Inhibition of heterotrimeric Gs proteins by FR900359 - more than a simple transfer of the inhibitor binding site

Abstract

A plethora of vital functions of the human body, including blood pressure, cell proliferation and metabolism are regulated by G protein-mediated signaling cascades. These cascades form a complex network of cause and effect that allows our bodies to adapt to, function and thrive in a wide range of environments and circumstances. However, this network of signals does not always run flawlessly, and when off balance, pathological changes and diseases are often the consequence such as cancer or hypertension. To treat these diseases, the thorough study of G protein-mediated signaling cascades is an important component of basic research, to understand their influence or contribution to certain cellular events. Such in depth investigations could be accomplished, for example, with specific G protein inhibitors. However, despite the discovery of G proteins about 30 years ago, only specific inhibition of Gi and Gq, two of the four G protein families, could be achieved by PTX or FR900359 (FR) / YM-25489 (YM), respectively. To bridge the lack of such highly cell permeable specific inhibitors of a third G protein family, Gas (Gs) proteins with artificial FR/YM-binding sites, such as Gα_s11 (Gs 11), Gα_s10 (Gs 10), respectively, have been developed. <br /> To this end, we investigated the suitability of Gs 11 and Gs 10 as chemogenetic tools for further possible studies. The idea behind chemogenetics is generally described as the approach in which engineered molecules interact with previously unrecognized molecules that are pharmacologically inert in the absence of the designed protein. Using CRISPR/Cas9-generated Gα_s-null or Gα_{s/olf/q/11/12/13/z} - null cells along with several cutting-edge tools such as label-free whole-cell biosensing, HTRF based cAMP accumulation or real-time BRET-based G protein activation, we determined conditions for the use of Gs 10 as a possible chemogenetic tool. <br /> However, intriguingly despite the transfer of the inhibitor binding site, FR´s inhibitory properties could not be entirely transferred. While Gq protein- dependent signaling events could be abolished completely, comparable conditions, such as the maximum activation of the overexpressed β2 adrenergic receptor (β2AR) resulted in partial inhibition for the artificial FR-sensitive Gs proteins. Moreover, we noted biologically distinct activities for FR and YM, despite close structural similarities. Thus, the lack of full inhibition was pronounced for the studies done with YM as compared with FR. <br /> Our results suggest that the simple transfer of the inhibitor binding site is not sufficient to transfer the properties of inhibition. Thus, possible FR scaffold-based inhibitors which mimic the interaction of FR with Ga proteins accommodating engineered FR-binding sites are not sufficient to perform studies on the same scale as would be possible with Gq. <br /> As a result, our study provides an important basis for the understanding of G protein activation and inhibition thus demonstrating the uniqueness of each individual G protein family since although they share an identical inhibitor binding site, Gq but not Gs 11 and Gs 10 could be fully inhibited by FR.