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subunits conserved across species but hypervariable among subunit isoforms
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina 29425, USA
Reprint requests to: John D. Hildebrandt, Department of Pharmacology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425; e-mail: hildebjd{at}musc.edu; fax: 843-792-2475.
(RECEIVED June 27, 2001; FINAL REVISION September 18, 2001; ACCEPTED September 18, 2001)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.26401.
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Abstract |
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, ß, and 
. There are 12 known mammalian subunit genes whose products are the smallest and most variable of the G protein subunits. Sequencing of the bovine brain 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 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 subunit sequences from four different species shows that the N-terminus of a given 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 is a functionally important part of the protein exhibiting substantial isoform-specific variation.
Keywords: G proteins; subunit; 10; mass spectrometry
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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, ß, and (Kuhn 1980; Bokoch et al. 1984; Hildebrandt et al. 1984). Seven transmembrane receptors activate G proteins by promoting the binding of GTP, which induces dissociation into an subunit and a ß dimer. These activated components independently regulate intracellular target proteins (Gilman 1987; Iniguez-Lluhi et al. 1993). There are multiple isoforms of each of the three G protein subunits. These isoforms allow a large number of possible heterotrimer combinations, which are likely to be important in the role of G proteins in signal integration in cells (Hildebrandt 1997).
G protein subunits are relatively small proteins of about 8 kD. Twelve subunit isoforms have been identified, all of which are predicted to be post-translationally modified by prenylation at the C-terminus of the mature proteins. This modification involves three sequential events: prenylation of the cysteine four residues from the C-terminus, proteolytic removal of the three C-terminal amino acids, and carboxymethylation of the new C-terminal prenylated cysteine (Clarke 1992). The type of prenyl group attached to the protein is determined by the C-terminal residue. If the C-terminal residue is Leu, the protein is geranylgeranylated (20-carbon group); however, if the C-terminal residue is Cys, Ser, Ala, Met, or Gln, the protein is farnesylated (15-carbon group) (Cox 1995). This modification is essential to the function of the G protein. Without prenylation, the resultant ß dimer does not associate with membranes, fails to permit coupling to receptors, and does not interact with downstream effector proteins such as adenyl cyclase (Iniguez-Lluhi et al. 1992; Muntz et al. 1993).
Notwithstanding the important role of subunit prenylation in G protein function, the role of subunit heterogeneity in signaling by G proteins remains to be clarified. The subunits are the least similar of the G protein subunits, with <30% identity among some isoforms, in contrast to the ß subunits, which have >90% identity for four of the five known isoforms (Hurowitz et al. 2000). Based upon these differences, it might have been predicted that the subunits would encode any specificity associated with ß dimers. Nevertheless, the ß subunit is best characterized as a determinant of downstream effector specificity (Yan and Gautam 1996, 1997 ;McIntire et al. 2001), and only the ß subunit interacts with in crystals of intact heterotrimers (Wall et al. 1995; Lambright et al. 1996). The subunit has been shown to be a determinant of receptor specificity (Kleuss et al. 1993; Kisselev and Gautam 1993), but this may in part be due to the C-terminal modifications of the subunits (Yasuda et al. 1996). One important strategy for determining the role of the specificity of G protein subunits would be to identify the structurally important elements of the subunits that might be required for their specific functions.
Here, we isolated and sequenced the bovine 10 subunit isoform from the brain and show that its sequence differs near the N-terminus from that of the cloned human protein (Ray et al. 1995). This finding led us to determine how conserved regions of the subunit isoforms are between different species. These comparisons showed that the N-terminus of is hypervariable among different subunit isoforms, but that these differences are highly conserved between different species. This identifies the N-terminus of as a likely site for isoform-specific functions of the subunits.
