CRISPR Breaks Into GPCR Research Again Delineating the Role of GRK2/3 in Opioid Receptor Internalization

Despite the plethora of publications citing CRISPR engineering, as of May 2020 a mere 37 papers involved GPCRs (1,2).  Even this limited number of reports clearly show the potential impact of CRISPR engineering on GPCR drug discovery (2).  The recent study published in Scientific Reports in October 2020 from Moller et al. at the University of Copenhagen describes the use of a comprehensive set of tools, including CRISPR cell engineering, to delineate the roles of GRK2/3 in µ-OR internalization and β-arrestin2 recruitment – processes which have been linked to the development of opioid tolerance (3).

Different µ-OR agonists stimulate specific patterns of receptor phosphorylation and lead to differential cellular outcomes.  Previous in vitro and in vivo studies link GRK2 and GRK3 phosphorylation to µ-OR internalization and β-arrestin recruitment.  These studies, however, relied on techniques such as short interfering RNA (si-RNA) or overexpression of dominant negative mutants which are susceptible to incomplete KO of GRK activity or undesired effects of overexpression. 

The Scientific Reports article describes CRISPR-mediated generation of HEK293 clonal cell lines with the complete KO of GRK2, GRK3 or both, to better understand µ-OR pharmacology.  To supplement the CRISPR KO findings, the Brauner-Osborne and Bouvier labs used GRK2 and GRK3 transient expression to reintroduce the deleted protein to show restored function.

Collectively, their studies demonstrate that GRK2 and GRK3 both contribute to agonist-induced β-arrestin2 recruitment and µ-OR internalization for 3 of 4 agonist tested. The exception was morphine, a full agonist of G protein signaling but only a partial agonist of µ-OR internalization and β-arrestin recruitment.  The effects of GRK2 and -3 KO were additive, however, receptor internalization was not completely abrogated in the GRK2/3 double KOs suggesting additional factors, such as other GRKs, are involved in µ-OR internalization. The authors also describe a GRK2/3 independent pathway of β-arrestin2 recruitment to the plasma membrane that is observed after prolonged stimulation (60 minutes), particularly at high agonist concentrations.

It’s tools like the clonally-derived, CRISPR engineered GRK2/3 KO cell lines and other innovative tools such as β-arrestin recruitment assays that don’t require GPCR tagging, that are poised to more definitively unravel GPCR signaling and its role in pathophysiology, and ultimately to the development of novel, more efficacious therapeutics.