Growth factor-dependent phosphorylation of Gα 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-coupled GPCR CXCR4 through the phosphorylation of Gα. Phosphomimicking mutations in two residues in the α helix of Gα (tyrosine-154/tyrosine-155) suppressed agonist-induced Gα activation while promoting constitutive Gβγ signaling. Phosphomimicking mutations in the P loop (serine-44, serine-47, and threonine-48) suppressed G activation entirely, thus completely segregating growth factor and GPCR pathways. As expected, most of the phosphorylation events appeared to affect intrinsic properties of Gα 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 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 but also how such cross-talk may generate signal diversity.