-tracking software.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript describes a CCKB receptor agonist ligand (MF-8) with a strong bias for beta-arrestin-dependent internalization compared to the activation of G protein-dependent signaling pathways. The authors use the compound to study the role of CCKB receptor-dependent long-term potentiation in electrophysiological experiments on brain slices. Please note that my comments are mainly for the BRET- and FRET-based experiments and the other GPCR signaling assays.\nMajor issues:\n- The FRET ligand-binding assay (Figure 1E,I, p.4) is unlikely to work as described. The Forster radius of the GFP-rhodamine FRET pair is 5-6 nanometers, far too short to reliably measure energy transfer from a fluorescently labeled ligand bound to the extracellular side of the receptor to a GFP fusion protein on the cytoplasmic side of the receptor. It is hard to understand why you did not use the N-terminal HaloTag-fusion with an appropriate donor fluorophore to measure ligand binding.\n- The use of the Gq/11 signaling pathway blockers U-73122 and 2-APB is not state-of-the-art to demonstrate the involvement of Gq/11 signaling. You should consider using the Gq/11 inhibitors YM-254890 of FR300569, which are highly specific to make your point.\n- The presentation of the results from the TRUPATH experiments in Figure 4 is very confusing. The radar plot shows Galpha13, Galphas, and Galphaq/11, but the dose-response curves show Galpha12 and Galpha13. Which data did you use for beta-arrestin, Galphaq/11, and Galphas in this plot?\n- What do you mean by \"MF-8 induces CCKBR internalization with high potency.\" (Figure S5)? The single-concentration internalization experiments do not allow us to conclude about the potency.\nMinor comments:\n- There is a typo \"th11e\" in line 229, p.11.\n- The description of the plasmid constructs (p.15) lacks the necessary details to infer their exact sequence. Consider providing the sequences or details of the final constructs, including primer sequences used to generate them.\n- What do you mean by \"would\" in the sentence line 373, p.15? Do you perform a second infection or not? In line 381, p.16, you write \"The second infection was performed...\"\n- What do you mean by \"calcium imaging assay\" in line 396, p.16? Do you \"image\" the wells or the plate? Or do you measure the total fluorescence for each well?", "reviewer_2": "The manuscript presents an analysis of β-arrestin biased agonism at the cholecystokinin B receptor (CCKBR) using CCK4-derived compounds. As β-arrestin bias has not been previously observed at the CCKBR, the manuscript contains significant novel findings and is therefore likely to be of interest to the Nature Communications readership. While the experimental approaches are comprehensive, the criteria for selection/derivatization of the ligands tested require justification. The authors present compelling evidence for their lead compound MF-8 as a β-arrestin-biased CCKBR agonist. It is initially identified by comparing calcium mobilization for G protein signaling and PRESTO-Tango assays for ß-arrestin signaling. In addition, the compound's binding properties are well validated through FRET-based studies, and its biased activity is demonstrated via BRET assays comparing CCKBR signaling pathways of MF-8 to the endogenous agonist CCK8 as a reference. Regarding selectivity, the authors have appropriately characterized MF-8's specificity for CCKBR versus CCKAR using both β-arrestin recruitment and calcium influx assays. However, the selectivity assessment against GPR173 is limited to calcium mobilization only. Given MF-8's proposed β-arrestin-bias, it would be valuable to include β-arrestin recruitment data for GPR173 to provide a more complete selectivity profile.\nThe authors provide convincing evidence that CCKBR-mediated long-term potentiation (LTP) exclusively relies on G protein signaling, with no apparent contribution from β-arrestin signaling pathways, which also represents a novel finding. This conclusion is supported by their comparison of low-frequency electrical stimulation-induced LTP in the presence and absence of their previously established β-arrestin biased agonist MF-8. Additionally, their mechanistic studies effectively demonstrate that MF-8 reduces G protein signaling through β-arrestin-dependent receptor internalization. This finding is well supported by several experimental approaches, including cellular imaging, calcium mobilization measurements, and cAMP accumulation assays.\nWhile the authors employ computational approaches to analyze the molecular determinants of MF-8-protein interactions, revealing numerous potential hydrogen bond interactions within the binding site, a definitive binding mode for MF-8 remains to be elucidated. The authors establish a mechanistic analysis of ß-arrestin bias at the CCKBR through mutational studies, which successfully identify Asn383 as a critical residue for β-arrestin recruitment through MF-8. The computational part is methodologically weaker than the experimental part and needs more substantial revision. Additional structural studies would be valuable to fully reveal the binding mechanism of MF-8 and thus also structural determinants for ß-arrestin biased signaling at the CCKBR.\nOverall, the manuscript provides valuable insights into the signaling pathways exhibited by CCKBR and the potential of the ß-arrestin-biased ligand MF-8. While the data presented is compelling, the manuscript would benefit from a revision to improve clarity and flow of writing.\nWe would recommend acceptance of the article, subject to the points below being addressed:\nMajor Points:\n1.) The manuscript needs more precise pharmacological terminology. The authors should provide clear definitions for the terms \"agonist\" and \"antagonist,\" as these appear to be used interchangeably for the same compounds throughout the text. In addition, the term \"biased antagonist\" needs to be either properly defined or removed to improve clarity. If the authors are unsure about the terminology, they should refer to, for example, https://doi.org/10.1111/bph.15811 and https://doi-org/10.1016/j.tips.2004.02.012.\nWe suggest that compound MF-8, which preferentially activates the β-arrestin pathway, should be classified as a β-arrestin-biased agonist. While the observed reduction in G protein signaling may result from β-arrestin-mediated receptor internalization, this represents a downstream consequence rather than direct antagonism of the G protein signaling pathway.\n2.) It is unclear why the compounds tested were selected for this study. They are certainly derivatives of CCK8, but how did the authors decide on which amino acids and which moieties to change? Was this done computationally? If so, more details should be included, if not, the rationale for the synthesis of these specific compounds should be given. Were previous studies performed that identified these compounds? If not, were they synthesized based on a binding hypothesis, or were they selected in an attempt to repurpose them? Understanding the reasoning behind the selection of the compound series is crucial for assessing the novelty of the presented ligands. Therefore, a detailed rationale for the selection of compounds tested would strengthen the story.\n3.) The computational aspects of this study require substantial revision. The docking analysis lacks a clear binding hypothesis, and the selection criteria for residues chosen for mutational studies appear to be based on general hydrogen bonds within the binding site, rather than specific docking predictions. Scoring-based selection using AUTODOCK4 is problematic and not state-of-the-art (see https://doi.org/10.1021/jm050362n and citing articles). The methodology would be strengthened by including essential details about ligand preparation, including protonation states, tautomerization, and initial conformer generation. The pose generation could also benefit from the generation of more binding modes, as the ligand is rather large and flexible, as it is derived from a tetrapeptide. Furthermore, the criteria and methods for hydrogen bond analysis should be described in full detail.\n4.) The determination of the selectivity of MF-8 for CCKBR would be enhanced by a ß-arrestin signaling assay of MF-8 on GPR173.\nMinor Points:\n1.) It would enhance clarity to include Ballesteros-Weinstein residue annotations when referencing amino acid positions.\n2.) Page 3: The section on screening for ligand bias would benefit from a table containing the screening results of all ligands, these should be moved from the SI to the main manuscript.\n3.) Chemical representations of the compounds could be of use in the SI.\n4.) The manuscript should be proof-read by a native English speaker and grammar-checked by a language tool.\nOur expertise lies in computational methodologies and the evaluation of overall pharmacological findings of biased GPCR ligands. Therefore, we can thoroughly evaluate the molecular modeling approaches and the pharmacological findings regarding biased agonism at CCKBR. However, the specific execution of wet-lab experiments, including the precise protocols for BRET, FRET, PRESTO-Tango and electrophysiology studies, falls outside our expertise and we cannot provide a detailed assessment of the methodological/technical execution of these assays. We do, however, understand the methodology and are able to interpret the results.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "In the manuscript entitled “Identification and characterization of a biased CCKBR agonist that blocksCCKBR-dependent long-term potentiation in a β-arrestin dependent manner”, Shi etal. screened out a novel β-arrestin biased CCKBR agonist, MF8.The authors showed that MF8 both activates CCKBR signaling and antagonizes the CCKBR-LTP in a β-arrestin-biasdmanner, and further illustrated the ligand binding sites of MF8 and CCKBR, with explaination of the mechanism.Most of the experiments were performed in vitro using HEK cells or ex vivo using mice brain slices, with minimal in vivo studies were performed. To confirm the β-arrestin bias is real ligand bias but not system bias, in vivo physiological contextusing tissue-specific β-arrestinknockout mice is necessary.Meanwhile, as the author mentioned in the introduction, that LTP is essential for the memory, emotion, and movement, detecting the neuron physiological phenotype in vivo with MF8 treated could enhance the translational potential of this study.\nOverall, this study illustrated the discovery of a novelβ-arrestin-biased CCKBR agonist which could be potentially serving as a drug target for the treatment of neurological diseases.This study could benefits general readers from the fields of GPCR regulation, drug design, and neurological disorder treatment. The data were solid, with the methods used in this study to besound. After solve the following major questions, this work could be accepted for publication.\nMajor：\n1. Page 4 line 120, the subtitle “The CCKBR-LTP in the AC is not determined by β-arrestin signaling”is confusing, that what “determined” means should be clarified here.\n2. Page 5 line 130, the author mentioned “Considering that MF-8 preferentially activates the β-arrestin signaling of CCKBR, the aboveresults demonstrate that the CCKBR-LTP in the AC is not determined by the β-arrestin signaling”, which lacks sufficent evidence to exclude other possible pathways leading to the same phenotype. Therefore, a tissue-specific β-arrestin knock-out is necessary here to confirm the selectivity.\n3. Page 6 line 138, why TBS is used here to induce the LTP in AC, instead of using LFS (Page 5, line 124)? The chosen of the stimulation should be explained in the maintext.\n4. Fig 3, to identify which signaling pathway is responsible for CCKBR-LTP, the authors used a series of signaling pathway blockers targeting CCKBR, Gαq/11, Gαs, and Gαi. But why blocker targeting β-arrestin was not detected here, as the author mentioned that “Previous reports indicated that CCKBR could activate a series of Gαi, Gαs, Gαq/11, and β-arrestinsignaling pathways”?The β-arrestin blocker data should also be added in this part.\n5. Fig 4, using BRET assay or cAMP assay, the author compared the agonist efficiency bewteen MF-8 and the balanced agonist control CCK8s in different CCKBR downstream signaling in a dose-dependent manner, and found MF-8 did not active downstream signaling to the same extent of CCK8, then drawed the conclusion that “MF-8 is a potent CCKBR agonistwith a strong bias for the β-arrestin signaling”. However, the activation of β-arrestin signaling was not shown in the same Figure panel, which will make it hard to understand where the data supporting for the β-arrestin bias.The β-arrestin bias may be detected in Fig 1C by PRESTO-Tango assay, but the a BRET assay should also be used to decect β-arrestin signaling in Fig 4, which would make all the data comparable.\n6. Fig 5, the activation of CCKAR stimulated by CCK8 and MF-8 should be combined into the same concentration-response curve (Fig 5A), Calcium (Fig 5B), and GPR173 (Fig 5C), respectively.\n7. For all the statistical analysis, bar charts with dots are recommend to illustrate the distribution of each sample. (Bar charts in Figure 6, and 7)\n8. Fig 6A2, the size and shape of the cell in MF-8+Rho-2CF3 group is significantly different from the other two groups. Was this cell getting apoptisis in this condition? A better representative image with more cells in a single view should be presented. And co-labeling of nucleus is recommended.\n9. Fig 7A and B, empty vector transfected 293T cells should be added as negative control along with 293T-CCKBR cells.\n10. Page 11, line 207, the author mentioned “we hypothesized that MF-8 could antagonize Gαq/11 andGαs-mediated CCKBR-LTP in the AC.”, but they did not validate the necessary roles of Gαq/11 andGαs in the AC. Accordingly, a rescue study using AAV-mediated Gαq/11 andGαs ex vivo overexpression could help to fulfil this gap.\n11. Fig 8, as the author wants to validate theβ-arrestin dependency in the MF-8 antagonizing CCKBR-mediated LTP, a β-arrestin tissue-specific knockout or knockdown in vivo study should be performed. The validation of the global mechnism in both CCKBR KO and β-arrestin KO mice could enhance the central discovery of this study.\n12. Given CCKBR-mediated LTP is essential for the memory, emotion, and movement, a preclinical study observing the neuron physiological phenotype in vivo with MF8 treatment couldfurther increase the translational value of this study.