Characterization of a Peptide Modulator of Gβγ Signaling Derived from Adenylyl Cyclase Type 2
Authors: Velasquez Monica Soto , Surve Chinmay R. , Perez Minervo , Parker Laurie L. , Smrcka Alan V. , Watts Val J. 
Published: November 2023

The objective of this study was to use a vector minigene strategy to explore the inhibitory action of a previously identified 20‐mer peptide on Gβγ‐mediated signaling. The 20‐mer peptide (C2–20) was identified from a set of lipidated peptides that were based on the conserved cytoplasmic domains of adenylyl cyclase (AC) 2, 4 and 7. Lipidated and TAT‐modified forms of the peptide derived from an uncharacterized domain of the C2a domain (i.e. C2–20), showed inhibitory activity on Gβγ potentiation of phorbol‐ester‐mediated stimulation of AC2, however, the peptides were associated with significant toxicity at high doses. Subsequently, a minigene strategy was proposed and used to characterize the identified C2–20 peptide as a modulator of Gβγ signaling. The minigene contained an N‐terminal CD8 domain to localize the C2–20 peptide to the membrane to facilitate Gβγ interactions. Expression of the CD8‐C2–20 minigene in HEK cells, transiently expressing AC2 and the D2 dopamine receptor (D2R), revealed inhibitory action on Gβγ potentiation of AC2 activity. The inhibitory effect was dose‐dependent and transient expression of a scrambled version of the CD8‐C2–20 minigene failed to reduce the Gβγ potentiation of AC2. Surface plasmon resonance to measure in vitro binding affinity of the unmodified C2–20 and the Gβγ subunit, indicated high binding affinity (ca. 2 nM). The C2–20 did not appear to bind Gαi and a scrambled peptide failed to bind Gβγ subunits. The inhibitory effects of the CD8‐C2–20 minigene were further evaluated on other Gβγ subunit‐mediated pathways. Given the importance of Gβγ subunits for the recruitment of G‐protein receptor kinases (GRK) to activated G protein‐coupled receptors, and subsequent β‐arrestin recruitment, the activity of the CD8‐C2–20 minigene was explored for β‐arrestin recruitment to the D2R using the DiscoveRx pathway cellular model. Transient expression of the CD8‐C2–20 minigene attenuated the recruitment of β‐arrestin to the activated D2R, whereas expression of the scrambled version of the CD8‐C2–20 minigene was inactive. We also evaluated the effects of the peptide on a Gβγ‐dependent response, heterologous sensitization of AC2. In contrast to acute modulation of AC2 and β‐arrestin recruitment, expression of the CD8‐C2–20 minigene failed to prevent agonist‐induced sensitization. To assess the amino acid specificity of the CD8‐C2–20 minigene, a series of mutant minigenes containing triple‐alanine substitutions were designed, and their ability to inhibit Gβγ potentiation of AC2 activity was examined. The resulting studies suggest that specific regions of the C2a domain could be used to selectively modulate Gβγ signaling. The robust modulation by CD8‐C2–20 was somewhat surprising considering the numerous previously identified sites for Gβγ modulation of AC2. These studies highlight further the complexities associated with AC signaling and the unique ways in which isoforms are modulated by Gβγ subunits.Support or Funding InformationPurdue University Research Incentive Award and NIH MH060397