Gα s is palmitoylated at the N‐terminal glycine

Covalent lipid attachments are essential co‐ and post‐translational modifications for signalling proteins. Gα s , the α‐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 Gα 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 (Gly 2 ). 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 Gα i are downregulated to <10%. Thereby, Gly 2 ‐palmitoylation of Gα s relieves cellular stimulation at the level of adenylyl cyclase whereas it renders the inhibitory modulation via Gα i more difficult.