\nMinor:\n1. Fig 1F, the proper control for CCKBR-EGFP should be the same virus vector harboring EGFP.\n2. Page 3, line 114. Please add necessary backround for the use of YF476 as a control in the FRET assay.\n3. Fig 2A, please add brief description of the pipeline for the LTP assay in the maintext.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear reviewers,\nOn behalf of all authors involved in this research, I would like to express my profound gratitude to\nall the five reviewers for your meticulous review of this article. Your keen observations have\nhighlighted several areas in need of improvement. Guided by your thoughtful recommendations, we\nhave undertaken a comprehensive revision of our initial manuscript. Below, we detail the extensive\namendments that we have implemented.\nReviewer #1\nMajor issues:\n- The FRET ligand-binding assay (Figure 1E,I, p.4) is unlikely to work as described. The Forster\nradius of the GFP-rhodamine FRET pair is 5-6 nanometers, far too short to reliably measure energy\ntransfer from a fluorescently labeled ligand bound to the extracellular side of the receptor to a GFP\nfusion protein on the cytoplasmic side of the receptor. It is hard to understand why you did not use\nthe N-terminal HaloTag-fusion with an appropriate donor fluorophore to measure ligand binding.\nResponse: When we developed the previous binding system, we were worried that the HaloTag at\nthe N-terminal of CCKBR would affect the expression efficiency of the receptor on the cell\nmembrane. According to your suggestion, we resynthesized fluorescein-labeled chloroalkane linker\n(Fluo-Halo) (Fig. S1C) as a donor to label the N-terminus HaloTag-labeled CCKBR and used SRB-\nCCK5 (Fig. S1B) as an acceptor. We got better results than the previous binding system (Fig. 1D,\n1E). Therefore, we replaced the previous results with the results of the new binding system. The\ncorresponding results were shown in Fig. 1D, Fig. 1E, and Fig. S1C. We have also included the\ncorresponding methods for synthesizing Fluo-Halo and testing new binding systems.\nFig.1D Fig.1E\n- The use of the Gq/11 signaling pathway blockers U-73122 and 2-APB is not state-of-the-art to\ndemonstrate the involvement of Gq/11 signaling. You should consider using the Gq/11 inhibitors\nYM-254890 or FR300569, which are highly specific to make your point.\nResponse: Here we used YM-254890 as a Gq/11 blocker for in vitro electrophysiological recordings.\nWe found that this blocker also significantly inhibited TBS-induced CCKBR-dependent LTP in the\nauditory cortex. The results have been added to Fig. 3E and Fig. 3I.\nFig.3E Fig.3I\n1\n- The presentation of the results from the TRUPATH experiments in Figure 4 is very confusing. The\nradar plot shows Galpha13, Galphas, and Galphaq/11, but the dose-response curves show Galpha12\nand Galpha13. Which data did you use for beta-arrestin, Galphaq/11, and Galphas in this plot?\nResponse: Previously, we used the β-arrestin results in Fig. 1, the calcium results in Fig. 1, and the\ncAMP results in Fig. 4 to calculate the relative activities of MF-8 against β-arrestin, Gαq/11, and\nGαs in the radar plot, respectively.\non the BRET experiments. These results have been added to Fig. 4C, 4D, and 4E. The new radar\nplot in Fig. 4F was calculated based on the BRET results of Fig. 4A-E, and it suggests that MF-8 is\na highly β-arrestin biased CCKBR agonist.\nMF-8 bias factor shown in Fig. S6B was calculated based on the results of cAMP (Fig. S6A),\ncalcium (Fig. 1B), and β-arrestin recruitment (PRESTO-Tango, Fig. 1C), and this result also\nindicates that MF-8 is biased towards β-arrestin signaling.\n- What do you mean by \"MF-8 induces CCKBR internalization with high potency.\" (Figure S5)?\nThe single-concentration internalization experiments do not allow us to conclude about the potency.\nResponse: We previously used such statements because the low concentration (100nM) of MF-8\ncan induce CCKBR internalization at a level similar to that of 100 nM CCK8s. As you said, it is not\nvery rigorous to say that “high potency” with a single concentration as potency determination\nrequires a concentration-dependent response analysis. Here we amended it to “MF-8 is able to\ninduce CCKBR internalization at a low concentration”.\nMinor comments:\n- There is a typo \"th11e\" in line 229, p.11.\nResponse: We have revised “th11e” to “the” in the paper.\n- The description of the plasmid constructs (p.15) lacks the necessary details to infer their exact\nsequence. Consider providing the sequences or details of the final constructs, including primer\nsequences used to generate them.\nResponse: The final sequence information inserted into the multiple cloning sites were\nsupplemented in Fig. S14 to Fig. S21.\n- What do you mean by \"would\" in the sentence line 373, p.15? Do you perform a second infection\nor not? In line 381, p.16, you write \"The second infection was performed...\"\nResponse: Sorry for that the word “would” was misused here. We did perform a second infection to\nenhance the receptor expression. The sentence “The second infection would be performed at the\ninterval of 24 h, and the virus supernatant mix would be replaced by the fresh normal medium of\nHEK293T after 6-8 h of each time infection” has been revised to “The second infection was\nperformed at the interval of 24 h, and the virus supernatant mix was replaced by the fresh normal\nmedium of HEK293T after 6-8 h of each time infection”.\n- What do you mean by \"calcium imaging assay\" in line 396, p.16? Do you \"image\" the wells or the\nplate? Or do you measure the total fluorescence for each well?\nResponse: Actually, the calcium mobilization assay is more accurate here. We have revised “calcium\nimaging assay” to “calcium mobilization assay”. In fact, the calcium mobilization assay of CHO-\nGPR173 cells is the same as that of other cells, and all calcium mobilization assays were performed\nwith a Microplate Reader to read the data well by well.\nMajor Points:\n1.) The manuscript needs more precise pharmacological terminology. The authors should provide\nclear definitions for the terms \"agonist\" and \"antagonist,\" as these appear to be used interchangeably\nfor the same compounds throughout the text. In addition, the term \"biased antagonist\" needs to be\neither properly defined or removed to improve clarity. If the authors are unsure about the\nterminology, they should refer to, for example, https://doi.org/10.1111/bph.15811 and https://doi-\norg/10.1016/j.tips.2004.02.012.\nWe suggest that compound MF-8, which preferentially activates the β-arrestin pathway, should be\nclassified as a β-arrestin-biased agonist. While the observed reduction in G protein signaling may\nresult from β-arrestin-mediated receptor internalization, this represents a downstream consequence\nrather than direct antagonism of the G protein signaling pathway.\nResponse: We strongly agree with your suggestion. MF-8 should be defined as a β-arrestin-biased\nagonist. We have removed the description of MF-8 as an antagonist, and also made additional\ncorrections to other misleading descriptions, as detailed below.