Receptor tyrosine kinases activate heterotrimeric G proteins via phosphorylation within the interdomain cleft of Gαi

The molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs crosstalk has been a long-standing question, but answers remain elusive. Using linear-ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator, GIV, dissociates G-alpha(i)/beta/gamma trimers, scaffolds monomeric G-alpha(i) with RTKs, and facilitates the phosphorylation on two tyrosines located within the inter-domain cleft of G-alpha(i). Phosphorylation triggers the activation of G-alpha(i) and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cellular decision to go vs. grow. These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes.