A Novel CRISPR/Cas9-Based Cellular Model to Explore Adenylyl Cyclase and cAMP Signaling

Abstract

Functional characterization of adenylyl cyclase (AC) isoforms has proven challenging in mammalian cells because of the endogenous expression of multiple AC isoforms and the high background cAMP levels induced by nonselective AC activators. To simplify the characterization of individual transmembrane AC (mAC) isoforms, we generated a human embryonic kidney cell line 293 (HEK293) with low cAMP levels by knocking out two highly expressed ACs, AC3 and AC6, using CRISPR/Cas9 technology. Stable HEK293 cell lines lacking either AC6 (HEK-ACΔ6) or both AC3 and AC6 (HEK-ACΔ3/6) were generated. Knockout was confirmed genetically and by comparing cAMP responses of the knockout cells to the parental cell line. HEK-ACΔ6 and HEK-ACΔ3/6 cells revealed an 85% and 95% reduction in the forskolin-stimulated cAMP response, respectively. Forskolin- and G-coupled receptor-induced activation was examined for the nine recombinant mAC isoforms in the HEK-ACΔ3/6 cells. Forskolin-mediated cAMP accumulation for AC1-6 and AC8 revealed 10- to 250-fold increases over the basal cAMP levels. All nine mAC isoforms, except AC8, also exhibited significantly higher cAMP levels than the control cells after G-coupled receptor activation. Isoform-specific AC regulation by protein kinases and Ca/calmodulin was also recapitulated in the knockout cells. Furthermore, the utility of the HEK-ACΔ3/6 cell line was demonstrated by characterizing the activity of novel AC1 forskolin binding-site mutants. Hence, we have developed a HEK293 cell line deficient of endogenous AC3 and AC6 with low cAMP background levels for studies of cAMP signaling and AC isoform regulation.

G protein-regulated endocytic trafficking of adenylyl cyclase type 9

“…The β2AR stimulates cAMP production primarily from the plasma membrane, with endosomal activation contributing a relatively small but functionally significant fraction (Irannejad et al, 2013; Tsvetanova and von Zastrow, 2014). In a similar vein, AC3 and AC6 are the most highly expressed AC isoforms and mediate most of global cAMP production in these cells, but AC1 and AC9 transcripts are expressed at somewhat lower levels and comparable to one another (Soto-Velasquez et al, 2018). This prompted us to ask whether AC1 and/or AC9 might be relevant to generating the portion of cellular cAMP initiated by β2AR activation in endosomes.…”

“…To test this, we applied the diterpene drug forskolin (FSK) to stimulate cytoplasmic cAMP production independently from the receptor or Gs. While AC9 is relatively insensitive to activation by FSK, other AC isoforms that are major contributors to cAMP production in HEK293 cells (such as AC3 and AC6) are sensitive, making FSK an effective stimulus of overall cAMP elevation (Baldwin et al, 2019; Soto-Velasquez et al, 2018). FSK did not cause detectable internalization of either HA-β2AR or Flag-AC9 assessed by imaging ( Fig S4A, S4C, S4D, S4l ) or surface biotinylation ( Fig S4E, S4F ).…”

“…As another test of this hypothesis, and to investigate selectivity under conditions of recombinant AC overexpression (which were necessarily used for the trafficking studies), we asked if a specific endosomal signaling effect is evident also using tagged AC isoforms. To do so, we used HEK293 cells depleted of both AC3 and AC6 (AC3/6 DKO) that were shown previously to provide a reduced background useful for assessing effects of recombinant AC expression on cellular cAMP (Soto-Velasquez et al, 2018). The increase of isoproterenol-induced cAMP accumulation produced by overexpression of Flag-AC9, defined as the ‘AC9 response’, was blocked by alprenolol but not CGP ( Fig 6E ).…”

Functional characterization of AC5 gain-of-function variants: Impact on the molecular basis of ADCY5-related dyskinesia

“…Current pharmacological therapies vary widely, ranging from benzodiazapines and barbiturates to carbidopa-levodopa with limited effectiveness (15). Here, we took advantage of a newly developed HEK cell line that used Crispr-Cas9 to eliminate AC3 and AC6, the two most abundant endogenous ACs in HEK cells to reduce the background AC activity and cAMP signal (17). Using this novel cell model, we examined the functional characteristics of five AC5 mutations identified in patients with ADCY5related dyskinesia, and further explore AC5 as a potential therapeutic target for this earlyonset pleiotropic movement disorder.…”

Immune-metabolic genesis of pathological processes

“…These compounds can be considered as secondary intracellular messengers mediating the action of extracellular factors. The formation of cyclic nucleotides is associated with the activity of enzymes -adenylyl and guanylate cyclase, being components of cell membranes (Aronsson et al 2014, Soto-Velasquez et al 2018.…”