\nThe sentence in the abstract “Both these two signaling pathways induced by CCKBR activation\nwere suppressed by MF-8 with high potency, suggesting that this agonist could be an antagonist for\nthe CCKBR-LTP” has been changed to “Both these two signaling pathways induced by CCKBR\nactivation were suppressed by MF-8 with high potency, suggesting that this agonist could suppress\nthe CCKBR-LTP”.\nThe sentence in the abstract “These results revealed the signal pathway preference of the CCKBR-\nLTP in the AC and identified a novel antagonist of the LTP” has been changed to “These results\nrevealed the signal pathway preference of the CCKBR-LTP in the AC and identified a novel blocker\nof the LTP”.\nThe sentence in the abstract “The concept that β-arrestin-biased agonists could function as\nantagonists for other signaling pathways might be extended to the development of other GPCR\ndrugs” has been changed to “The concept that β-arrestin-biased agonists could function as blockers\nfor other signaling pathways might be extended to developing other GPCR drugs”.\nThe sentence in the last paragraph of the introduction “This biased agonist could antagonize other\nCCKBR signaling pathways” has been changed to “This biased agonist could reduce other CCKBR\nsignaling pathways”.\n……\n2.) It is unclear why the compounds tested were selected for this study. They are certainly derivatives\nof CCK8, but how did the authors decide on which amino acids and which moieties to change? Was\nthis done computationally? If so, more details should be included, if not, the rationale for the\nsynthesis of these specific compounds should be given. Were previous studies performed that\nidentified these compounds? If not, were they synthesized based on a binding hypothesis, or were\nthey selected in an attempt to repurpose them? Understanding the reasoning behind the selection of\nthe compound series is crucial for assessing the novelty of the presented ligands. Therefore, a\ndetailed rationale for the selection of compounds tested would strengthen the story.\nResponse: These peptides were not designed computationally. There is currently no good way for\n4\nus to achieve computer-based peptide design, especially for biased peptide agonists, as peptide\nmolecules are so flexible. On the other hand, we did not find any reports on the biased agonist\nbinding mode of CCKBR, so it is difficult to predict which parts of CCK8s and CCK8ns determine\nthe signaling pathway bias of CCKBR. However, we found from previously reported literatures that\nCCK4 should be the shortest CCK fragment that activates CCKBR1. CCK4 contains 4 amino acids\nat the carboxyl terminus of CCK8s and CCK8ns. Our current results also support that CCK4 is the\nsmallest fragment that can activate CCKBR, because several peptides containing only the three\namino acids at the amino terminus of CCK4, including MF-2 and MF-4, can only weakly activate\ncalcium and β-arrestin signals of CCKBR (Table. 1 & S1 ). Instead, CCK4 can fully activate\nCCKBR β-arrestin and calcium signal with the potency similar to CCK8ns and CCK8s (Table. 1 &\nS1). In fact, in our previous studies we have systematically synthesized more than two hundred\npeptides based on CCK4 to obtain CCKBR agonists with higher stability and potency2. However,\nwe only performed calcium mobilization assay for cell-based screening in our previous work. When\nconducting calcium mobilization assays, we found a very interesting phenomenon. Although the\ntwo compounds HT-178 and MF-12 (The structures are shown below) only differ by one methyl\ngroup, their activation ability to CCKBR in the calcium response is very different. HT-178 showed\na very strong activation ability to CCKBR with an EC50 below nM (See the figure shown as below),\nwhile the EC50 of MF-12 was ~1000 of times higher than that of HT-178 (Table. 1). Therefore, we\nspeculated whether such a small change in molecular structure might lead to a change in the bias of\nthe CCKBR downstream signaling pathway rather than a change in binding ability. For this reason,\nwe tested the peptides in our hands listed in the Table. 1 & S1, except for MF-8. After comparing\ncalcium response and β-arrestin signals, we were surprised to find the MF-7, MF-12, and MF-13 all\nhad strong biased towards the β-arrestin signaling pathway. And they all have one thing in common,\nthey are all modified at the same position as indicated in the red box of MF-12 below. So we\nsynthesized MF-8 with glutamate modification at the same site, and we also found that this\ncompound had a high β-arrestin bias, and the bias was even higher than MF-7, MF-12, and MF-13.\nWhereafter, we used MF-8 for the subsequent tests.\nI am very sorry that I did not state these contents in the previous manuscript, because the HT-178\ninvolves some future commercial interests. I hope that you will understand that we do not include\nthis part in the manuscript.\nMF-12\n5\n3.) The computational aspects of this study require substantial revision. The docking analysis lacks\na clear binding hypothesis, and the selection criteria for residues chosen for mutational studies\nappear to be based on general hydrogen bonds within the binding site, rather than specific docking\npredictions. Scoring-based selection using AUTODOCK4 is problematic and not state-of-the-art\n(see https://doi.org/10.1021/jm050362n and citing articles). The methodology would be\nstrengthened by including essential details about ligand preparation, including protonation states,\ntautomerization, and initial conformer generation. The pose generation could also benefit from the\ngeneration of more binding modes, as the ligand is rather large and flexible, as it is derived from a\ntetrapeptide. Furthermore, the criteria and methods for hydrogen bond analysis should be described\nin full detail.\nResponse: We previously attempted to find a site in CCKBR that has a significant effect on MF-8-\nmediated β-arrestin signaling but little effect on CCK8s-mediated calcium signaling. In this way,\nwe could overexpress CCKBR with the mutation in CCKBR knockout mice to prove that MF-8\nblocks CCKBR-LTP in a β-arrestin dependent manner. Therefore, we did not focus on the specific\nbinding mode before.\nYour opinions and suggestions are very important and can enrich our story. The article you\nrecommended also provided a lot of guidance for our molecular simulation. We have already cited\nthis article in our paper. Here, we used autodock vina to perform molecular docking of CCKBR and\nMF-8, and then we selected the docking results based on the conformation and orientation of gastrin-\n17 in the Cyro-EM gastrin-17-CCKBR complex3. Afterwards, molecular dynamics simulations with\nGROMACS were used to evaluate the binding mode of CCKBR and MF-8. By analyzing the\nformation of the complex structure after the stabilization of the simulation, we determined hydrogen\nbonds and hydrophobic interactions formed by MF-8 and CCKBR. Point mutations in CCKBR\ncombined with a BRET-based β-arrestin assay and PRESTO-Tango assay were applied to verify\nhydrogen bond interaction of MF-8 and CCKBR. As shown in Fig. 8A, multiple sites of CCKBR,\nincluding V1383.36, M1343.32, V1303.28, V3496.51, L2225.42, P1142.64, H3647.27, Y3807.43, C205ECL2,\nI3727.35, A3636.55, V206ECL2, A3526.54, could form hydrophobic interactions with MF-8. Among\nthem, V1383.36, M1343.32, V3496.51, L2225.42, H3647.27, Y3807.43, I3727.35 were reported to form\nhydrophobic interactions with the CCK4 part of gastrin-17. The remaining hydrophobic interactions\nwere not reported in gastrin17-CCKBR. The formation of more hydrophobic interactions in\nCCKBR-MF-8 may explain why MF-8 has better potency than CCK4 in the PRESTO-Tango assay.