Effects of the G protein ß3-subunit gene C825T polymorphism: should hypotheses regarding the molecular mechanisms underlying enhanced G protein activation be revised? Focus on "A splice variant of the G protein ß3-subunit implicated in disease states does not modulate ion channels"

Winfried Siffert

Department of Pharmacology, University Hospital, D-45147 Essen, Germany

HETEROTRIMERIC G PROTEINS consist of a large family of {alpha}-, ß-, and {gamma}-subunits (3). They are coupled to heptahelical receptors and mediate signal transduction to intracellular effectors (2). On receptor stimulation, {alpha}- and ß{gamma}-subunits, the latter existing always in a dimeric form (5), dissociate from the stimulated receptor and modulate the activities of ion channels, the adenylyl cyclase, and phospholipases C, to name but a few. Somatic mutations in the gene GNAS1, which encodes the G{alpha}s subunit of heterotrimeric G proteins, have been implicated in some tumors and rare genetic disorders (4). Following studies on immortalized cell lines (27) and primary skin fibroblasts (17) with enhanced G protein signaling, a C825T polymorphism was detected in exon 10 of the gene GNB3, which encodes the ß3-subunit of heterotrimeric G proteins (28). The 825T allele was found to be associated with the expression of a short splice variant termed Gß3-s. This splice variant, which lacks 123 nucleotides, hypothetically gives rise to a protein with a deletion of 41 amino acids. By means of RT-PCR experiments, expression of this alternatively spliced mRNA variant was confirmed in B lymphoblasts (28), neutrophils (34), and T lymphocytes (13). While homozygous 825C allele carriers express only the wild-type gene product, homo- and heterozygous 825T allele carriers apparently express both wild-type Gß3 as well as Gß3-s mRNA. Initially it was unclear how a remote polymorphism located in exon 10 could affect splicing of exon 9, leading to the generation of Gß3-s (8). Additional polymorphisms were detected in the intron between exons 9 and 10, in the promoter region, and in the 3'-untranslated region. Interestingly, these were found to be in unusually tight linkage disequilibrium (18, 21). Hence, there exist two complex haplotypes associated with the 825T and the 825C allele which are expected to have an impact on the structure of the Gß3 pre-mRNA and may, therefore, affect splicing (21). A second aberrant splicing process was also identified which affects exon 10 (in which the C825T polymorphism is located) giving rise to a splice variant referred to as Gß3-s2. (20). By means of RT-PCR this splice variant was detected in human heart, T cells, neutrophils, peripheral blood lymphocytes, and bladder carcinoma predominantly in cells and tissues from 825T allele carriers (20).

Since G protein activation is the key event in intracellular signal transduction, it is not too surprising that the C825T polymorphism has an impact on a variety of disease processes, signal transduction in human cells and tissues, as well as responses to common drugs. In most studies, the 825T allele was shown to increase the risk for obesity (6, 25, 26), hypertension (7, 24), coronary heart disease (16, 35), stroke (15, 36), and depression (1, 38) in the white human population of European descent. By contrast, the association of the 825T allele with these disorders in other, non-Caucasian ethnicities remains controversial (911, 30, 31). Furthermore, the 825T allele serves as a pharmacogenetic marker to predict responses to diuretics (32), antidepressants (38), sildenafil (29), clonidine (14), angiotensin II (37), endothelin-1 (37), and responses to vaccination against hepatitis B (12), to name but a few.

These associations have been reported and confirmed by independent investigators and it appears that the C825T polymorphism represents a very interesting and potentially clinically relevant marker to predict the risk of disorders and drug responses. However, the molecular and biochemical mechanisms underlying these associations have largely remained obscure. There exist a number of questions whose clarification appears to be of utmost importance: Can the splice variants Gß3-s and Gß3-s2 be detected as protein by means of Western blot analysis, and what is their tissue distribution? Are truncated Gß subunits stable and can they dimerize with G{gamma} subunits and form coordinated G{alpha}ß{gamma} complexes capable of combining with heptahelical receptors? Can Gß3-s/G{gamma} complexes modulate intracellular effectors or ion channels? In fact, answering these pending questions would shed light on the mechanisms and potentially explain the contrasting phenotypes associated with the C- and T-haplotypes.

