Abstract 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 Gαi•βγ trimers, scaffolds monomeric Gαi with RTKs, and facilitates the phosphorylation on two tyrosines located within the inter-domain cleft of Gαi. Phosphorylation triggers the activation of Gαi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cell’s decision to ‘go’ vs . ‘grow’. These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes. Significance Statement Growth factors and heterotrimeric G proteins are two of the most widely studied signaling pathways in eukaryotes; their crosstalk shapes some of the most fundamental cellular responses in both health and disease. Although mechanisms by which G protein pathways transactivate growth factor RTKs has been well-defined, how the reverse may happen is less understood. This study defines the key steps and cellular consequences of a fundamental mechanism of signal crosstalk that enables RTKs to transactivate heterotrimeric G protein, Gαi. Mutations found in tumors shed light on how derailing this mechanism impacts tumor cell behavior. Thus, findings not only show how cells integrate extracellular signals via pathway crosstalk, but also demonstrate the relevance of this pathway in cancers.

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