\nIn addition, CCKBR could form stable hydrogen bond interactions with MF-8 through residues\nT1112.61, H3767.39, N1152.65, Q204ECL2, H207ECL2, Y1894.60, R3566.58, N3536.55, which is similar to\nthe bonds formed by gastrin-17-CCKBR3. Among them, Q204ECL2 and N1152.65 could not form\nstable hydrogen bond interactions with the CCK4 part of gastrin-173, which may explain why MF-\n8 can act as a potent agonist of CCKBR.\nConsidering the drastic effect of N3536.55 on MF-8-mediated β-arrestin signaling (Fig. 8B, S12) and\nthe weak effect of this site on the Gq signaling pathway3, we speculated that mutation of this site\nwould not affect TBS-induced CCKBR-LTP, but would eliminate the blockade of MF-8 on the TBS-\ninduced CCKBR-LTP since the LTP mainly depends on the CCKBR-Gαq/11-mediated signaling\npathway. Therefore, we overexpressed the CCKBR receptor with mutation at N3536.55 in the\nauditory cortex of CCKBR-KO mice to verify our hypothesis.\nThe detailed molecular docking and dynamics simulation process has been added to the methods\nsection. The results of this section have also been added to the paper. We look forward to your\n6\ncomments on this part and we will continue to optimize our calculation results based on your\nopinions.\nFig. 8A Fig. 8B\n4.) The determination of the selectivity of MF-8 for CCKBR would be enhanced by a β-arrestin\nsignaling assay of MF-8 on GPR173.\nResponse: We here used BRET-based β-arrestin assays to detect the activation of MF-8 on the β-\narrestin1 and β-arrestin2 signals downstream of GPR173. PNX is a previously reported ligand for\nGPR1734. We used CCK8s and PNX-14 as positive controls. Although both CCK8s and PNX-14\ncan activate β-arrestin1 and β-arrestin2 signals downstream of GPR173, we did not detect changes\nin these two signals with increasing MF-8 concentrations (Fig.5D1&D2). In contrast, the CCKBR\ncan respond to MF-8 in these two β-arrestin assays in a concentration-dependent manner (Fig. 4E1\n& E2). These results indicate that MF-8 cannot activate the β-arrestin1 and β-arrestin2 signals\ndownstream of GPR173. The experimental results are shown in Fig 5D1&D2 and Fig 4E1&E2, and\nthe method part is also included in the paper.\n1.) It would enhance clarity to include Ballesteros-Weinstein residue annotations when referencing\namino acid positions.\nResponse: We have marked the annotations in the upper right corner of the corresponding amino\nacid in the paper.\n2.) Page 3: The section on screening for ligand bias would benefit from a table containing the\nscreening results of all ligands, these should be moved from the SI to the main manuscript.\nResponse: We have integrated the original Table. S2, S3, and S4 into Table1. Please see Table 1 in\nthe main manuscript for details.\n3.) Chemical representations of the compounds could be of use in the SI.\nResponse: We have moved the chemical representations of MF-8 and SRB-CCK5 to SI. Please see\nFig. S1.\n4.) The manuscript should be proof-read by a native English speaker and grammar-checked by a\nlanguage tool.\nResponse: A native English speaker, Dr. Stephen Temitayo Bello, has helped us polish the paper.\nWe have also checked the manuscript with grammar. If it is still not good enough, we will find\nacademic proofreading services to polish it in the next round.\nReviewer #4 (Remarks to the Author):\nMajor：\n1. Page 4 line 120, the subtitle “The CCKBR-LTP in the AC is not determined by β-arrestin signaling”\nis confusing, that what “determined” means should be clarified here.\nResponse: Here we changed “determined by” to “dependent on”. We also revised “determined by”\nto “dependent on” in the title of Fig. 2. In Fig. 1, we proved that MF-8 can activate β-arrestin\nsignaling of CCKBR with the EC50=4.54 nM. In Fig. 2B, we found that neither 20nM nor 200nM\nMF-8 could induce LTP like CCK8s. Therefore, we preliminarily concluded that the CCKBR-LTP\nin AC does not depend on β-arrestin signaling. Meanwhile, we performed additional experiments in\nthe past few months to further verify this conclusion. Please see the next reply for detailed results.\n8\n2. Page 5 line 130, the author mentioned “Considering that MF-8 preferentially activates the β-\narrestin signaling of CCKBR, the above results demonstrate that the CCKBR-LTP in the AC is not\ndetermined by the β-arrestin signaling”, which lacks sufficient evidence to exclude other possible\npathways leading to the same phenotype. Therefore, a tissue-specific β-arrestin knock-out is\nnecessary here to confirm the selectivity.\nResponse: We currently do not have β-arrestin knock-out mice, and if we order this mouse line, it\nwill take about one year before we can start to use the mouse in Hong Kong. Alternatively, we\napplied two other methods to confirm this conclusion.\n1). We used a β-arrestin blocker barbadin to incubate the brain slices for one hour, and then induced\nLTP with LFS together with CCK8s. We found that the slices incubated with the blocker could still\nproduce the LTP of the same magnitude as the control group (Fig. 2C). 2). We knocked down β-\narrestin1 and β-arrestin2 in the auditory cortex using shRNA. Although both forms of β-arrestin\nwere significantly down-regulated (Fig. 2D1), CCK8s and LFS still induced LTP of the same\nmagnitude in the auditory cortex of these knockdown mice as in the scramble shRNA group (Fig.\n2D2, D3). All of the above results prove that the CCKBR-LTP is not dependent on the β-arrestin\nsignaling pathway.\n3. Page 6 line 138, why TBS is used here to induce the LTP in AC, instead of using LFS (Page 5,\nline 124)? The chosen of the stimulation should be explained in the maintext.\nCCKBR-dependent LTP, while low-frequency electrical stimulation requires the addition of CCK8s\nto induce CCKBR-dependent LTP5. Considering that antagonists need to be added during in vitro\nelectrophysiological recording in Fig. 3, using LFS and CCK8s to induce the CCKBR-LTP will\nincrease the complexity of the operation. However, if high-frequency electrical stimulation is used\nfor the CCKBR-LTP induction, only blockers need to be added during the recording process, which\nwill make the recording process simpler. TBS is a type of high-frequency electrical stimulation, so\nhere we used TBS to directly induce CCKBR-dependent LTP without the addition of CCK8s.\nWe added the explanation on page 8 line502-503: As theta-burst electrical stimulation (TBS) is an\nalternative method to induce the CCKBR-LTP.\n4. Fig 3, to identify which signaling pathway is responsible for CCKBR-LTP, the authors used a\nseries of signaling pathway blockers targeting CCKBR, Gαq/11, Gαs, and Gαi. But why blocker\ntargeting β-arrestin was not detected here, as the author mentioned that “Previous reports indicated\nthat CCKBR could activate a series of Gαi, Gαs, Gαq/11, and β-arrestin signaling pathways”? The\nβ-arrestin blocker data should also be added in this part.