Study on the Mechanism of CCKBR-dependent Long-term Potentiation and Identification of a Biased CCKBR Agonist that Blocks the Potentiation in a β-arrestin Dependent Manner

Abstract

G protein-coupled receptors (GPCRs) transmit signaling through diversified downstream pathways involving distinctive biological mechanisms and assorted disease-related effects. CCKBR, as a GPCR, its activation induces neocortex long-term potentiation (LTP) of excitatory transmission and enhances memory formation, while its antagonists weaken the LTP in the amygdala and are used to alleviate depression-like behaviours in mice. However, the mechanism of CCKBR dependent-LTP (CCKBR-LTP) remains elusive. There is also no highly signaling pathway-biased and CCKBR-selective agonist to modulate the CCKBR-LTP. Here, we discovered a β-arrestin biased and CCKBR selective agonist with IC₅₀ = 15.3nM, and we named the agonist 5R15. Full activation of the β-arrestin signaling pathway with 5R15 couldn’t induce the auditory cortex (AC) CCKBR-LTP in the mouse brain slice but efficiently induced the endocytosis of CCKBR in cell-based assays. Further Multi-Electrode Array (MEA) results demonstrated that the CCKBR-LTP was dependent on Gα_q/11-Ca²⁺ and Gα_s-cAMP signaling pathways. Both these two signaling pathways induced by CCKBR activation were suppressed by 5R15 with high potency, suggesting that this agonist could be an antagonist for the CCKBR-LTP. Accordingly, the LTP was completely blocked by 5R15 at a low concentration in a β-arrestin dependent manner. The blockade was abolished by mutating the key site Asn353, which determines the β-arrestin signaling bias induced by 5R15. These results revealed the signal pathway preference of the CCKBR-LTP in the AC and BLA and identified a novel “antagonist” of the LTP, which provides us with broader insights into developing new drugs targeting CCKBR. The concept that β-arrestin-biased agonists could function as antagonists for other signaling pathways might be extended to the development of other GPCR drugs.

Date of Award	23 Dec 2024
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Jufang HE (Supervisor)