In this release of Physiological Genomics, Ruiz-Velasco and Ikeda (Ref. 22; see page 85 in this release) have used a microinjection technique to demonstrate the potential functional significance of Gß3 and Gß3-s in rat sympathetic neurons in terms of modulating N-type Ca2+ or G-protein-gated inwardly rectifying K+ channels. Moreover, these authors investigated whether Gß3-s can couple to a pertussis toxin-insensitive G{alpha}i2 mutant or to {alpha}2-adrenoceptors and whether Gß3-s can form a heterodimer with G{gamma}2. Dimerization of G protein ß-subunits with G{gamma} is required for interaction with all known effectors, as the ß{gamma}-subunits form a functional monomer (5). Their findings, at first glance, suggest that Gß3-s is functionally inactive. First, in contrast to Gß3/G{gamma}5 and Gß3/G{gamma}2, neither Gß3-s/G{gamma}5 nor Gß3-s/G{gamma}2 evoked any basal facilitation of Ca2+ currents or enhancement of Ca2+ current inhibition (Fig. 2 of Ref. 22). Second, no functional effect of Gß3-s on K+ channels could be found.

These findings are in contrast to those of others that showed a significant effect on various cell functions and signal transduction components due to overexpressing Gß3-s. Overexpression of Gß3-s in COS-7 (African green monkey kidney) cells stimulated with lysophosphatidic acid was associated with a significantly increased chemotactic migration index (compared with the expression of Gß3) (33). Functional expression of Gß3-s in COS-7 cells (confirmed by Western blot analysis) evoked a significantly enhanced activation of G proteins as monitored by GTP{gamma}S binding over that observed in mock- or Gß3-transfected cells. When Sf9 insect cells were infected with Gß3 and Gß3-s along with G{alpha}i2, G{gamma}5, and the m2-muscarinergic receptors, carbachol-stimulated binding of GTP{gamma}S was enhanced 10-fold in those cells expressing Gß3-s compared with cells expressing Gß3. These differences were not explained by large differences in the expression levels of Gß3 or Gß3-s (19). These findings would suggest that Gß3-s is capable of forming a functional heterodimer with G{gamma}5. In our own research, when Gß3-s was overexpressed in HEK-TS cells, we observed a spontaneous activation of the mitogen-activated protein kinase (MAP kinase) pathway, which was three times more pronounced than that following overexpression of Gß1, Gß3, or Gß4 (20). A straightforward interpretation of these results suggests that the truncated Gß3-s not only interacts with G{gamma} subunits but is also capable of activating intracellular effector pathways with a greater efficacy than wild-type Gß3. Hence, these latter findings are in apparent contrast to those reported by Ruiz-Velasco and Ikeda (22).

While only speculation can explain such divergent results, it must be emphasized that findings from our laboratory may be regarded as artificial as they were all obtained in systems in which Gß3-s was strongly overexpressed, e.g., in Sf9 insect cells or COS-7 and HEK TS cells, respectively. It appears reasonable to assume that such high expression levels greatly exceed those occurring under physiological conditions. An alternative explanation would be that Gß3-s, due to its structural deletion, has lost its ability to interact with Ca2+ and K+ channels but retains its ability to stimulate the MAP kinase pathway. This latter hypothesis can seemingly be ruled out, as Ruiz-Velasco and Ikeda also reported lack of dimerization of labeled Gß3-s with G{gamma} subunits and lack of formation of a G protein heterotrimer using fluorescence resonance energy transfer (FRET), which is a rather indirect though well-established method (22). We have recently expressed HA-tagged G{gamma}5, G{gamma}8, and G{gamma}12 subunits together with His-tagged Gß3 or Gß3-s subunits in HEK TSA cells. Following labeling with [35S]methionine and immunoprecipitation, we observed specific complexes of Gß3-s/G{gamma}5, Gß3-s/G{gamma}8c, and Gß3-s/G{gamma}12 suggesting dimerization. Coprecipitation experiments of in vitro translated Gß subunits and epitope-tagged G{gamma} subunits suggested dimerization of Gß3-s with G{gamma}12 and G{gamma}5 (19). However, the amounts of these latter products were significantly reduced compared with those obtained after in vitro translation of wild-type Gß3 together with these tagged G{gamma} subunits. It can, therefore, not be ruled out that Gß3-s/G{gamma} dimers form exclusively in artificial systems in which all components are strongly overexpressed. As correctly mentioned by Ruiz-Velasco and Ikeda (22), a definitive proof for the dimerization and functional activity of Gß3-s with G{gamma} subunits would require the purification and functional reconstitution of Gß3-s.