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Results |
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10 subunit2 from bovine brain (Wilcox et al. 1995), as well as two different variants of 5, which differ in their pattern of modification at the C-terminus (Cook et al. 1998). One technique used to characterize these proteins, acid hydrolysis, took advantage of the presence of a single acid-sensitive Asp–Pro bond in all known subunit isoforms except 10 (Cook et al. in press). Acid hydrolysis of this bond produces N-terminal (
5 kD) and C-terminal (2.5 kD) fragments characteristic of each subunit isoform. During further analysis of the potential subunits found in bovine brain extracts, many of them could be shown to produce Asp–Pro fragments typical of subunit isoforms (data not shown). In fact, when HPLC fractions from the separation of subunits in purified bovine brain G proteins were analyzed by MALDI MS after acid treatment, only one prominent protein in the subunit range, at m/z 7134.9 ± 0.7, n = 11 (average [M+H]+) was insensitive to acid treatment (data not shown). This protein was a likely candidate for the bovine brain 10 subunit, which lacks an Asp–Pro bond, but the predicted mass of the [M+H]+ ion of the human 10 protein is 7106.3 Daltons (Ray et al. 1995) and not 7134.9 Daltons.
The above results could be explained by species differences in the protein sequence of the 10 subunit isoforms found in cows versus humans. To test this possibility, HPLC fractions containing the suspected 10 isoform, as well as 2 and 3 (Fig. 1
), were analyzed on a Finnigan LCQ (ESI-ion trap) mass spectrometer (Fig 2). This instrument provides more accurate protein molecular weight estimates than MALDI-MS, and can be used to determine the sequence of the protein. MS/MS data of intact 10 ([M+6H]6+ selected at m/z 1190.0) contained two series of y ions (y13, y14, y15, y16, y17 and y44, y45, y46, y47, y48, y49, y50, y51) compatible with the known sequence of 10 with the predicted C-terminal prenylation pattern. However, the series of b ions in the spectra (b30, b31, b32, b33 and b46, b47, b48, b49, b50, b51) indicated a mass increase ranging from 27 to 29.6 Daltons greater than that predicted from the human sequence (average increase = 27.9 ± 0.16 Daltons) compatible with the mass difference observed between the human and bovine 10 proteins. These data localized the difference in mass to the first 13 residues of 10.
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10 were analyzed. The HPLC fraction containing 10 was digested with trypsin, and peptides generated from this digest were analyzed. The sample was separated by HPLC on a C-18 column in line with a Finnigan LCQ-Ion trap mass spectrometer, allowing collection of MS and MS/MS data for each eluting peptide. A mass compatible with an acetylated (minus methionine) N-terminal peptide (residues 2–12 at m/z 1104.5) 28 Daltons higher in mass than that predicted from the human sequence was identified. To determine the position of the increased mass in the N-terminal peptide, this fragment was sequenced by MS/MS analysis (Fig. 3). The b2–b5 ions and y2–y5 ions are identical to those predicted for the human 10 isoform assuming the N-terminus is acetylated after removal of methionine, as would be predicted for this protein. However, the b6–b10 ions and y6–y10 ions are all 28 mass units higher than what is predicted from the human 10 sequence. The change in mass occurred at the sixth (seventh in protein sequence) amino acid, which is Ala in the human sequence. The increase in 28 mass units at this position would be explained by a Val residue. Thus, the 7134.9 mass represents the bovine equivalent of 10, which is acetylated at the amino terminus, as predicted, with a substitution of Val7 for Ala7.
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10 isoforms had a sequence difference at the N-terminus, we wanted to determine if this was also true of other subunit isoforms. To augment human data available to us at the time of these experiments, and to answer this question, we screened the human dbEST (Expressed Sequence Tag) database for previously unreported human sequences homologous to subunits cloned from other species. From these analyses we found two possible sequences: one homologous to bovine 12 (dbEST clone ID no. 270914, accession number: AF365871) (Morishita et al. 1995), and another homologous to bovine 9 (dbEST clone ID no. 190321, accession number: AF365870) (Fig. 4) (Ong et al. 1995). Clones for these cDNAs were obtained from ATCC (American Type Culture Collection) and resequenced to verify the dbEST sequences. The human 9 sequence contained 10 amino acid differences from the published bovine sequence (Ong et al. 1995). Most importantly, these differences appeared to be randomly dispersed throughout the protein when subunits from different species were compared. The human 12 sequence had four amino acid differences from the published bovine sequence (Morishita et al. 1995), and these, too, were found dispersed throughout the protein sequence. (These conclusions about the human subunit sequences have also been reached by others independently [Ong et al. 1997; Hurowitz et al. 2000] while our work was in progress.) These data suggest that the amino acid differences in a single subunit between different species are random, and are not localized to any particular region of the protein.
