Development and application of a high-content virion display human GPCR array

Guan-Da Syu,Xinzhong Dong,Nongjian Tao,Shuang Liu,Jie Xiao,Prashant J Desai,Pedro Ramos,Lien-Chun Weng,Guangzhong Ma,Kwang Sik Kim,Ignacio Pino,Shih-Chin Wang,Donna Pearce,Devlina Ghosh,Brandon Henson,Shaopeng Wang,Atish Prakash,Heng Zhu,Daniel Eichinger

doi:10.1038/s41467-019-09938-9

Abstract

Human G protein-coupled receptors (GPCRs) respond to various ligands and stimuli. However, GPCRs rely on membrane for proper folding, making their biochemical properties difficult to study. By displaying GPCRs in viral envelopes, we fabricated a Virion Display (VirD) array containing 315 non-olfactory human GPCRs for functional characterization. Using this array, we found that 10 of 20 anti-GPCR mAbs were ultra-specific. We further demonstrated that those failed in the mAb assays could recognize their canonical ligands, suggesting proper folding. Next, using two peptide ligands on the VirD-GPCR array, we identified expected interactions and novel interactions. Finally, we screened the array with group B Streptococcus, a major cause of neonatal meningitis, and demonstrated that inhibition of a newly identified target, CysLTR1, reduced bacterial penetration both in vitro and in vivo. We believe that the VirD-GPCR array holds great potential for high-throughput screening for small molecule drugs, affinity reagents, and ligand deorphanization.

Highlights

Human G protein-coupled receptors (GPCRs) respond to various ligands and stimuli
STOP codons of the GPCR open reading frames (ORFs) were removed via PCR reactions and the modified ORFs subcloned into the Gateway Entry vector
At least two colony PCR reactions were performed using a primer pair that annealed to the viral sequences flanking the cloning site of the GPCR ORFs, and gel electrophoresis was employed to examine whether the amplicon was of the expected size of the GPCR cloned

Summary

Introduction

Human G protein-coupled receptors (GPCRs) respond to various ligands and stimuli. GPCRs rely on membrane for proper folding, making their biochemical properties difficult to study. By displaying GPCRs in viral envelopes, we fabricated a Virion Display (VirD) array containing 315 non-olfactory human GPCRs for functional characterization. Of all annotated human GPCRs, 370 are non-odorant and play crucial roles in many different aspects of cellular processes. They are the most preferred drug targets because their dysregulation often lead to disease or cancer. Perhaps most importantly, 116 (31%) of the ~370 non-odorant GPCRs are orphans, without known physiological agonists or antagonists, which precludes study of their activity. To demonstrate the power of VirD technology in this study, we fabricated a high-content VirD-GPCR array, comprised of 315 non-odorant GPCRs, and demonstrated that the VirD-GPCRs were folded and functional using a variety of biochemical assays

Methods

Results

Conclusion