A Novel CRISPR/Cas9‐Based Cellular Model to Explore Adenylyl Cyclase and Cyclic AMP Signaling

Abstract

Functional characterization of adenylyl cyclase (AC) isoforms has proven to be challenging in mammalian cell systems due to the endogenous expression of multiple AC isoforms, and the high background cyclic AMP (cAMP) responses mediated by AC activators such Gαs or forskolin. Hence, the objective of this study was to generate an isolated HEK293 cell line with low cAMP levels by knocking out the two abundantly expressed AC isoforms using CRISPR/Cas9 technology. Based on qPCR studies examining AC isoform gene expression in HEK293 cells, we chose single guide RNA (sgRNA) sequences that target AC3 and AC6, and generated stable HEK293 cell lines lacking either AC6 (HEK‐AC6 KO) or both AC3 and AC6 (HEK‐AC3/AC6 KO). Loss of function of AC3 and AC6 was evaluated genetically and by comparing the forskolin‐ and Gαs‐coupled receptor‐stimulated cAMP responses in the knockout cells and the parental HEK293 cell line. AC activity in response to 50 μM forskolin was reduced by more than 80% in the HEK‐AC6 KO cells, and by an additional 10% when AC3 was also knocked out resulting in a total reduction of cAMP levels by 95% in the HEK‐AC3/AC6 KO cells compared to the HEK293 parental cell line. For Gαs‐coupled receptor‐mediated AC activation, cAMP accumulation was evaluated for the endogenous β2‐adrenergic receptors (β2AR) and prostaglandin E2 receptors (PGE2R). HEK‐AC6 KO cells produced 50% less cAMP at maximal β2‐adrenergic receptor stimulation (10 μM isoproterenol), whereas the HEK‐AC3/AC6 KO cells displayed >75% lower cAMP accumulation than the HEK293 parental cell line. The cAMP response induced by the PGE2R agonist, PGE‐2, was >85% lower in the HEK‐AC6 KO than that observed in the parental cell line, but no additional decrease in cAMP accumulation was detected in the HEK‐AC3/AC6 KO cells. We subsequently used the HEK‐AC3/AC6 KO cell line to pharmacologically characterize the individual membrane‐bound AC isoform responses to forskolin, β2AR, and PGE2R by reintroducing each individual isoform by transient transfection. Forskolin‐mediated cAMP accumulation for AC1‐6 and AC8 revealed 2‐ to 50‐fold increases over the cAMP levels of the control/venus‐transfected cells. In contrast, AC7 and AC9 showed little or no forskolin response, respectively. The cAMP responses for the nine AC isoforms following β2AR and PGE2R activation were also significantly higher than that observed for the control‐transfected cells. Isoform specific AC regulation by other regulatory mechanisms such as Gαi‐coupled receptors, protein kinases, and Ca+2/calmodulin was also recapitulated in the HEK‐AC3/AC6 KO cell line. We have used CRISPR/Cas9 to develop a HEK293 cell line deficient of endogenous AC3 and AC6 that exhibits very low basal and drug‐stimulated cAMP background levels. These HEK‐AC3/AC6 KO cells will facilitate the selective characterization of AC isoform activity and regulation in a mammalian cellular background.Support or Funding InformationNIH MH101673, Department of Medicinal Chemistry and Molecular Pharmacology (MCMP), and Purdue UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.