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subunit sequences subunits vary between isoforms, or between species, we sought to compare all available subunit sequences. We used the sequence for the bovine 10 protein described here, and retrieved 37 partial or full G protein subunit sequences from Genbank and the mouse genome database. The relationship of the nucleotide coding region sequences of these subunits is shown in Figure 5, indicating five major subfamilies.
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10 focused our attention on the differences at the N-termini for these proteins. Comparison of all 38 subunit sequences demonstrated that overall the subunits have 62% identities, but that the N-terminus of the subunit isoforms differs substantially more than other parts of the protein (Fig.6, solid black line). The N-terminus of is the most variable part of the protein. The degree of variability is striking compared to the rest of the protein, as indicated in Figure 6.
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isoforms from different species could imply that this part of the protein is relatively unimportant to its function. Conversely, conservation of isoform differences between species could indicate that this is a primary site in the protein for determining the specific functional role for the subunit isoforms. To evaluate these ideas we analyzed all 12 different subunit isoforms cloned from as many as four mammalian species. We examined the conservation of different regions of the protein for a single isoform in several species. The results, shown in Figure 6
(protein indicated by broken gray lines, and nucleotide coding region indicated by solid gray lines), indicate that the protein and DNA sequences are highly conserved for a single isoform across species. This is just as true of the N-terminus of the protein even though it varies greatly among isoforms; and to a much greater extent than other regions of the protein. For example, the sequence of a given subunit (e.g., 2) will be very conserved over the entire length of the protein when 2 subunits from several species are compared. However, when the 2 subunit is compared to other subunits, the variation in sequence will be much greater at the N-terminus of the protein. Thus, this variation of sequence at the N-terminus for each isoform suggests that this region of the protein might be important for the specialized function of that isoform. The N-terminus is as important as other parts of the protein that are more conserved, such as regions that are involved in interactions with the ß subunit. |
Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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10 proteins. The bovine brain protein has a single amino acid difference of Ala to Val at position 7, compared to that of the human cloned 10 isoform (Ray et al. 1995). This single amino acid change can account for the increase in mass seen for bovine 10, compared to the human protein. In the DNA sequence of the genes for these proteins, this is a single base difference of C to T, indicating that the sequence of this isoform is relatively conserved. Although this conclusion is compatible with our data, we cannot be certain that the Val-to-Ala substitution at position 7 is the only difference between the human and bovine proteins. We were also able to obtain MS/MS sequencing data on the sixth trypsin fragment corresponding to residues 46–60 of the protein. No differences were evident from the analysis of this peptide. We cannot rule out the possibility that there may be other substitutions in the proteins involving Ile and Leu, or Lys and Gln, because these amino acid pairs have equivalent nominal masses and would not be differentiated by MS/MS sequencing.
This high degree of variability at the N-terminus, obvious when different isoforms were compared, was not evident when the sequences of a single isoform were compared from different species. In fact, when comparing the maximum percent identities for a single isoform from different species, the N-terminus of the protein is just as conserved as the rest of the protein at both the protein and the nucleotide (coding region) level (Fig. 6). The variation of the N-terminus for single isoforms could mean that this region is important for the specialized function of different subunits. From this, it can be hypothesized that the N-terminus of the subunits may have an important function in the cell. Some G protein subunits have known isoform-specific functions or localization. For example, 5, which has been found in focal adhesions (Hansen et al. 1996), may be regulated differently than other isoforms, possibly by unique interactions of the N-terminus of 5 with other proteins. The 12 isoform has previously been shown to be phosphorylated at a serine residue in the N-terminus by protein kinase C. This phosphorylation increases the ß dimer's affinity for subunits, but not effectors, because the unphosphorylated and phosphorylated ß12 dimer interacts with effectors to the same extent (Morishita et al. 1995). This implies that the subunit may play a role in interacting with subunits.
