The G protein ␤␥-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 ␥ 7 -subunit in mice. Notably, deletion of Gng7 caused behavioral changes that were associated with reductions in the ␣ olf -subunit content and adenylyl cyclase activity of the striatum. These data demonstrate that an individual ␥-subunit contributes to the specificity of a given signaling pathway and controls the formation or stability of a particular G protein heterotrimer.The heterotrimeric G proteins control diverse biological processes by conveying signals from cell-surface receptors to intracellular effectors. Although function was originally ascribed to the GTP-bound ␣-subunit, it is now well established that the ␤␥-dimer plays active roles in the signaling process through upstream recognition of receptors and downstream regulation of effectors (1). Molecular cloning has identified at least 5 ␤-and 12 ␥-subunit genes in the mouse and human genomes. Structurally, ␥-subunits are the most diverse, with four subgroups that show less than 50% identity to each other (2). Moreover, ␥-subunits exhibit very different temporal (3, 4) and spatial (5) patterns of expression. These characteristics suggest that ␥-subunits have heterogeneous functions. However, comparison of their biochemical properties has revealed only modest differences (6 -8), perhaps because of the inherent limitations of transfection and reconstitution approaches. Gene ablation in mice has proven to be a powerful approach to identifying the functional roles of several G protein ␣-subunits (9). We report the first use of a gene targeting strategy to identify a unique function for a member of the ␥-subunit family.The G protein ␥ 7 -subunit (G␥ 7 ) was originally cloned from bovine brain (10). In situ hybridization of rat brain sections revealed that mRNA for G␥ 7 is most highly expressed in the striatum (5), where it is found in 40 -50% of medium sized neurons in the caudate putamen (11). The regional expression of mRNA for G␥ 7 in the brain mirrors that of the striatumenriched D 1 dopamine receptor (D1R), 1 G␣ olf , and adenylyl cyclase Type V (12), suggesting involvement of G␥ 7 in the G␣ olf -mediated stimulation of adenylyl cyclase by dopamine. Single cell RT-PCR analysis confirms that D1R and G␥ 7 are expressed in the same subset of rat neurons (13). Ribozyme suppression studies support a role for G␥ 7 in the endogenous ␤-adrenergic receptor pathway (14) and the heterologously expressed D1R pathway in human embryonic kidney cells (13).

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.

Vascular endothelial growth factor (VEGF) is a major mediator of pathologic angiogenesis, a process necessary for the formation of new blood vessels to support tumor growth. Historically, VEGF has been thought to signal via receptor tyrosine kinases, which are not typically considered to be G protein dependent. Here, we show that targeted knockdown of the G protein gng2 gene (G␥ 2 ) blocks the normal angiogenic process in developing zebrafish embryos. Moreover, loss of gng2 function inhibits the ability of VEGF to promote the angiogenic sprouting of blood vessels by attenuating VEGF induced phosphorylation of phospholipase C-gamma1 (PLC␥ 1 ) and serine/threonine kinase (AKT). Collectively, these results demonstrate a novel interaction between G␥ 2 -and VEGF-dependent pathways to regulate the angiogenic process in a whole-animal model. Blocking VEGF function using a humanized anti-VEGF antibody has emerged as a promising treatment for colorectal, non-small lung cell, and breast cancers. However, this treatment may cause considerable side effects. Our findings provide a new opportunity for cotargeting G protein-and VEGFdependent pathways to synergistically block pathologic angiogenesis, which may lead to a safer and more efficacious therapeutic regimen to fight cancer. IntroductionThe zebrafish has emerged as one of the leading vertebrate models to study human diseases. 1 The significant similarity in protein sequences, conservation of developmental processes leading to organogenesis, and common appearance of pathophysiologic mechanisms all contribute to the significant advantages of using zebrafish in biomedical research. Particularly relevant to this study, zebrafish offer additional benefits for the study of angiogenesis, the process whereby new blood vessels develop from the existing vasculature. Zebrafish eggs are externally fertilized. Hence, various reagents (eg, morpholino antisense oligonucleotides and mRNA) can be readily introduced to manipulate gene expression, and an analysis of the resulting phenotype can provide a rapid survey of gene function in this system. Moreover, since developing embryos are transparent, blood vessels can be stained and visualized microscopically as a primary screen for the identification of novel genes affecting this process. Furthermore, since blood circulation is not required for the first several days of development, even those embryos showing severe defects can survive long enough for morphologic identification. To date, study of gene knockdown and ENU (N-ethyl-N-nitrosourea) mutants in this model system has revealed that blood-vessel formation is a multistep process, which is highly dependent upon growth factors such as vascular endothelial growth factor (VEGF). 2-4 Loss of VEGF or its receptor VEGFR-2 (Flk-1/KDR) leads to abnormal angiogenesis, which is characterized by loss of intersomitic vessels even though the initial establishment of the axial vasculature appears normal. [2][3][4] Here, we demonstrate that zebrafish is a viable whole-animal model for identifying ...

Here, we report the identification and expression analysis of the zebrafish G protein gammaT1 subunit gene (gngT1) during development. Similar to its human and mouse homologs, we confirm zebrafish gngT1 is expressed in the developing retina, where its transcription overlaps with the photoreceptor cell-specific marker, rhodopsin (rho). Surprisingly, we also show zebrafish gngT1 is expressed in the dorsal diencephalon, where its transcription overlaps with the pineal specific markers, arylalkylamine N-acetyltransferase-2 (annat-2) and extra-ocular rhodopsin (exorh). Analysis of the proximal promoter sequence of the zebrafish gngT1 gene identifies several conserved binding sites for the cone-rod homeobox/orthodenticle (Crx/Otx) homeodomain family of transcription factors. Using a morpholino anti-sense approach in zebrafish, we show that targeted knockdown of otx5 potently suppresses gngT1 expression in the pineal gland, whereas knockdown of crx markedly reduces gngT1 expression in the retina. Taken together, these data indicate that pineal- and retinal-specific expression of the gngT1 gene are controlled by different transcription factors and exogenous signals.