The role of palmitoylation of the guanine nucleotide binding protein G11α in defining interaction with the plasma membrane

Abstract

Mutations of Cys-9 to serine, Cys-10 to serine and a combination of both alterations were produced in a cDNA encoding murine G11 alpha to potentially interfere with the ability of the expressed polypeptides to act as substrates for post-translational palmitoylation. Each of these mutants and the wild-type protein were expressed in simian COS-1 cells. Mutation of either cysteine-9 or cysteine-10 decreased the degree of palmitoylation of the protein by some 80% compared with the wild-type, while the double mutant totally failed to incorporate [3H]palmitate. By contrast, in all transfections the endogenously expressed simian G11 alpha incorporated [3H]palmitate to similar levels. Particulate and cytoplasmic fractions from these cells were subjected to SDS/PAGE under conditions which allow resolution of primate and rodent forms of G11 alpha. Immunoblotting of these fractions demonstrated that in all cases the endogenously expressed simian G11 alpha was exclusively associated with the particulate fraction, as was the transfected and expressed wild-type murine G11 alpha. By contrast, each of the mutated forms of murine G11 alpha displayed a distribution in which approx. 70% of the expressed protein was present in the particulate fraction and 30% in the supernatant. To examine the conformation of the particulate expressed forms of murine G11 alpha, these fractions were treated with various concentrations of sodium cholate and immunoblots were subsequently performed on the solubilized and remaining particulate proteins. Whereas essentially all of the endogenous simian G11 alpha was solubilized by treatment with 1% (w/v) sodium cholate and some 50% with 0.32% cholate, expressed wild-type murine G11 alpha was more recalcitrant to solubilization. However, that fraction of wild-type murine G11 alpha which was solubilized behaved identically to the endogenous simian G11 alpha on Superose-12 gel-exclusion chromatography. The particulate fraction of the C9S/C10S double mutant of murine G11 alpha was highly resistant to solubilization by sodium cholate, whereas the particulate fractions of the two single cysteine to serine mutants were intermediate to the wild-type and double mutant in their ability to be solubilized by this detergent. These data demonstrate that the palmitoylation status of the cysteine residues at positions 9 and 10 in murine G11 alpha plays a central role in defining membrane association of this G-protein and indicate that much of the particulate fraction of the expressed palmitoylation-resistant mutants is likely to represent non-functional rather than correctly folded protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Coordinated Agonist Regulation of Receptor and G Protein Palmitoylation and Functional Rescue of Palmitoylation-deficient Mutants of the G Protein G11α following Fusion to the α1b-Adrenoreceptor

“…2). Both of these mutants have been shown to partition between membrane and cytosol much less effectively than wild type G␣ 11 (20,21), and this thus restricts their relative membrane concentration compared with the receptor.…”

“…We have previously demonstrated that G␣ 11 can be post-translationally acylated on both Cys 9 and Cys 10 (20) and that mutation of both of these amino acids to Ser prevents membrane association of the G protein (20,21). Co-transfection of EF88 cells with the ␣ 1b -adrenoreceptor and C9S,C10S G␣ 11 thus also failed to result in a phenylephrine-mediated elevation of [Ca 2ϩ ] i (Fig.…”

“…In the first step, the coding sequence of each form of G␣ 11 (20,21) was modified by PCR amplification using the amino-terminal primer 5Ј-GAGGACGGTACCACTCTG-GAGTCCATG-3Ј the initiating Met of G␣ 11 was removed, and both a KpnI restriction site (underlined) and a two-amino acid spacer (GlyAsn) were introduced. Using the C-terminal primer 5Ј-TTGTGCGGC-CGCCGGTCACACCAGGTT-3, a NotI restriction site (underlined) was introduced downstream of the stop codon of G␣ 11 .…”

“…This has been best examined for the adenylyl cyclase stimulatory G protein G s , where activation mediated by GPCRs or cholera toxin has been shown to alter [ 3 H]palmitoylation of the G protein (17)(18)(19). G␣ 11 possesses two adjacent cysteine residues at positions 9 and 10, which have been established to be sites of acylation (20,21). Mutation of these two cysteine residues results in production of a soluble polypeptide.…”

Site-specific analysis of protein S-acylation by resin-assisted capture

“…3B . These sites include Cys 9 and Cys 10 within the ␣ -subunit of the heterotrimeric G-protein, G 11 ( 32 ), Cys 181 and Cys 184 of H-Ras ( Fig. 2A, B, and see reference [ 27 ]), as well as 6 Cys residues within SNAP23, of which 4 are conserved in its betterstudied homolog SNAP25, a known S -palmitoylated protein ( 33 ).…”