The N-terminus of the subunit has not yet been shown to have a specific function. It has been implicated in interactions with subunits and protects from tryptic digestion (Rahmatullah et al. 1995). The subunit also lies near one of the regions of the ß subunits that interact with the effectors (Wall et al. 1995; Lambright et al. 1996; Sondek et al. 1996), the -helical N-terminal domain of the ß subunit. Therefore, the subunit may influence interactions with those effectors binding at this region, but not with others that bind to a different site on the ß subunit. In addition, the N-terminus may be involved in uncharacterized interactions of the heterotrimer or ß dimer with other proteins. Variation of the N-terminal sequence among subunits may be an important determinant of their isoform-specific functions.
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Materials and methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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subunits subunit isoforms were separated from each other and from their associated and ß subunits in purified G protein heterotrimer by reverse-phase HPLC over a 220 x 4.6-mm Aquapore 7-µm phenyl column (Brownlee) eluted in line with a Finnigan LCQ (ESI-Ion Trap) mass spectrometer (Cook et al. 1998; Cook et al. in press). The flow was split postcolumn so that a small part of the eluent was sent to the ESI source while the remainder was collected as fractions and stored at -20°C until further analysis. Electrospray mass spectra (MS) and MS/MS mass spectra were obtained during the run.
MALDI and aspartate–proline bond cleavage
MALDI MS and acid hydrolysis of the D-P bond was performed as described previously on a PerSeptive Biosystems Voyager-DE MS instrument (Cook et al. 1998; Cook et al. in press). The flow was split postcolumn so that a small part of the eluent was sent to the ESI source while the remainder was collected as fractions and stored at -20°C until further analysis. Electrospray mass spectra (MS) and MS/MS mass spectra were obtained during the run.
MALDI and aspartate–proline bond cleavage
MALDI MS and acid hydrolysis of the D-P bond was performed as described previously on a PerSeptive Biosystems Voyager-DE MS instrument (Cook et al. 1998; Cook et al. in press).
Nanospray
Nanospray (ESI-MS/MS) was performed as described (Cook et al. in press).
Trypsin digestion of 10 and HPLC separation of tryptic peptides
Approximately 425 µL of pooled 10 fractions were dried under vacuum and resuspended in 8.4 µL 10 mM NH4HCO3 with 0.032 µg of sequencing grade trypsin (Pierce) (1:200 w/w) and incubated at 32°C for at least 12 h. The sample was again dried under vacuum and resuspended in 10 µL of 0.1 M acetic acid. Tryptic peptides were separated by HPLC as described previously in line with a Finnigan LCQ for electrospray analysis (Cook et al. in press).
Analysis of nucleotide and amino acid sequences of subunits
Sequences of cloned subunit cDNAs were identified using the GCG computer programs to search the GenBank database. A total of 37 subunit sequences were identified in the database. A dendogram was constructed for these 37 nucleotide coding sequences using Pileup (GCG Wisconsin Package). The optimal alignment of protein sequences was used to calculate the percent homology at each position of the aligned proteins. The principle adjustment made involved aligning the prenylation sequence motif at the C-terminus of the protein. Alignment of the N-terminal sequences made little difference in the overall homology scores. The percent homology was taken to be the percent of time the most frequent amino acid or nucleotide was found in a group of sequences. To account for differences in the length of the proteins at the N-terminus, we considered the denominator for percent identities only the number of proteins with an amino acid at that position. To average variation across the protein, we averaged these identity scores across a window of seven residues. This, with the exception of a very few positions, normalized any effect of difference in length of the proteins at the N-terminus. To calculate homology between isoforms, we analyzed all protein sequences in our data set for a single species and then averaged the data for all species. To calculate homology within species, we analyzed all the sequences for a given subunit isoform from different species and then averaged the values for all the isoforms.
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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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 
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 (
