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Receptor-mediated changes at the myristoylated amino terminus of G α il proteins †

GPCRs catalyze nucleotide release in heterotrimeric G proteins, the slow step in G protein activation. G i/o family proteins are permanently, cotranslationally myristoylated at the extreme amino terminus. While myristoylation of the amino terminus has long been known to participate in anchoring G i proteins to the membrane, the role of myristoylation in regards to interaction with activated receptors is not known. Previous studies have characterized activation-dependent changes in the amino terminus of G α proteins in solution [Medkova, M. (2002) Biochemistry 41 , 9963-9972; Preininger, A.M. (2003) Biochemistry 42, 7931-7941 ], but changes in the environment of specific residues within the G α i1 amino terminus during receptor-mediated G i activation has not been reported. Using site-specific fluorescent labeling of individual residues along a stretch of the G α il amino terminus, we found specific changes in the environment of these residues upon interaction with activated receptor and following GTP γ S binding. These changes map to a distinct surface of the amino-terminal helix opposite the G βγ binding interface. The receptor-dependent fluorescent changes are consistent with a myristoylated amino terminus in close proximity to the membrane and/or receptor. Myristoylation affects both the rate and intensity of receptor activation-dependent changes detected at several residues along the amino terminus (with no significant effect on the rate of receptor-mediated GTP γ S binding). This work demonstrates that the myristoylated amino terminus of G α il proteins undergoes receptor-mediated changes during the dynamic process of G protein signaling. The properly localized (unmyristoylated) G α t fraction in the ROS was able to bind G βγ and undergo receptor-mediated GTP γ S binding similar to native G α t , but had defects in deactivational processes such as GTPase activity and PDE-mediated GAP activity (21). These data point to a role for myristoylation beyond that of simple membrane localization and G βγ binding. Using biochemical and biophysical studies of purified, fluorescently labeled myristoylated (and unmyristoylated G α il proteins) and membrane bound receptors, we demonstrate here that specific amino-terminal residues of G α il proteins undergo receptor-mediated changes during the dynamic process of G protein activation. To examine changes at individual positions along the amino terminus, residues of interest in the amino terminus of a G α il protein lacking solvent-exposed cysteines (G α il Hexa I) were mutated to cysteine, followed by thiol-directed fluorescent labeling. The receptor-mediated environmental changes in individual amino-terminal G α il residues reported here, and the effect of myristoylation on those changes, together shed light on receptor-mediated changes that are communicated through the amino terminus of G α il proteins.