Growth factor-dependent phosphorylation of Gα i shapes canonical signaling by G protein-coupled receptors.

A long-standing question in the field of signal transduction is how distinct signaling pathways interact with each other to control cell behavior. Growth factor receptors and G protein-coupled receptors (GPCRs) are the two major signaling hubs in eukaryotes. Given that the mechanisms by which they signal independently have been extensively characterized, we investigated how they may cross-talk with each other. Using linear ion trap mass spectrometry and cell-based biophysical, biochemical, and phenotypic assays, we found at least three distinct ways in which epidermal growth factor affected canonical G protein signaling by the G i -coupled GPCR CXCR4 through the phosphorylation of Gα i . Phosphomimicking mutations in two residues in the α E helix of Gα i (tyrosine-154/tyrosine-155) suppressed agonist-induced Gα i activation while promoting constitutive Gβγ signaling. Phosphomimicking mutations in the P loop (serine-44, serine-47, and threonine-48) suppressed G i activation entirely, thus completely segregating growth factor and GPCR pathways. As expected, most of the phosphorylation events appeared to affect intrinsic properties of Gα i proteins, including conformational stability, nucleotide binding, and the ability to associate with and to release Gβγ. However, one phosphomimicking mutation, targeting the carboxyl-terminal residue tyrosine-320, promoted mislocalization of Gα i from the plasma membrane, a previously uncharacterized mechanism of suppressing GPCR signaling through G protein subcellular compartmentalization. Together, these findings elucidate not only how growth factor and chemokine signals cross-talk through the phosphorylation-dependent modulation of Gα i but also how such cross-talk may generate signal diversity.