\nResponse: That’s a valuable idea. When we were working on the data in Fig. 3, we did not find any\ncommercially available β-arrestin inhibitors, so we did not test the effects of this type of inhibitor\non the CCKBR-LTP before. Here we purchased the previously reported β-arrestin inhibitor\nbarbadin6, whose IC50 for β-arrestin1 and β-arrestin2 were 19.1 μM and 15.6 μM, respectively. We\nused a final concentration of 100 μM to test its inhibitory effect on the LTP, and we found that the\ninhibitor did not affect the TBS-induced CCKBR-LTP. We have added this part of the results to Fig.\n3F&3I, and have also added the result description to the paper.\nFig. 3F Fig. 3I\n5. Fig 4, using BRET assay or cAMP assay, the author compared the agonist efficiency between\nMF-8 and the balanced agonist control CCK8s in different CCKBR downstream signaling in a dose-\ndependent manner, and found MF-8 did not active downstream signaling to the same extent of\nCCK8, then drawed the conclusion that “MF-8 is a potent CCKBR agonist with a strong bias for\nthe β-arrestin signaling”. However, the activation of β-arrestin signaling was not shown in the same\nFigure panel, which will make it hard to understand where the data supporting for the β-arrestin bias.\nThe β-arrestin bias may be detected in Fig 1C by PRESTO-Tango assay, but a BRET assay should\nalso be used to decect β-arrestin signaling in Fig 4, which would make all the data comparable.\nResponse: That’s a very insightful suggestion. We used the BRET-based principle7 to measure the\nactivation of CCK8s and MF-8 to β-arrestin1 and β-arrestin2 downstream of CCKBR. We found\n10\nthat MF-8 has a higher potency than CCK8s in recruiting these two forms of β-arrestin (Fig. 4E).\nMeanwhile, to maintain consistent within the same figure panel, we removed the previous PRESTO-\nTango assay results and replaced it with the BRET results to creat a radar chart of the bias of MF-8\nrelative to CCK8s, and we found that MF-8 is indeed clearly biased towards β-arrestin signaling\npathways (Fig. 4F). These results are shown in Fig. 4 and described in the main text. The method of\nBRET-based β-arrestin assay is also described in the method section.\n6. Fig 5, the activation of CCKAR stimulated by CCK8 and MF-8 should be combined into the\nsame concentration-response curve (Fig 5A), Calcium (Fig 5B), and GPR173 (Fig 5C), respectively.\nResponse: According to your suggestion, we have merged previous Fig. 5A1 and Fig. 5A2 into Fig.\n5A, Fig. 5B1 and Fig. 5B2 into Fig. 5B, and Fig. 5C1 and Fig. 5C2 into Fig. 5C.\n7. For all the statistical analysis, bar charts with dots are recommended to illustrate the distribution\nof each sample. (Bar charts in Figure 6, and 7)\nResponse: Sorry for the dots missing, Fig6 and Fig7D have been changed to bar charts with dots.\n8. Fig 6A2, the size and shape of the cell in MF-8+Rho-2CF3 group is significantly different from\nthe other two groups. Was this cell getting apoptisis in this condition? A better representative image\nwith more cells in a single view should be presented. And co-labeling of nucleus is recommended.\nResponse: It is more likely that we just happened to pick a cell with this morphology. Of course,\nthis cell may indeed undergo apoptisis. Here we replaced the cell with normal morphology as an\nexample (Fig. 6A2). We also co-stained DAPI based on Fig 6A2 and displayed multiple cells in a\nsingle view in the supplementary file (Fig S10), and most cells maintain normal morphology.\n9. Fig 7A and B, empty vector transfected 293T cells should be added as negative control along with\n293T-CCKBR cells.\nResponse: We tested the responses of CCK8s and MF-8 in mock-infected 293T cells (293T-puro)\nand found that 293T-puro cells showed no response to the two compounds in either calcium\nmobilization (Fig. S11A) or cAMP (Fig. S11B) assays. We attached the corresponding results in Fig.\nS11.\n12\n10. Page 11, line 207, the author mentioned “we hypothesized that MF-8 could antagonize Gαq/11\nand Gαs-mediated CCKBR-LTP in the AC.”, but they did not validate the necessary roles of Gαq/11\nand Gαs in the AC. Accordingly, a rescue study using AAV-mediated Gαq/11 and Gαs ex vivo\noverexpression could help to fulfil this gap.\nResponse: If Gαq/11 and Gαs are expressed exogenously using AAV, we currently do not have a\ngood way to manipulate their real-time expression. Alternatively, chemogenetics is an excellent\napproach to achieve real-time control of G protein activation. Therefore, we applied CNO to activate\nGαq/11 and Gαs in real-time through hM3D(Gq)8 and rM3D(Gs)9 respectively. We found that MF-\n8 couldn’t inhibit TBS-induced LTP in the presence of CNO-induced Gαq and Gαs activation (Fig.\nS5). These results demonstrate the necessity of Gαq and Gαs in the CCKBR-LTP. We have placed\nthe corresponding results in Fig. S5, and the corresponding method section has also been added into\nthe paper.\n13\n11. Fig 8, as the author wants to validate the β-arrestin dependency in the MF-8 antagonizing\nCCKBR-mediated LTP, a β-arrestin tissue-specific knockout or knockdown in vivo study should be\nperformed. The validation of the global mechanism in both CCKBR KO and β-arrestin KO mice\ncould enhance the central discovery of this study.\nResponse: That is a fantastic idea, but we do not have β-arrestin knockout mice. It will take at least\none year from ordering to using this mouse in City University of Hong Kong. Alternatively, we\nchose two other reliable methods for further verification.\n1) We used a β-arrestin inhibitor, barbadin6, to verify that MF-8 could not inhibit TBS-induced\nCCKBR-LTP once blocking β-arrestin signaling through the inhibitor. As you see in Fig. 8F, the\ninhibitor alone didn’t affect the TBS-induced CCKBR-LTP. Although MF-8 could inhibit the LTP,\nTBS could induce the LTP normally after adding barbadin together with MF-8 (Fig. 8F).\n2) We used AAV-mediated shRNA to knock down β-arrestin1 and β-arrestin2. We found that after\nthe two β-arrestins were knocked down (Fig. 2D1), the inhibitory effect of MF-8 on the CCKBR-\nLTP was significantly reduced (Fig. 8G).\nAlthough we don’t have β-arrestin knockout mice, the above results prove that MF-8’s blockade of\nthe CCKBR-LTP depends on the β-arrestin signaling pathway. All the relevant results and methods\nwere added to the paper.\n14\n12. Given CCKBR-mediated LTP is essential for the memory, emotion, and movement, a preclinical\nstudy observing the neuron physiological phenotype in vivo with MF8 treatment could further\nincrease the translational value of this study.\nResponse: Enhanced fear memories are thought to be closely linked to anxiety, depression, and post-\ntraumatic stress disorder10-12. In our previous study, we found that the CCKBR-LTP in the auditory\ncortex can enhance cue-to-cue fear memory, while CCKBR antagonists could inhibit the formation\nof fear memory5. In this study, we found that the CCKBR-LTP in the auditory cortex is mainly\nmediated by the Gαq/11 and Gαs signaling pathways. MF-8 as a β-arrestin biased CCKBR agonist\ncan inhibit the CCKBR-LTP. Therefore, MF-8 may also be able to inhibit the CCKBR-LTP mediated\ncue-to-cue fear memory. Here we used the previous behavioral paradigm and found that MF-8 can\nindeed inhibit cue-to-cue fear memory (Fig 9), which highlights the translational value of MF-8 in\nfear memory-related disorders. All the relevant results and methods were added to the paper.