Novel Tools to Study Modulation of Adenylyl Cyclase Isoforms
Adenylyl cyclases (AC) are a major component of the cAMP signaling pathway. The differential regulatory properties and tissue expression patterns of the nine transmembrane AC isoforms provide unique mechanisms to regulate cAMP signaling in a precise and organized manner. However, the understanding of the individual roles of AC isoforms in the overall cellular response presents considerable challenges due to the expression of multiple AC isoforms in cells, and the lack of available tools to accurately detect isoform-specific AC expression or selectively modulate its catalytic activity. For these reasons, the research aims of this study were to develop a series of tools to characterize isoform-specific AC responses. In the first approach, a cell line with low cAMP levels in response to drug-stimulated conditions was developed by disrupting the expression of two of the most abundant ACs (i.e. AC3 and AC6) expressed in HEK293 cells using the CRISPR-Cas9 gene editing technology. Our HEK-ACΔ3/6 cell line displayed a substantially reduced cAMP response to forskolin (less than 95%) and to endogenous Gαs-coupled receptors, β2AR and EP 2R (75% and 85% reduction respectively), compared to the cAMP responses of the parental HEK293 cells. Characterization of the cAMP responses of the nine membrane-bound AC isoforms to stimulatory and inhibitory paradigms in the HEK-ACΔ3/6 knockout cell line indicated that the regulatory properties of the AC isoforms previously reported in the literature were recapitulated. Furthermore, a comparison of the cAMP responses of a series of AC1 mutants demonstrated that the HEK-ACΔ3/6 cell line provided an enhanced signal window over the parental HEK293 cells to characterize AC constructs with reduced catalytic activity. A second approach to better understand the individual role of AC isoforms in biological responses is by exploiting isoform-specific interactions with AC regulators to modulate AC activity. Thus, it was also determined in these studies that juxtamembrane domains derived from AC2 could modulate Gβγ-mediated AC activity. Based on the high degree of sequence homology between the juxtamembrane region of the C2a domain of Gβγ-stimulated cyclases, AC2, AC4, and AC7, together with surface plasmon resonance analysis with a C2a derived peptide (C2–20), it was demonstrated that Gβγ subunits bind with high affinity to this C2a juxtamembrane region. In addition, a minigene expressing the C2-20 peptide downstream of a membrane-anchoring domain, CD8, abolished Gβγ-mediated potentiation of PMA-stimulated AC2 activity. Mutagenesis studies indicated that several residues towards the middle of the sequence of the C2–20 peptide mediated the inhibitory activity on Gβγ-signaling, and the inhibitory effects of the minigene appeared to be selective for Gβγ-mediated stimulation of AC2. In the last approach to study isoform-specific AC responses, a series of structure activity relationship (SAR) studies were carried out towards the development of potent and selective AC1 and AC8 inhibitors. Initial SAR studies included analogs of the selective AC1 inhibitor, ST034307, that revealed a structure relationship between AC1 and AC8 selective inhibition. Furthermore, two new scaffolds were identified from a 10,000-compound screening campaign from the Life Chemicals compound collection, that showed dual inhibitory activity of calcium-stimulated AC1 and AC8 activity or selective inhibition of AC1 activity with sub-micromolar potency. Preliminary SAR analysis of various analogs of the dual AC1/AC8 inhibitory scaffold led to analogs with differential selectivity profiles for AC1 and AC8 and improved potency on both Ca2+/calmodulin-stimulated AC isoforms. In conclusion, throughout this work, a cellular model, a peptide/minigene, and a series of inhibitor scaffolds were developed as cellular and pharmacological tools to facilitate the study of the individual responses and respective roles of AC isoforms in cellular signaling.
Watts, Purdue University.
Off-Campus Purdue Users:
To access this dissertation, please log in to our