Phosphorylation of Gαi shapes canonical Gα(i)βγ/GPCR signaling

A long-standing question in the field of signal transduction is to understand the interplay between distinct signaling pathways that control cell behavior. For growth factors and heterotrimeric G proteins, the two major signaling hubs in eukaryotes, the mechanisms of independent signal transduction have been extensively characterized; however, if/how they may cross talk remains obscure. Here we use linear-ion-trap mass spectrometry in combination with cell-based biophysical, biochemical, and phenotypic assays to chart at least three distinct ways in which growth factors may impact canonical G(i){beta}{gamma} signaling downstream of a GPCR (CXCR4) via phosphorylation of Gi. Phosphomimicking mutations in a cluster of residues in the E helix (Y154/Y155) result in the suppression of agonist-induced G(i) activation while promoting constitutive G{beta}{gamma} signaling; others in the P-loop (Ser44, Ser47, Thr48) suppress Gi activation entirely thus completely segregating the growth factor and GPCR pathways. While most phosphoevents appear to impact, as expected, the core properties of G(i) (conformational stability, nucleotide binding, G{beta}{gamma} association and release, etc.), one phosphomimicking mutation promoted mislocalization of Gi from the plasma membrane: a novel and unexpected mechanism of GPCR signal suppression. A phosphomutation of C-terminal Y320 was sufficient to orchestrate such suppression by protein compartmentalization. Findings not only elucidate how growth factor and chemokine signals crosstalk through phosphomodulation of Gi, but also how such crosstalk may generate signal diversity.