\nMinor:\n1. Fig 1F, the proper control for CCKBR-EGFP should be the same virus vector harboring EGFP.\nResponse: According to your suggestion, here we added 293T-EGFP cells that use the same viral\nvector but only contain EGFP as a control. Although most cells express EGFP, these EGFPs are\nexpressed inside the cells, not on the cell membrane. In 293T-CCKBR-EGFP cells, we can clearly\nsee that EGFP is expressed in large quantities on the cell membrane. Since one of the reviewers\nsuggested that we established another binding system, and we also obtained better data with the new\nbinding system (Fig. 1D, 1E, S1A, S1C), we removed the previous binding data from the article.\n2. Page 3, line 114. Please add necessary background for the use of YF476 as a control in the FRET\nassay.\nResponse: YF476 is a selective and potent CCKBR antagonist with a Ki value of 0.19nM for human\nCCKBR13. Therefore, we selected YF476 as a positive control in the previous binding system. Since\nwe removed the previous binding results, the background knowledge of YF476 was not added here.\n3. Fig 2A, please add brief description of the pipeline for the LTP assay in the maintext.\nResponse: Coronal slices (300 μm) containing AC were recovered in aCSF at 28°C for 1.5 hours,\nthen positioned in a MED64 probe with continuous aCSF perfusion. Minutely fEPSPs were\nrecorded at 30% maximal response until stable, then after 10 minutes of drug-treated aCSF perfusion,\nLFS was applied at 1 Hz for 60 cycles at 70% maximal response. Finally, minutely fEPSPs were\nrecorded again for over 60 minutes. This part has been added to the maintext of this paper.\nReference:\n1. Steigerwalt, R.W. & Williams, J.A. Binding specificity of the mouse cerebral cortex receptor\nfor small cholecystokinin peptides. Regulatory peptides 8, 51-59 (1984).\n2. JF, T.M.H. Polypeptide and application thereof as cck receptor agonist/antagonists.\nCN113929737A (Granted) filed October 20, 2021. PCT/CN2022/102744 filed April 27, 2023. .\n3. Zhang, X. et al. Structures of the human cholecystokinin receptors bound to agonists and\nantagonists. Nature Chemical Biology 17, 1230-1237 (2021).\n4. Treen, A.K., Luo, V. & Belsham, D.D. Phoenixin Activates Immortalized GnRH and Kisspeptin\nNeurons Through the Novel Receptor GPR173. Molecular endocrinology (Baltimore, Md.) 30,\n872-888 (2016).\n5. Chen, X. et al. Cholecystokinin release triggered by NMDA receptors produces LTP and sound-\nsound associative memory. Proceedings of the National Academy of Sciences of the United\nStates of America 116, 6397-6406 (2019).\n6. Beautrait, A. et al. A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay\nbetween GPCR internalization and signalling. Nature communications 8, 15054 (2017).\n7. Avet, C. et al. Effector membrane translocation biosensors reveal G protein and βarrestin\ncoupling profiles of 100 therapeutically relevant GPCRs. eLife 11 (2022).\n8. Armbruster, B.N., Li, X., Pausch, M.H., Herlitze, S. & Roth, B.L. Evolving the lock to fit the\nkey to create a family of G protein-coupled receptors potently activated by an inert ligand.\nProceedings of the National Academy of Sciences of the United States of America 104, 5163-\n5168 (2007).\n9. Guettier, J.M. et al. A chemical-genetic approach to study G protein regulation of beta cell\nfunction in vivo. Proceedings of the National Academy of Sciences of the United States of\nAmerica 106, 19197-19202 (2009).\n10. Coutellier, L. & Usdin, T.B. Enhanced long-term fear memory and increased anxiety and\ndepression-like behavior after exposure to an aversive event in mice lacking TIP39 signaling.\nBehavioural brain research 222, 265-269 (2011).\n11. Hori, H. et al. Fear memory regulation by the cAMP signaling pathway as an index of\nreexperiencing symptoms in posttraumatic stress disorder. Molecular psychiatry 29, 2105-2116\n16\n(2024).\n12. Careaga, M.B.L., Girardi, C.E.N. & Suchecki, D. Understanding posttraumatic stress disorder\nthrough fear conditioning, extinction and reconsolidation. Neuroscience and biobehavioral\nreviews 71, 48-57 (2016).\n13. Takinami, Y. et al. YF476 is a new potent and selective gastrin/cholecystokinin-B receptor\nantagonist in vitro and in vivo. Alimentary pharmacology & therapeutics 11, 113-120 (1997).\n17"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript resolved the main issues from the original version. In particular, the new experiments with the Gq-blocker, the extracellular-side FRET assay, and BRET-based bias plots strenghtening the pharmacological conclusions.", "reviewer_2": "We would like to thank the authors for their detailed and constructive responses to our comments. Our minor points were all addressed with great attention to detail. While we appreciate the effort put into revising the manuscript, we anticipate that our concerns regarding two major points were only partially resolved. We respond for each point from our original review comments below:\nMajor points:\n1. Resolved.\n2. While we generally understand the author's concerns about future commercialization, the reader of the manuscript should still understand the rationale for selecting the ligands. We therefore recommend including at least some reasoning behind ligand selection in the main text of the manuscript.\n3. Greatly improved, especially the inclusion of the MD part. However, we believe the following points should yet be addressed:\n- Including a 3D visualization of the binding poses would enhance the interpretability of the docking results.\n- Lines 946-947: The definition of \"stabilization of the simulation” remains unclear. Please elaborate on what metrics were used to assess stabilization (e.g., ligand RMSD, key interactions, etc.).\n- Tracking specific bonding interactions (e.g., hydrogen bonds, salt bridges, hydrophobic contacts) throughout the MD simulation would strengthen the mechanistic interpretation of the binding hypothesis. We would suggest using tools such as ProLIF (https://github.com/chemosim-lab/ProLIF) or similar.\n- Lines 1664–1666: Please specify how many 3D conformers were generated prior to docking, to clarify the extent of sampling.\n- No clear explanation was provided regarding how protonation states of the ligands and protein were determined. A short justification would help support the accuracy of the docking and MD setup.\n- A single 50 ns simulation may be insufficient to capture conformational changes to assess the stability of the docked peptide ligand. We recommend the authors run longer simulations at least in triplicates and acknowledge problems potential drawbacks in the discussion.\n- There is still no elaboration on hydrogen bond tracking. Please describe the method used (e.g., distance/angle criteria or software) to identify and evaluate H-bonds.\n4. The addition of the additional experiments has resolved this point.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "All my questions have been solved properly with no further comments.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear reviewers,\nThank you very much for your recognition and constructive comments and suggestions. We have learned a lot from your suggestions, especially your opinions on the calculation part, which are of great guiding significance to us. For the second point, we have added necessary explanations about the ligand selection in the main text and appendix. For the third point, we have explained or calculated the results one by one according to your suggestions. We hope that our work can address all your comments and suggestions. At the same time, if you have any new suggestions and comments, please feel free to let us know to improve the quality of our work.