Gγ identity dictates efficacy of Gβγ signaling and macrophage migration.

G protein βγ subunit (Gβγ) is a major signal transducer and controls processes ranging from cell migration to gene transcription. Despite having significant subtype heterogeneity and exhibiting diverse cell- and tissue-specific expression levels, Gβγ is often considered a unified signaling entity with a defined functionality. However, the molecular and mechanistic basis of Gβγ's signaling specificity is unknown. Here, we demonstrate that Gγ subunits, bearing the sole plasma membrane (PM)-anchoring motif, control the PM affinity of Gβγ and thereby differentially modulate Gβγ effector signaling in a Gγ-specific manner. Both Gβγ signaling activity and the migration rate of macrophages are strongly dependent on the PM affinity of Gγ. We also found that the type of C-terminal prenylation and five to six pre-CaaX motif residues at the PM-interacting region of Gγ control the PM affinity of Gβγ. We further show that the overall PM affinity of the Gβγ pool of a cell type is a strong predictor of its Gβγ signaling-activation efficacy. A kinetic model encompassing multiple Gγ types and parameterized for empirical Gβγ behaviors not only recapitulated experimentally observed signaling of Gβγ, but also suggested a Gγ-dependent, active-inactive conformational switch for the PM-bound Gβγ, regulating effector signaling. Overall, our results unveil crucial aspects of signaling and cell migration regulation by Gγ type-specific PM affinities of Gβγ.