Ruiz-Velasco and Ikeda come up with an interesting hypothesis for a novel mechanism potentially explaining the alternative splicing of GNB3 associated with the 825T allele and the biological effects and phenotypes observed so far; they suggest that "the pathophysiological effects of the C825T allele may result from a ‘functional knockout’ of Gß3, i.e., something missing rather than something with too much activity" (22). This could mean that Gß3-s and Gß3-s2 mRNA is formed due to alternative splicing of GNB3 in 825T allele carriers. However, since no functional protein is formed in vivo, Gß3-s mRNA is degraded and 825T allele carriers would lack the functional wild-type Gß3. This effect should be stronger in homozygous compared with heterozygous 825T allele carriers in whom "junk Gß3-s mRNA" is formed from only one allele. Do we have any evidence for such a hypothetical scenario ? Unfortunately, Gß3 tissue distribution has rarely been studied on the protein level and available antibodies may lack sensitivity and specificity. However, the difficulties of demonstrating Gß3-s expression by means of Western blot analysis compared with the ease by which Gß3-s mRNA is detected by means of RT-PCR adds supports to the scenario implicated by Ruiz-Velasco and Ikeda. Interestingly, Ryden et al. (23) studied adrenoceptor-mediated lipolysis in fat cells from individuals with GNB3 TT, TC, and CC genotypes. A blunted response toward the lipolytic effects of compounds activating ß1- or ß2-adrenoceptors was found in fat cells from 825T allele carriers despite unchanged levels of adrenoceptors and Gi/Gs proteins. These functional studies support an association between 825T allele carrier status and an increased risk for obesity. Even more interesting is the authors’ finding regarding the protein expression of Gß3. By means of Western blot analysis, Ryden et al. (23) found significant expression of Gß3 but a lack of Gß3-s in fat cells regardless of GNB3 genotype. The major finding of their studies, however, is that the amount of wild-type Gß3 is lowest in fat cells from individuals with the TT genotype and highest in cells from individuals with the CC genotype, with cells from TC individuals displaying intermediate levels. These findings would be in full agreement with the hypotheses raised by Ruiz-Velasco and Ikeda (22).

What these findings do not yet explain is why the 825T allele is, in most cases, associated with increased rather than decreased G protein activation and intracellular signal transduction. A consistent interpretation would strongly imply that the expression level of wild-type Gß3 correlates relatively strictly with, or may even determine the efficacy of, signal transduction via a variety of G protein-coupled receptors. More precisely, higher expression of Gß3 wild-type protein as observed in 825 CC homozygous individuals would act as a "brake" for G protein activation. To this author’s knowledge, such an effect has so far not been described for Gß subunits. Nevertheless, the amount of expressed Gß3 wild-type protein may have an impact on the delicate balance between the different G protein ß- and {gamma}-subunits and their specific combination with heptahelical receptors. The findings of Ruiz-Velasco and Ikeda therefore also suggest some relatively simple experiments to be done in the near future. One way to assess the hypotheses mentioned above would be to express increasing amounts of Gß3 in cells and to measure the effect of this manipulation on specific cell responses or signal transduction events. If the hypothesis were right, then one would expect reduced cell responses with increased Gß3 expression. In our own laboratory, we have performed experiments to determine cell migration following expression of Gß3 and Gß3-s (33). Although the fold change in migrated cells was always higher in cells transfected with Gß3-s compared with Gß3, we observed in some but not all experiments that the absolute number of migrated cells was reduced in Gß3-transfected compared with mock-transfected cells. At that time this effect was not systematically investigated, since Gß3-transfected cells were regarded as the proper control for Gß3-s-transfected cells.

It would not be too surprising if alternative splicing generating "mRNA junk" would result in a reduced amount of functional wild-type protein. That such a lack of expression would, on the other hand, generate a "gain-of-function" phenotype comes as a surprise. It is the merit of Ruiz-Velasco and Ikeda to revitalize the discussion of how the GNB3 C825T polymorphism ultimately establishes a phenotype of increased G protein activation and signal transduction.

FOOTNOTES

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: W. Siffert, Dept. of Pharmacology, Univ. Hospital, Hufelandstr. 55, D-45147 Essen, Germany (E-mail: winfried.siffert{at}uni-essen.de).

10.1152/physiolgenomics.00031.2003

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