Characterization of the Major Bovine Brain Go α Isoforms: MAPPING THE STRUCTURAL DIFFERENCES BETWEEN THE α SUBUNIT ISOFORMS IDENTIFIES A VARIABLE REGION OF THE PROTEIN INVOLVED IN RECEPTOR INTERACTIONS

William E Mcintire,Jane Dingus,Kevin L Schey,John D Hildebrandt

doi:10.1074/jbc.273.50.33135

Abstract

Go is the major G protein in bovine brain, with at least three isoforms, GoA, GoB, and GoC. Whereas alphaoA and alphaoB arise from a single Goalpha gene as alternatively spliced mRNAs, alphaoA and alphaoC are thought to differ by covalent modification. To test the hypothesis that alphaoA and alphaoC have different N-terminal lipid modifications, proteolytic fragments of alphao isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The major masses observed in immunoprecipitates were the same for all three alphao isoforms and corresponded to the predicted mass of a myristoylated N-terminal fragment. Structural differences between alphaoA and alphaoC were also compared before and after limited tryptic proteolysis using SDS-polyacrylamide gel electrophoresis containing 6 M urea. Based upon the alphao subunit fragments produced under activating and nonactivating conditions, differences between alphaoA and alphaoC were localized to a C-terminal fragment of the protein. This region, involved in receptor and effector interactions, implies divergent signaling roles for these two alphao proteins. Finally, the structural difference between alphaoA and alphaoC is associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry.

Highlights

Go is the major G protein in bovine brain, with at least three isoforms, GoA, GoB, and GoC
To test the hypothesis that ␣oA and ␣oC have different N-terminal lipid modifications, proteolytic fragments of ␣o isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry
The structural difference between ␣oA and ␣oC is associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry

Summary

EXPERIMENTAL PROCEDURES

Purification of G Protein Isoforms—G proteins were purified from bovine brain using a modification [33, 34] of the method of Sternweis and Robishaw [2]. One to two ␮g of purified Go or ␣o isoform was incubated with 20 mM Tris, pH 8.0, 0.1% Thesit, 1 mM EDTA, 1 mM DTT, 150 mM NaCl, 25 mM MgCl2, either 1 ␮M GTP␥S (Boehringer Mannheim) or 100 ␮M GDP (Sigma), and trypsin (1:25 (w/w) for GTP␥S and 1:50 (w/w) for GDP). Antiserum (1:25) in 300 ␮l of 20 mM Tris, pH 8.0, 1 mM EDTA, 1 mM DTT, 20 mM NaCl, and 0.01% Thesit (TEDNT) was incubated with 5 ␮l of beads at room temperature for 1 h. 1. Linear map of ␣o, and immunoreactivity and SDS-polyacrylamide gel electrophoretic mobility of ␣o isoforms. MacBioSpec version 1.0.1 (PE SCIEX Instruments, Thornhill, Canada) was used to generate predicted ion m/z values

RESULTS

DISCUSSION

Methods