A Highly Effective Dominant Negative α_s Construct Containing Mutations That Affect Distinct Functions Inhibits Multiple G_s-coupled Receptor Signaling Pathways*

Abstract

To investigate the subcellular organization of receptor-G protein signaling pathways, a robust dominant negative α_s mutant containing substitutions that alter distinct functions was produced and tested for its effects on G_s-coupled receptor activity in HEK-293 cells. Mutations in the α3β5 loop region, which increase receptor affinity, decrease receptor-mediated activation, and impair activation of adenylyl cyclase, were combined with G226A, which increases affinity for βγ, and A366S, which decreases affinity for GDP. This triple α_s mutant can inhibit signaling to G_s from the luteinizing hormone receptor by 97% and from the calcitonin receptor by 100%. In addition, this α_s mutant blocks all signaling from the calcitonin receptor to G_q. These results lead to two conclusions about receptor-G protein signaling. First, individual receptors have access to multiple types of G proteins in HEK-293 cell membranes. Second, different G protein α subunits can compete with each other for binding to the same receptor. This dominant negative α_s construct will be useful for determining interrelationships among distinct receptor-G protein interactions in a wide variety of cells and tissues.