Disruption of G-protein γ5 subtype causes embryonic lethality in mice.

Heterotrimeric G-proteins modulate many processes essential for embryonic development including cellular proliferation, migration, differentiation, and survival. Although most research has focused on identifying the roles of the various αsubtypes, there is growing recognition that similarly divergent ßγ dimers also regulate these processes. In this paper, we show that targeted disruption of the mouse Gng5 gene encoding the γ5 subtype produces embryonic lethality associated with severe head and heart defects. Collectively, these results add to a growing body of data that identify critical roles for the γ subunits in directing the assembly of functionally distinct G-αßγ trimers that are responsible for regulating diverse biological processes. Specifically, the finding that loss of the G-γ5 subtype is associated with a reduced number of cardiac precursor cells not only provides a causal basis for the mouse phenotype but also raises the possibility that G-ßγ5 dependent signaling contributes to the pathogenesis of human congenital heart problems.

Synergistic roles for G-protein γ3 and γ7 subtypes in seizure susceptibility as revealed in double knock-out mice.

The functions of different G-protein αßγ subunit combinations are traditionally ascribed to their various α components. However, the discovery of similarly diverse γ subtypes raises the possibility that they may also contribute to specificity. To test this possibility, we used a gene targeting approach to determine whether the closely related γ(3) and γ(7) subunits can perform functionally interchangeable roles in mice. In contrast to single knock-out mice that show normal survival, Gng3(-/-)Gng7(-/-) double knock-out mice display a progressive seizure disorder that dramatically reduces their median life span to only 75 days. Biochemical analyses reveal that the severe phenotype is not due to redundant roles for the two γ subunits in the same signaling pathway but rather is attributed to their unique actions in different signaling pathways. The results suggest that the γ(3) subunit is a component of a G(i/o) protein that is required for γ-aminobutyric acid, type B, receptor-regulated neuronal excitability, whereas the γ(7) subunit is a component of a G(olf) protein that is responsible for A(2A) adenosine or D(1) dopamine receptor-induced neuro-protective response. The development of this mouse model offers a novel experimental framework for exploring how signaling pathways integrate to produce normal brain function and how their combined dysfunction leads to spontaneous seizures and premature death. The results underscore the critical role of the γ subunit in this process.

Adenosine A2A receptor signaling and golf assembly show a specific requirement for the gamma7 subtype in the striatum.

The adenosine A(2A) receptor (A(2A)R) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α(olf) subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the ßγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct ßγ dimers could arise from combinatorial association of the five known ß and 12 γ subunit genes. However, key questions regarding which ßγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ(7) subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ(7) protein controls the hierarchial assembly of a specific G-protein α(olf)ß(2)γ(7) heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A(2A)R activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ(7) subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A(2A)R G-protein α(olf)ß(2)γ(7) interface as a possible therapeutic target for Parkinson disease.

Mice lacking the G protein gamma3-subunit show resistance to opioids and diet induced obesity.

Contributing to the obesity epidemic, there is increasing evidence that overconsumption of high-fat foods may be analogous to drug addiction in that the palatability of these foods is associated with activation of specific reward pathways in the brain. With this perspective, we report that mice lacking the G protein gamma(3)-subunit (Gng3(-/-) mice) show resistance to high-fat diet-induced weight gain over the course of a 12-wk study. Compared with Gng3(+/+) controls, female Gng3(-/-) mice exhibit a 40% reduction in weight gain and a 53% decrease in fat pad mass, whereas male Gng3(-/-) mice display an 18% reduction in weight gain and no significant decrease in fat pad mass. The basis for the lowered weight gain is related to reduced food consumption for female and male Gng3(-/-) mice of 13% and 14%, respectively. Female Gng3(-/-) mice also show a lesser preference for high-fat chow than their female Gng3(+/+) littermates, suggesting an attenuated effect on a reward pathway associated with overconsumption of fat. One possible candidate is the micro-opioid receptor (Oprm1) signaling cascade. Supporting a defect in this signaling pathway, Gng3(-/-) mice show marked reductions in both acute and chronic morphine responsiveness, as well as increases in endogenous opioid mRNA levels in reward-related regions of the brain. Taken together, these data suggest that the decreased weight gain of Gng3(-/-) mice may be related to a reduced rewarding effect of the high-fat diet resulting from a defect in Oprm1 signaling and loss of the G protein gamma(3)-subunit.

Genetic and molecular aspects of McCune-Albright syndrome.

