Virion display (VirD) approach to characterizing non‐odorant GPCRs in Humans

Approximately one‐third of the human proteome is comprised of membrane proteins that belong to protein families with a wide variety of biochemical activities, such as transporters, channels, receptors, recognition molecules, and adhesion molecules. Among them, the GPCR protein family is of particular importance because they exhibit a wide variety of signaling pathways depending on ligand binding specificity and various coupling proteins during activation. In deed, they are most preferred drug targets. However, GPCRs are notoriously difficult to study because they have to be embedded in a membrane to maintain a native conformation and many require proper posttranslational modifications (PTMs), such as glycosylation. To facilitate high‐throughput biochemical analyses of GPCRs, we have recently developed a VirD array technology by which a GPCR is displayed in the membrane envelop of herpes simplex (i.e., HSV‐1) virions to maintain their native conformation. To date, we have displayed 315 non‐odorant GPCRs on the virions and formed a GPCR VirD array by spotting down the purified viruses. To characterize the virion‐displayed GPCRs, we first tested the orientation and ligand binding activities of selected GPCRs. Of the 20 commercial mAbs targeting ectodomains of GPCRs, 11 could specifically recognize their intended targets on the VirD arrays. For the nine GPCRs that failed to be recognized by the commercial mAbs, we showed that they could readily recognize their canonical ligands using single molecule imaging. Moreover, using an SPR‐based technology, we could obtain real‐time binding kinetics and deduce binding affinity for selected ligand‐GPCR pairs, indicating that they were all folded correctly. To determine whether VirD technology could help de‐orphanize GPCRs, ~80 fluorescently labeled orphan GPCR virions were individually probed to HuProt array, each comprised of 20,240 human proteins. Many secreted and transmembrane proteins were identified as specific targets, providing an important stepping‐stone for further characterization of these orphan GPCRs. Taken together, the VirD technology provides a high‐throughout platform for screening for drugs, affinity reagents, ligands, and beyond. Support or Funding Information NCI IMAT R33 and CDI Labs This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .