Kinetic changes in Ga cycling can increase cAMP accumulation while decreasing G protein‐coupled receptor kinase‐mediated receptor desensitization
Authors: Sharma Garima , Yeon Jae Kyung , Bastepe Murat , Jackson Edwin K. , Masuho Ikuo , Martemyanov Kirill A. , Andresen Bradley T. 
Published: June 2021

A G protein‐coupled receptor interacts with the GDP‐bound Gαβγ trimer, G protein‐coupled receptor kinases (GRKs), and in part arrestins through the cytosolic surface of the transmembrane helixes, suggesting that there is either competition for binding to the same surface or an orchestrated interchange between binding partners allowing for proper interactions and signaling. Experimentally induced competition for the intracellular Gαβγ trimer binding site results in a reduction in GRK and Gα function. Here we present a Gαs chimera, called sis, containing most of the helical domain of Gαi1. Initially, sis was designed to study GRK4γ interaction with Gαs and GRK4γ‐mediated receptor desensitization. However, sis prevented various GRK‐mediated reductions in cAMP accumulation. Therefore, we tested the hypothesis that sis disrupts normal orchestration of the Gαβγ trimer and GRK/arrestin function, resulting in an apparent Gα‐bias. To exclude any background effects of endogenous Gαs, we used GnasE2−/E2−‐MEFs re‐expressing Gαs (control) or sis and measured cAMP accumulation, receptor‐Gs kinetics, GRK function, and receptor internalization. The sis helical domain did not significantly affect the GEF activity of endogenously expressed β2‐AR or Gαβγ trimer formation; however, GRK function and cAMP production were altered. GRK2, 4γ, 5, and 6 failed to attenuate isoproterenol‐mediated cAMP accumulation in sis‐expressing cells, but all GRKs attenuated cAMP accumulation in Gs‐expressing cells. Expectedly, the rate of receptor endocytosis was reduced in sis‐expressing cells. Additionally, the production of cAMP in the Gs and sis cells was not equal. With phosphodiesterases inhibited, isoproterenol generated approximately 2.5‐fold more cAMP in sis cells compared to controls; moreover, in the same conditions forskolin was 50% less effective in sis cells. The original classification of adenylyl cyclase indicates that the effects of Gαs and forskolin are synergistic leading to greater cAMP production than either component alone. Therefore, our cAMP data suggests that interaction between sis and adenylyl cyclase is compromised, and sis likely cycles back to the receptor rapidly blocking GRK association and allowing itself to be reactivated. Supporting this conclusion, the rate of Gαs‐mediated cAMP accumulation was marginally faster than sis‐mediated cAMP accumulation (0.404 ± 0.127 s−1 vs. 0.178 ± 0.022 s−1) and the rate of cAMP degradation was greatly attenuated, resulting in a much larger area under the curve (2.4‐fold). These data mirror the previous end‐point cAMP measurements and further support the conclusion that sis allows the β2‐AR to remain active for a longer duration, resulting in greater cAMP accumulation.

Support or Funding Information

WesternU MSPS program funds and NIH grants DK073911 (MB), DK079307 (EKJ) and HL109002 (EKJ) supported this work.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.