Receptor- and nucleotide exchange-independent mechanisms for promoting G protein subunit dissociation

Mechanisms for heterotrimeric G protein activation that do not rely on G protein coupled receptor activation are becoming increasingly apparent. We recently identified betagamma subunit-binding peptides that we proposed bound to a hot spot on betagamma subunits, stimulating G protein dissociation without stimulating nucleotide exchange and activating G protein signaling in intact cells. AGS3, a member of the activators of G protein signaling family of proteins, also activates G protein signaling in a nucleotide exchange-independent manner, and AGS3 homologues are involved in asymmetric cell division during development. Here we demonstrate that a consensus G protein regulatory (GPR) peptide from AGS3 and related proteins is sufficient to induce G protein subunit dissociation and that both the GPR and hot spot-binding peptides promote dissociation to extents comparable with a known G protein activator, AMF. Peptides derived from adenylyl cyclase 2 and GRK2 prevented formation of the heterotrimeric complex but did not alter the rate of alpha subunit dissociation from betagamma subunits. These data indicate that these nucleotide exchange-independent G protein activator peptides do not simply compete for alpha interactions with betagamma subunits, but actively promote subunit dissociation. Thus, we propose two novel mechanisms for nucleotide exchange independent activation of G protein signaling, one that involves conformational changes in the alpha subunit and one that involves conformational changes in the betagamma subunits.