Abstract

Covalent lipid attachments are essential co- and post-translational modifications for signalling proteins. G_s, the -subunit of the heterotrimeric G protein that activates adenylyl cyclase, is known to be palmitoylated at the third N-terminal amino acid, a cysteine. Palmitoylation is involved in anchoring G_s to the membrane by increasing its intrinsic hydrophobicity. We identified by mass spectrometry a second, functionally even more important, covalent modification. It consists of another palmitoyl residue attached to the preceding glycine (Gly²). Palmitoylation at this position has profound consequences for levels of signal transduction. It sensitizes the cell up to 200-fold for adenylyl cyclase-stimulating agents. The inhibitory inputs mediated by G_i are downregulated to <10%. Thereby, Gly²-palmitoylation of G_s relieves cellular stimulation at the level of adenylyl cyclase whereas it renders the inhibitory modulation via G_i more difficult.