Galpha(s) is palmitoylated at the N-terminal glycine

Abstract

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

Fig. 1. Mass spectrometric analysis of Gα_s. Native or recombinant Gα_s was in-gel digested and the resulting peptides analysed by MALDI-MS. Mass peaks with m/z 973.5 and 735.3 corresponded to the palmitoylated and non-palmitoylated N-terminal sequence G²CLGNSK of Gα_s, respectively. (A) LC chromatogram recorded for the native protein at m/z 973.5 and the recombinant protein at m/z 735.3. (B) MS/MS spectrum of m/z = 973.58 of native Gα_s. Relevant ions were labelled according to the accepted nomenclature and listed in Table II.

Fig. 2. LC/MS analysis. Mass chromatogram (m/z 973.5) of the peptide mixture generated by in-gel digestion of native Gα_s with Lys-C (A) and of the peptide palm-GCLGNSK prepared by solid-phase peptide synthesis (B). Samples were separated by HPLC and detected on-line by electrospray mass spectrometry. Indicated are signal intensities in arbitrary units (u).

Fig. 3. Autopalmitoylation of Gα_s at Cys³. Gα_s (2.5 µM) purified from recombinant bacteria (open circle) or native source (filled triangle for Gα_s-short, filled diamond for Gα_s-long) were incubated with 25 µM [9,10–³H]palmitoyl–coenzyme A (20 000 c.p.m./pmol) for the indicated times at 20°C. (A) Shown are ³H-images of reaction products after electrophoretic separation and transfer onto nitrocellulose membranes (upper panel), and immunostains with Gα_s-specific antiserum C-18 (lower panel). (B) Relative ³H-incorporation into each protein was calculated from band intensities of the ³H-imaging normalized to the intensity of the corresponding immunostain. Recombinant Gα_s was palmitoylated with a stoichiometry of ∼0.03 as determined by the filter binding assay described previously (Duncan and Gilman, 1996), implying >40% of native Gα_s being palmitoylated after the 2 h incubation.

Fig. 4. Autopalmitoylation of peptides at Cys. Synthetic peptides representing the N-terminus of Gα_s (P, M, O: GCLGNSK) or Gα_i (I: GCTLSAEDK) were chemically modified at the N-terminal glycine by palmitate (P), myristate (M and I) or nothing (O). Peptides were subjected to autopalmitoylation and separated by thin-layer chromatography. Radioactivity was detected by fluorography. Shown is the fluorogram of a representative developed TLC plate. Arrows indicate the position of the bi-acylated N-terminal peptide of Gα_s (lipid-s), bi-acylated N-terminal peptide of Gα_i (lipid-i1), and the substrate palmitoyl–coenzyme A near to the origin. X denotes a non-identified spot that is already present in the substrate palmitoyl–coenzyme A and is located just above the preadsorbent/silica gel interface of the plate.

Fig. 5. Stimulation of adenylyl cyclase constructs by Gα_s. Membranes (2 µg protein) containing particulate adenylyl cyclase or soluble protein (20 µg, partially purified by Ni²⁺–NTA agarose) were used. Increasing amounts of GTPγS-activated Gα_s purified from bovine brain (native; closed circle) or bacterially expressed protein (recombinant; open circles) were added. Shown are adenylyl cyclase activities of full-length particulate adenylyl cyclase type I (top), soluble adenylyl cyclase halves anchored to the plasma membrane by fusion to the transmembrane span of the CD8-receptor (CD8-C₁ + CD8-C₂; center), and soluble adenylyl cyclase (bottom). The cartoons depict the putative topology of expressed adenylyl cyclase constructs; the CD8-span of adenylyl cyclase fusion construct is coloured grey. Values are representative of one experiment out of three similar assays performed in duplicates, SD is indicated.

Fig. 6. Inhibition of adenylyl cyclase. Recombinant adenylyl cyclase type V in insect cell membranes (10 µg protein) was reconstituted with GTPγS-activated myristoylated Gα_i to give the indicated final concentrations in the presence of 0.1 nM GTPγS-bound native Gα_s (filled squares) or 20 nM GTPγS-bound recombinant Gα_s (open squares). Please note, we determined the inhibitory action of myristoylated Gα_i at comparable levels of adenylyl cyclase pre-stimulated activity by using only 1/200 the amount of Gly²-palmitoylated than non-palmitoylated Gα_s. Pre-stimulated adenylyl cyclase activity in the absence of Gα_i (100% activity) was 3.8 nmol/min/mg protein for recombinant Gα_s and 4.2 nmol/min/mg protein for native Gα_s. Values are representative of one experiment out of three similar assays, mean SEM of duplicates was below 1%.