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. 1999 Apr 13;96(8):4268-72.
doi: 10.1073/pnas.96.8.4268.

A mutation in the heterotrimeric stimulatory guanine nucleotide binding protein alpha-subunit with impaired receptor-mediated activation because of elevated GTPase activity

D R Warner  1 , L S Weinstein
Affiliations

A mutation in the heterotrimeric stimulatory guanine nucleotide binding protein alpha-subunit with impaired receptor-mediated activation because of elevated GTPase activity

D R Warner et al. Proc Natl Acad Sci U S A. .

Abstract

It has been reported that substitution of Arg258, a residue within the GTPase domain of the heterotrimeric guanine nucleotide binding protein (G protein) alpha-subunit (alphas), to alanine (alphas-R258A) results in decreased activation by receptor or aluminum fluoride (AlF4-) and increased basal GDP release. Arg258 interacts with Gln170 in the helical domain, and, presumably, loss of this interaction between the GTPase and helical domain leads to more rapid GDP release, resulting in decreased activation by AlF4- and increased thermolability. In this study, we mutate Gln170 to alanine (alphas-Q170A) and demonstrate that this mutant, like alphas-R258A, has decreased activation by AlF4-, increased thermolability (both reversed in the presence of excess guanine nucleotide), and an increased rate of GDP release. However, unlike alphas-R258A, alphas-Q170A does not have impaired receptor-mediated activation. Therefore, this interdomain interaction is critical to maintain normal guanine nucleotide binding (and hence normal activation by AlF4-) but is not important for receptor-mediated activation. In single turnover GTPase assays, the catalytic rate for GTP hydrolysis of alphas-R258A was 14-fold higher than normal whereas that of alphas-Q170A was unaffected. Examination of the alphas crystal structure suggests that Arg258, through interactions with Glu50, might constrain the position of Arg201, a residue critical for catalyzing the GTPase reaction. This is an example of a mutation in a heterotrimeric G protein that results in an increased intrinsic GTPase activity and provides another mechanism by which G protein mutations can impair signal transduction.

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Figures

Figure 1
Figure 1
Trypsin protection of [35S]methionine-labeled in vitro translates of αs-WT (Upper) and αs-Q170A (Lower). In each gel, the full-length undigested αs (52 kDa) is shown in the first lane, and complete digestion in the absence of activators is shown in the second lane. The next four lanes show the results after preincubation at the temperatures and in the presence of various agents indicated below. The smaller products in the left lane are attributable to initiation of protein synthesis at downstream methionine codons. Gs, stimulatory G protein.
Figure 2
Figure 2
Time course of GTPγS binding to purified αs. Bovine αs-WT and -Q170A, each with a C-terminal hexahistidine extension, were purified from E. coli, and the rate of GTPγS binding for each was determined. αs-WT (●) and -Q170A (▴) were incubated with 1 μM [35S]GTPγS (≈10,000 cpm/pmol) at 20°C for varying times, and the amount of bound GTPγS was determined (18). Each data point is the mean ± SD of triplicate determinations. The apparent on rates for GTPγS (kapp) are shown (mean ± SE of six experiments).
Figure 3
Figure 3
Single turnover GTP hydrolysis rate of purified αs isoforms. GTP hydrolysis of αs-WT (■), αs-Q170A (▴), and αs-R258A (●) at 0°C is shown as the amount of phosphate (Pi) released as a function of time. The calculated catalytic rates (kcat) also are shown. Each data point represents the mean ± SE of three experiments. Baseline counts at time zero ranged from 30 to 55% of maximum counts released. The majority of baseline counts is attributable to free inorganic phosphate contamination of the stock [32P-γ]GTP.
Figure 4
Figure 4
Detailed view of interactions between the side chains of Arg258 in switch 3, Glu50, and Arg201 based on the crystal structure of GTPγS-αs. The interaction between the side chain of Arg258 and the backbone oxygen of Gln170 also is shown. Hydrogen bonds are shown as dotted lines. The figure was generated with look 3.0 (Molecular Applications Group, Palo Alto, CA) by using coordinates for the short form of bovine GTPγS-αs [Protein Data Bank accession code 1AZT (14)], although the numbering in the figure corresponds to the long form of αs.
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