Abstract
Endothelin receptor A (ETA), a class A G-protein-coupled receptor (GPCR), is involved in the progression and metastasis of colorectal, breast, lung, ovarian, and prostate cancer. We overexpressed and purified human endothelin receptor type A in Escherichia coli and reconstituted it with lipid and membrane scaffold proteins to prepare an ETA nanodisc as a functional antigen with a structure similar to that of native GPCR. By screening a human naive immune single-chain variable fragment phage library constructed in-house, we successfully isolated a human anti-ETA antibody (AG8) exhibiting high specificity for ETA in the β-arrestin Tango assay and effective inhibitory activity against the ET-1-induced signaling cascade via ETA using either a CHO-K1 cell line stably expressing human ETA or HT-29 colorectal cancer cells, in which AG8 exhibited IC50 values of 56 and 51 nM, respectively. In addition, AG8 treatment repressed the transcription of inhibin βA and reduced the ETA-induced phosphorylation of protein kinase B and extracellular regulated kinase. Furthermore, tumor growth was effectively inhibited by AG8 in a colorectal cancer mouse xenograft model. The human anti-ETA antibody isolated in this study could be used as a potential therapeutic for cancers, including colorectal cancer.
Highlights
G-protein-coupled receptors (GPCRs), the largest superfamily of membrane receptors in the human genome, transduce extracellular signals to the intracellular space through binding of their cognate ligands
Preparation of functional Human ETA (hETA) antigens mimicking the structure of native hETA on the cell membrane For screening of monoclonal human antibodies against hETA, it is necessary to prepare a sufficient amount of functional antigen structurally similar to native hETA
It is well known that the complex structure of GPCRs, with seven transmembrane αhelices, is difficult to express in heterologous hosts[31]
Summary
G-protein-coupled receptors (GPCRs), the largest superfamily of membrane receptors in the human genome, transduce extracellular signals to the intracellular space through binding of their cognate ligands. Intracellular signaling events triggered by conformational changes in GPCRs and interactions with intracellular proteins regulate numerous cellular functions, such as growth, motility, and differentiation[1,2]. Endothelin receptor type A (ETA) is a class A GPCR that belongs to the endothelin receptor family It regulates blood vessel constriction, cell growth, and differentiation through several downstream signaling pathways activated by the binding of ligands such as ET-1, ET-2, and ET-35,6. ETA, which undergoes a conformational change due to ligand binding, is involved in a variety of downstream signaling pathways through its interaction with G-protein alpha subunits (Gαs, Gαq/11, and Gαi) in the intracellular space[7,8,9]. All ETA antagonists that have been evaluated for antitumor efficacy are small-molecule drugs
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