McCune-Albright syndrome (MAS) is characterized by the clinical triad of polyostotic fibrous dysplasia, café-au-lait pigmented skin lesions and endocrinopathy (1,2) The molecular lesion in MAS is a postzygotic mutation in the GNAS gene that leads to activation of Gsalpha, the alpha chain of the heterotrimeric G protein, Gsalpha. Cells that carry the activating mutation are distributed in a mosaic pattern. A clinical diagnosis of MAS can be made when a patient is found to have at least two features of the classical triad (3). Because of the restricted pattern of distribution of the GNAS mutation, termed gsp, initial molecular analyses were limited to lesional tissue, but recent techniques such as peptide nucleic acid clamping have improved the sensitivity of current assays and now enable the detection of gsp mutations in circulating cells from many patients with MAS.

Transcriptional profile of Rous Sarcoma Virus transformed chicken embryo fibroblasts reveals new signaling targets of viral-src.

Transformation of chicken fibroblasts in vitro by Rous Sarcoma Virus represents a model of cancer in which a single oncogene, viral src, uniformly and rapidly transforms primary cells in culture. We experimentally surveyed the transcriptional program affected by Rous Sarcoma Virus (RSV) in primary culture of chicken embryo fibroblasts. As a control, we used cells infected with non-transforming RSV mutant td106, in which the src gene was deleted. Using Affymetrix GeneChip Chicken Genome Arrays, we report 811 genes that were modulated more than 2.5 fold in the virus transformed cells. Among these, 409 genes were induced and 402 genes were repressed by viral src. From the repertoire of modulated genes, we selected 20 genes that were robustly changed. We then validated and quantified the transcriptional changes of most of the 20 selected genes by real-time PCR. The set of strongly induced genes contains vasoactive intestinal polypeptide, MAP kinase phosphatase 2 and follistatin, among others. The set of strongly repressed genes contains TGF beta 3, TGF beta-induced gene, and deiodinase. The function of several robustly modulated genes sheds new light on the molecular mechanism of oncogenic transformation.

Mice with deficiency of G protein gamma3 are lean and have seizures.

Emerging evidence suggests that the gamma subunit composition of an individual G protein contributes to the specificity of the hundreds of known receptor signaling pathways. Among the twelve gamma subtypes, gamma3 is abundantly and widely expressed in the brain. To identify specific functions and associations for gamma3, a gene-targeting approach was used to produce mice lacking the Gng3 gene (Gng3-/-). Confirming the efficacy and specificity of gene targeting, Gng3-/- mice show no detectable expression of the Gng3 gene, but expression of the divergently transcribed Bscl2 gene is not affected. Suggesting unique roles for gamma3 in the brain, Gng3-/- mice display increased susceptibility to seizures, reduced body weights, and decreased adiposity compared to their wild-type littermates. Predicting possible associations for gamma3, these phenotypic changes are associated with significant reductions in beta2 and alphai3 subunit levels in certain regions of the brain. The finding that the Gng3-/- mice and the previously reported Gng7-/- mice display distinct phenotypes and different alphabetagamma subunit associations supports the notion that even closely related gamma subtypes, such as gamma3 and gamma7, perform unique functions in the context of the organism.

Loss of G protein gamma 7 alters behavior and reduces striatal alpha(olf) level and cAMP production.

The G protein beta gamma-dimer is required for receptor interaction and effector regulation. However, previous approaches have not identified the physiologic roles of individual subtypes in these processes. We used a gene knockout approach to demonstrate a unique role for the G protein gamma(7)-subunit in mice. Notably, deletion of Gng7 caused behavioral changes that were associated with reductions in the alpha(olf)-subunit content and adenylyl cyclase activity of the striatum. These data demonstrate that an individual gamma-subunit contributes to the specificity of a given signaling pathway and controls the formation or stability of a particular G protein heterotrimer.

Ribozyme-mediated suppression of G protein gamma subunits.

Efforts to determine the sequence of the human genome have resulted in sequence information on thousand of genes. Now, the challenge is to determine the functions of this myriad of genes, including those encoding the G protein subunit families. In this chapter, we describe the successful use of ribozymes to inactivate mRNAs expressed from the G protein gamma subunit genes. Ribozymes are unique in that they can inactivate specific gene expression, and thereby can be used to help identify the function of a protein or the role of a gene in a functional cascade. Compared to other means of identifying the role of a gene (i.e., transgenic or knockout animals), ribozymes are specific and relatively easy to use. Moreover, ribozymes are able to discriminate closely related, or even mutated, sequences within gene families. Thus, in addition to elucidating functions, ribozymes have the potential to be used in treating genetic disorders associated with mutations of G protein subunits.