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. 1997 Jun 10;94(12):6116-20.
doi: 10.1073/pnas.94.12.6116.

Gsalpha contains an unidentified covalent modification that increases its affinity for adenylyl cyclase

C Kleuss  1 , A G Gilman
Affiliations

Gsalpha contains an unidentified covalent modification that increases its affinity for adenylyl cyclase

C Kleuss et al. Proc Natl Acad Sci U S A. .

Abstract

Many G protein alpha subunits are dually acylated with myristate and palmitate or are palmitoylated on more than one cysteine residue near their N termini. The Galpha protein that activates adenylyl cyclase, alphas, is not myristoylated but can be reversibly palmitoylated. It appears that alphas contains another, as-yet-unidentified covalent modification that decreases its apparent dissociation constant for adenylyl cyclase from 50 nM to <0. 5 nM. This modification is at or near the N terminus of the protein and is hydrophobic. Palmitoylation of native alphas does not account for its high affinity for adenylyl cyclase.

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Figures

Figure 1
Figure 1
Activation of adenylyl cyclase. αs was purified from rabbit liver membranes (•) or after expression in Sf9 cells (▴) or E. coli (▪). The proteins were activated with GTP[γS] and reconstituted with membranes (5 μg) from Sf9 cells expressing type V adenylyl cyclase. Adenylyl cyclase activity was assayed as described. Values shown are the average of triplicate determinations and are representative of at least two experiments.
Figure 2
Figure 2
Inhibitory effect of β1γ2 on steady-state GTP hydrolysis by αs. Purified αs from rabbit liver (•, 0.39 nM) or recombinant αs synthesized in E. coli (▪, 0.5 nM) was mixed with the indicated concentrations of β1γ2 and then assayed for GTPase activity (20 min, 30°C). Rates of GTP hydrolysis in the absence of β1γ2 were 0.12 and 0.25 pmol/pmol of αs per min for the rabbit liver and recombinant protein, respectively.
Figure 3
Figure 3
Activation of adenylyl cyclase by N-terminally cleaved αs. αs was purified from rabbit liver (•) or after expression in Sf9 cells (▴) or E. coli (▪), activated with GTP[γS], and digested with 0.01 μg of LysC in 150 μl of 20 mM NaHepes, pH 8.0/1 mM EDTA/2 mM MgCl2/1 mM dithiothreitol/0.05% C12E10 containing 100 μg/ml bovine serum albumin at 30°C. Aliquots were withdrawn at the indicated times, Nα-(p-tosyl)lysine chloromethyl ketone (TLCK; 0.8 mg/ml) and aprotinin (0.4 mg/ml) were added, and protein was diluted and mixed with membranes from Sf9 cells expressing type V adenylyl cyclase (A). The proteins in duplicate aliquots were resolved electrophoretically, blotted on nitrocellulose, and visualized using an antibody specific for αs (B). (A) Adenylyl cyclase activities of the reconstituted mixtures. The final concentrations of αs during the assay were 8 nM for the rabbit liver protein and 56 nM and 67 nM for the proteins expressed in Sf9 cells and E. coli, respectively. The data shown are the average of duplicates and are representative of at least two such experiments. In the Inset, activity is expressed as a percentage of the value observed prior to exposure of αs to LysC. (B) The immunoblot was probed with antiserum 584 (29). Arrows show the position of migration of full-length αs protein (f) or the N-terminally cleaved product that accumulates during the incubation with LysC (p). Nondigested αs protein was loaded onto the gel in every other slot (appears as dots).
Figure 4
Figure 4
Phase partitioning of Gα proteins in Triton X-114. The proteins utilized were a mixture of myristoylated and nonmyristoylated αi1 and αs purified from liver or synthesized (untagged) in E. coli or Sf9 cells. Note that myristoylated and nonmyristoylated αi1 can be resolved by sodium dodecylsulfate/polyacrylamide gel electrophoresis. Approximately 1 μg of purified protein in 20 mM Tris⋅HCl, pH 7.5/150 mM NaCl was activated with GTP[γS], and a portion of each sample was then digested with 0.1 μg of LysC for 15 min at room temperature where indicated. Samples were diluted to 100 μl with 20 mM Tris⋅HCl, pH 7.5/150 mM NaCl and supplemented with 25 μl of 10% Triton X-114. The mixtures were then separated into detergent-rich (D) and aqueous (A) phases by incubation for 1 min at 30°C, followed by centrifugation at room temperature for 0.5 min at 13,000 × g. The separated phases were extracted two more times each with 10% Triton X-114 or buffer, adjusted to contain equal total volumes, and analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis and immunoblotting, using αs- or αi-specific antisera. The positions of full-length αs (f), the proteolyzed αs fragment (p), nonmyristoylated αi1 (i1), and myristoylated αi1 (myr-i1) are marked.
Figure 5
Figure 5
Treatment of native and recombinant αs with hydroxylamine. (A) αs was purified from rabbit liver (○, •) or after expression in E. coli (□, ▪). Proteins were activated with GTP[γS] and then incubated with 1 M hydroxylamine at pH 7.0 (•, ▪) or 1 M Tris⋅HCl, pH 7.0 (○, □), for 30 min at room temperature. Proteins were gel filtered prior to reconstitution at the indicated concentrations with membranes (10 μg) from Sf9 cells expressing type V adenylyl cyclase and assay of αs-stimulated adenylyl cyclase activity. (B) His6-tagged (N terminus) p21ras, αs-H6, and αs-CH6 were synthesized in Sf9 cells and labeled in vivo with [3H]palmitic acid. Proteins were enriched by Ni2+-NTA chromatography and treated with 1 M hydroxylamine (HA) or 1 M Tris⋅HCl (Tris) as described for A. Samples were then subjected to sodium dodecylsulfate/polyacrylamide gel electrophoresis and fluorography. An untreated sample is shown in the first lane for each protein. (C) Purified rabbit liver αs was mixed in 20 mM Tris⋅HCl, pH 7.0/150 mM NaCl with p21ras that had been metabolically labeled with [3H]palmitic acid; this starting material is shown in lanes 1, 2, and 6. Aliquots of this mixture were treated with palmitoyl-protein thioesterase (lane 3), 1 M Tris⋅HCl, pH 7.0 (lane 4), or 1 M hydroxylamine, pH 7.0 (lane 5) and then subjected to Triton X-114 phase partitioning as described in the legend of Fig. 4. Proteins were resolved by electrophoresis, transferred to nitrocellulose, and detected by immunoblotting (with an αs-specific antibody), autoradiography, or protein staining (with Ponceau S).
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