Identification of Novel Cholecystokinin Receptors and their Functions

Abstract

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the central nervous system (CNS), is involved in reward, memory, and epilepsy and mediates its actions via two G-protein-coupled receptors (GPCRs), CCKAR (cholecystokinin type 1 receptor, also known as CCK1R) and CCKBR (cholecystokinin type 2 receptor, also known as CCK2R). However, we recently discovered that high-frequency laser stimulation (HFS)-induced CCK release in GABAergic neurons could depress the local auditory response in CCKA/BR knockout mice, suggesting the presence of novel CCK receptor(s) in the CNS. Based on previous reports, we also found that a possible novel CCK receptor(s) presenting in the pmAcb (the posteromedial nucleus accumbens). Therefore, based on our previous findings, we searched for a novel CCK receptor(s) and explored its (their) functions in the CNS.

We initially investigated the novel CCK receptor located at the CCK-positive GABAergic terminal in the auditory cortex. Secondary structure comparison of the extramembrane GPCRs suggested higher similarity scores in several GPCRs compared to CCKAR and CCKBR. To verify the predicted receptors, we first constructed stable candidate GPCR-overexpressing monoclonal CHO (Chinese hamster ovary cells) cell lines using a lentivirus expression system and named these cell lines CHO-GPCRs. Consequently, we performed a calcium imaging assay for these CHO-GPCR cells and found that CCK8s (the main active form of CCK in the CNS) could elicit an intracellular calcium rise in CHO-GPR173 cells (GPR173, also named SREB3) with an EC50 (half maximal effective concentration) = 1.9 nM. In addition, calcium release in CHO-GPR173 cells could be decreased by shRNA (small hairpin RNA) targeting GPR173, which indicated that the calcium release induced by CCK8s in CHO-GPR173 cells was mediated by GPR173. Hence, we speculated that GPR173 is a novel CCK receptor.

We designed several other cell-based assays to confirm GPR173 as a novel CCK receptor. The first assay was a cell surface binding assay, in which we found that HA-tagged CCK8s could directly bind to Flag-tagged GPR173, and the HA-tagged CCK8s binding signal could be blocked by coincubation with CCK8s. Moreover, although we detected no β-arrestin2 recruitment to GPR173 after stimulation by CCK8s, we found that GPR173 could recruit β-arrestin1 in our optimized β-arrestin recruitment assay system. Both the cell surface binding assay and the optimized β-arrestin recruitment assay provided us with powerful evidence that GPR173 is a novel CCK receptor.

In addition to the cell-based assays, our anatomical exploration by immunohistochemistry frozen (IHC-Fr) staining showed that the colocalization density of GPR173 with CCK-positive GABAergic terminals was significantly denser than that of other candidate CCK receptors, which provided anatomical evidence that GPR173 should function at CCK-positive GABAergic terminals. Additionally, devazepide (also known as L364,718), a potent antagonist of GPR173, completely inhibited CCK8s-potentiated GABAergic inhibition at 60 nM, indicating that GPR173 could be a novel CCK receptor that strengthens GABA function in the auditory cortex.

Next, we established an in vivo model by overexpressing GPR173 (using AAV-PHP.eB) in the intrahippocampal kainate (KA)-injected epilepsy model of C57BL/6J mice. Our data suggested that overexpression of GPR173 could alleviate seizure symptoms in the long term, providing good prospects for treating epilepsy in the clinic.

Consequently, we examined which CCK receptor is in the pmAcb where is known for the CCK and DA (dopamine) interaction. VTA (ventral tegmental nucleus) CCK⁺DA⁺ neurons are the main source of pmAcb CCK⁺DA⁺ fibers. We found that 49% of CCK neurons and 30% of DA neurons were colabeled in the VTA; moreover, all CCK neurons in the VTA were costained with the GABA antibody, indicating the complicated GABA/CCK/DA corelease mechanism of CCK⁺DA⁺ neurons in the VTA. In addition, VTA CCK⁺DA⁺ neurons were mainly projected to the pmAcb. When AAV-dio-ChrimsonR-mCherry was injected into the VTA of CCK-ires-Cre mice, colocalization of most anti-mCherry and anti-TH signals in the pmAcb suggested that CCK⁺DA⁺ instead of CCK⁺DA^- neurons were projected into the pmAcb.

To further explore which CCK receptor was located at the CCK⁺DA⁺ terminal in the pmAcb, we searched the Allen Brain in situ hybridization (ISH) data (freely available multimodal atlas of anatomy and gene expression comprising a comprehensive 'all genes-all structures' array-based dataset of gene expression and complementary ISH gene expression studies targeting selected genes in specific brain regions) and reanalyzed published scRNA-seq data of the mouse Acb combined with IHC-Fr staining. Briefly, no CCKAR, CCKBR, GPR173, or GPR1 was found. However, GPR85 (also known as SREB2) and GPR83 were observed. Subsequently, GPR85 and GPR83, as two novel CCK receptors, were further confirmed by our previously designed cell surface binding or optimized β-arrestin recruitment assays. HA-tagged CCK8s directly bound to Flag-tagged GPR85. CCK8s could induce β-arrestin recruitment to GPR85 and GPR83. All these data suggested that GPR85 and GPR83 are two novel CCK receptors located at the CCK and TH positive areas in the pmAcb projected from the VTA CCK⁺DA⁺ neurons.

In summary, we built a multidisciplinary experimental platform and identified GPR173 as a novel CCK receptor that could potentiate GABAergic inhibition in the auditory cortex and relieve epilepsy by widespread expression of GPR173 in mice. GPR85 and GPR83 were also proven to be possible CCK receptors located at the CCK and TH positive areas in the pmAcb projected from the VTA CCK⁺DA⁺ neurons, providing an anatomical foundation for a deep understanding of the CCK-DA interaction system.

Date of Award	10 Oct 2022
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Jufang HE (Supervisor), Micky TORTORELLA (External Co-Supervisor) & Xin DENG (Co-supervisor)