Generation of Comprehensive GPCR-Transducer-Deficient Cell Lines to Dissect the Complexity of GPCR Signaling

Abstract

G-protein-coupled receptors (GPCRs) compose the largest family of transmembrane receptors and are targets of approximately one-third of FDA approved drugs owing to their involvement in almost all physiological processes. GPCR signaling occurs through the activation of heterotrimeric G-protein complexes and β-arrestins, both of which serve as transducers resulting in distinct cellular responses. Despite seeming simple at first glance, accumulating evidence indicates that activation of either transducer is not a straightforward process, as a stimulation of a single molecule has the potential to activate multiple signaling branches. The complexity of GPCR signaling arises from the aspects of G-protein-coupling selectivity, biased signaling, interpathway crosstalk, and variable molecular modifications generating these diverse signaling patterns. Numerous questions relative to these aspects of signaling remained unanswered until the recent development of CRISPR-Cas9 genome editing. Such genome editing technology presents opportunities to chronically eliminate the expression of G-protein subunits, β-arrestins, GRKs, and many other signaling nodes in the GPCR pathways at one's convenience. Here, we review the practicality of using CRISPR-Cas9-derived knockout (KO) cells in the experimental contexts of unravelling the molecular details of GPCR signaling mechanisms. To mention a few, KO cells have revealed the contribution of β-arrestins in ERK activation, Gα protein selectivity, GRK-based regulation of GPCRs, and many more, hence validating its broad applicability in GPCR studies.