\nReviewer #2 (Remarks to the Author):\nMajor points:\n2. While we generally understand the author's concerns about future commercialization, the reader of the manuscript should still understand the rationale for selecting the ligands. We therefore recommend including at least some reasoning behind ligand selection in the main text of the manuscript.\nResponse: Thank you for your understanding. We totally agree with your opinion. Adding necessary explanations can indeed increase the completeness and readability of our article.\nWe added the following explanation at the beginning of the results section: CCK4 (Table. S1) contains four amino acids at the carboxyl terminus of CCK8s, and it is considered the shortest CCK fragment that activates CCKBR1. In our previous screening campaign, we found that a CCK4-modified compound HT-178 (Fig. S1D) exhibited a good activation effect on CCKBR downstream calcium signaling. However, we found a very interesting phenomenon. A slight change in the substituent on the amino group in the red box of HT-178 (Fig. S1D) greatly affected the activation of calcium signals downstream of CCKBR. Since the downstream signaling pathway of CCKBR could be intricate2-5, we speculated that this slight change might not change the affinity for CCKBR, but only could change the bias for the downstream signaling pathway of CCKBR. Therefore, we selected compounds with different substituents at the amino position, as well as some other compounds based on CCK4 modification, for systematic biological evaluation (Table. S1).\nWe have also added the structural formula of HT-178 in the attachment (Fig. S1D).\n3. Greatly improved, especially the inclusion of the MD part. However, we believe the following points should yet be addressed:\n- Including a 3D visualization of the binding poses would enhance the interpretability of the docking results.\nResponse: Figure 8A now presents a representative 3D structure of the complex (extracted from one frame during the MD simulation) in which most key binding interactions are preserved. Hydrogen bonds are highlighted to facilitate interpretation of the docking and MD results. We have also made corresponding changes to the validation results of the cell tests. Please see Fig. 8B and Fig. S15 for details.\n- Lines 946-947: The definition of \"stabilization of the simulation” remains unclear. Please elaborate on what metrics were used to assess stabilization (e.g., ligand RMSD, key interactions, etc.).\nResponse: To assess simulation stability, we monitored the RMSD change of both the protein-ligand complex and the ligand itself. In all three simulation replicates, the ligand and complex RMSD stabilized within 50–100 ns (Fig. S12-14).\n- Tracking specific bonding interactions (e.g., hydrogen bonds, salt bridges, hydrophobic contacts) throughout the MD simulation would strengthen the mechanistic interpretation of the binding hypothesis. We would suggest using tools such as ProLIF (https://github.com/chemosim-lab/ProLIF) or similar.\nResponse: Binding interactions including hydrogen bonds, salt bridges, and hydrophobic interactions were tracked over the 200 ns MD simulations using GROMACS, PLIP, and ProLIF. The temporal distribution of interactions across residues is visualized as heat maps in the Fig. S12-14, supporting the proposed binding mechanism.\n- Lines 1664–1666: Please specify how many 3D conformers were generated prior to docking, to clarify the extent of sampling.\nResponse: As described in the methods section, we generated 1, 10, and 10 conformers respectively using MarvinSketch, Open Babel, and RDKit. The conformer with the lowest energy from each method was selected for docking to ensure sufficient conformational sampling.\n- No clear explanation was provided regarding how protonation states of the ligands and protein were determined. A short justification would help support the accuracy of the docking and MD setup.\nResponse: Ligand protonation states were assigned at pH 7.0 using MarvinSketch, and protein protonation was conducted in ChimeraX by adding hydrogens at the same pH. Additionally, the pH was set to 7.0 during the membrane system setup for MD simulations, ensuring consistency across all stages.\n- A single 50 ns simulation may be insufficient to capture conformational changes to assess the stability of the docked peptide ligand. We recommend the authors run longer simulations at least in triplicates and acknowledge problems potential drawbacks in the discussion.\nResponse: We have extended the MD simulations to 200 ns and performed three independent replicates to improve the reliability. Binding modes were largely consistent across replicates, though minor conformational variations were observed. Although the 200 ns triplicate MD simulations showed largely consistent binding modes, we acknowledge that that certain slow conformational changes may not be fully captured within this timescale. Additionally, the simplified lipid composition used in the membrane system may not fully recapitulate the native membrane environment, which could affect protein-ligand dynamics. Therefore, we acknowledge the problems in the last of the discussion part:\nOn the other hand, although we have proposed a binding mode of MF-8 and CCKBR through computational methods coupled with cell-based assays, this might not reveal the precise activation mechanism of the β-arrestin signaling pathway downstream of CCKBR. Decoding the structure of the MF-8 and CCKBR complex using cryo-electron microscopy may reveal the precise binding mechanisms and provide new insights into the development of biased drugs that target specific CCKBR signaling pathways.\n- There is still no elaboration on hydrogen bond tracking. Please describe the method used (e.g., distance/angle criteria or software) to identify and evaluate H-bonds.\nResponse: Hydrogen bond analysis was performed using all three tools (GROMACS, PLIP, and ProLIF). Results are presented in the Fig. S12-14.\nReferences\n1. Steigerwalt, R.W. & Williams, J.A. Binding specificity of the mouse cerebral cortex receptor for small cholecystokinin peptides. Regulatory peptides 8, 51-59 (1984).\n2. Inoue, A. et al. Illuminating G-Protein-Coupling Selectivity of GPCRs. Cell 177, 1933-1947.e1925 (2019).\n3. Masuho, I. et al. Rules and mechanisms governing G protein coupling selectivity of GPCRs. Cell reports 42, 113173 (2023).\n4. He, L. et al. A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition. The Journal of neuroscience : the official journal of the Society for Neuroscience 43, 2305-2325 (2023).\n5. Zeghal, M., Laroche, G., Freitas, J.D., Wang, R. & Giguère, P.M. Profiling of basal and ligand-dependent GPCR activities by means of a polyvalent cell-based high-throughput platform. Nature communications 14, 3684 (2023)."}, {"round": 3, "reviewer_comments": {"reviewer_2": "All comments have been adequately addressed.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter