Lack of N terminal palmitoylation of G protein alpha subunits reduces membrane association.

Heterotrimeric G proteins (composed of a , p and y subunits) mediate extracellular signals by coupling membrane bound receptors to intracellular second messenger systems [ 11. Despite the lack of obvious hydrophobic domains [2], G a subunits are associated with the cytoplasmic face of the membrane. This association has been proposed to be mediated by Py association [3], however studies of Goa, G i l a and Giza demonstrated that even after activation with non-hydrolyzable GTP analogues (resulting in a-&dissociation) the a subunits remained attached to the membrane [4]. Furthermore, greater amounts of a subunits are thought to be present in the cell than py [5], and the expression of a 25 fold excess of a sub-units cDNA over py still resulted in the a subunits being predominantly associated with the membrane [6]. These studies suggest that Py association alone is insufficient for membrane attachment. The N'terminus of the a subunit has been demonstrated to be involved with membrane association, as proteolytic removal of the 21 "terminal amino acids from activated Goa by trypsinization results in the release of the a subunit from the membrane [4]. Co-translational myristoylation of the "terminal glycine of some, but not all, a subunits has been shown to be required for membrane attachment [7]. A second lipid modification of palmitoylation on a subunits has recently been reported on a cysteine residue close to the N'terminal [8]. This palmitoylation is distinct from myristoylation as it is a potentially dynamic post-translational modification and as such may regulate G a localization and function [8]. We report in this communication the reduced ability of G o a to associate with the membrane when the N'terminal cysteine contained within the palmitoylation motif Met-Gly-Cys [8] is mutated to serine. The cDNAs for wild type and mutated Cys 3 to Ser 3 (C3S) G o a l expressed in the mammalian expression vector pcEXV-3 [8] were transfected into Rat-1 cells using DOTAP (Boehringer Mannheim) generating two stable cell lines named C5B and D3 respectively. Membrane associated fractions (P200) and soluble cytoplasmic fractions (S200) were generated by centrifugation at 200,OOOg for 30 minutes at 4OC. Expression of G o a l w a s detected by immunoblotting with the antibody IMl which has previously been demonstrated to specifically recognise Gocc [9]. The resulting immunoblots were densitometrically scanned which indicated that 4.6 f 4.6% of the total G o a expressed in the C5B …