Identification of a region in G protein gamma subunits conserved across species but hypervariable among subunit isoforms

Abstract

The heterotrimeric GTP binding proteins, G proteins, consist of three distinct subunits: alpha, beta, and gamma. There are 12 known mammalian gamma subunit genes whose products are the smallest and most variable of the G protein subunits. Sequencing of the bovine brain gamma(10) protein by electrospray mass spectrometry revealed that it differs from the human protein by an Ala to Val substitution near the N-terminus. Comparison of gamma isoform subunit sequences indicated that they vary substantially more at the N-terminus than at other parts of the protein. Thus, species variation of this region might reflect the lack of conservation of a functionally unimportant part of the protein. Analysis of 38 gamma subunit sequences from four different species shows that the N-terminus of a given gamma subunit isoform is as conserved between different species as any other part of the protein, including highly conserved regions. These data suggest that the N-terminus of gamma is a functionally important part of the protein exhibiting substantial isoform-specific variation.

Fig. 1.

MALDI mass spectrum of a representative HPLC fraction containing γ₁₀, γ₂, and γ₃. This spectrum was internally calibrated with insulin and cytochrome c and is the average of 111 scans.

Fig. 2.

MS/MS of a possible γ₁₀ subunit mass by nanospray on a Finnigan LCQ mass spectrometer. (A) MS/MS spectrum of the bovine γ₁₀ [M+6H]⁶⁺ ion, m/z 1190.0 selected, average of 22 scans with labeled b (italics) and y (bold) ions. (B) Table of b and y ions predicted for the human γ₁₀ protein and the ions observed by MS/MS from (A). Those masses in bold are 27–29.6 mass units higher than that predicted for bovine γ₁₀. The † indicates ions with more than one assignment. Ions with no charge state indicated are in the 1⁺ charge state. ^az = charge state of ion. The asterisk in the spectrum is the parent ion minus the geranylgeranyl group. The bovine γ₁₀ sequence was assumed to be identical to the human γ₁₀ sequence, except for the substitution described here, for the purposes of our analysis. Predicted ions are determined from the Sherpa 3.3.1 program (Taylor et al. 1996) and monoisotopic masses are used below mass 1500.

Fig. 3.

Electrospray ionization MS/MS spectrum of a γ₁₀ tryptic peptide. (A) MS/MS spectrum of bovine γ₁₀ trypsin fragment containing amino acids 2–12, [M+2H]²⁺, m/z 552.84 selected, 1 scan, with labeled b (italics) and y (bold) ions indicating the sequence variation. (B) Table of predicted and observed ions for MS/MS spectrum of bovine γ₁₀ trypsin fragment containing amino acids 2–12. Those marked by an asterisk indicate ions which are 28 mass units higher than that predicted for bovine γ₁₀. The † indicates ions with more than one assignment. Those marked by Δ indicate ions that have lost 1 water molecule. Ions with no charge state indicated are in the 1⁺ charge state. Note: the ions labeled as 2+ are unlikely to be generated in this experiment. ^az = charge state of ion. Predicted ions are determined from the Sherpa 3.3.1 program (Taylor et al. 1996) and monoisotopic masses are used below mass 1500.

Fig. 4.

Sequence of human γ₉ (A) and human γ₁₂ (B) derived from an EST database. (A) Sequence of human γ₉ obtained from dbEST clone ID no. 190321 (accession no. AF365870) sequenced with T7 primer. The coding region for the published sequence of γ₉ (accession no. U20085) was used to search The Institute for Genomic Research (TIGR) Human cDNA Database (HCD). (B) Sequence of human γ₁₂ obtained from dbEST clone ID no. 270914 (accession no. AF365871). The coding region for the published sequence of γ₁₂ (accession no. U37561) was used to search The Institute for Genomic Research (TIGR) Human cDNA Database (HCD).

Fig. 5.

G Protein γ subunit nucleotide coding region alignment. Accession numbers for nucleotide sequences are as follows: γ₁ bovine K02436, γ₁ human S62027, γ₁ mouse AK020863, γ₂ bovine M37183, γ₂ human AA868346, γ₂ mouse NM_010315, γ₃ bovine M58349, γ₃ human AF092129, γ₃ rat frg AF022088, γ₃ mouse NM_010316, γ₄ rat frg AF022089, γ₄ mouse NM_010317, γ₄ human U31382, γ₅ rat M95780, γ₅ mouse BC002316, γ₅ bovine M95779, γ₅ human AF038955, γ₇ rat L23219, γ₇ mouse frg U38499, γ₇ bovine M99393, γ₇ human AB010414, γ₈ rat L35921, γ₈ mouse AF188180, γ₈ human AF188179, γ_9(8cone) bovine U20085, γ_9(8cone) human AF365870, γ_9(8cone) mouse AK010554, γ₁₀ rat frg AF022090, γ₁₀ mouse NM_025277, γ₁₀ human HSU31383, γ₁₁ human HSU31384, γ₁₁ mouse NM_025331, γ₁₂ rat frg AF022091, γ₁₂ mouse NM_025278, γ₁₂ bovine U37561, γ₁₂ human AF365871, γ₁₃ human AB030207 and γ₁₃ mouse AB030194. The coding regions for human γ₉ and human γ₁₂ are those for the EST clones we resequenced. (frg) Partial sequences.

Fig. 6.

Percent identity of a given G protein γ subunit isoform among species (amino acids and DNA among species) and among all γ subunit isoforms at the protein level (amino acids among isoforms). See Materials and Methods section for details on how this figure was generated. The shaded area about the line for variation in protein sequence among isoforms represents the 95% confidence interval for the average percent identies when data for each species are analyzed separately and then averaged (n = 3 or 4).