Human HEK 293 cells present a simple and tractable system to directly test the hypothesis that the G protein ␥ subunits contribute to the specificity of receptor signaling pathways in vivo. To begin to elucidate the functions of the individual ␥ subunits in these cells, a ribozyme strategy was used to specifically inactivate the mRNA encoding the ␥ 7 subunit. A phosphorothioated DNA-RNA chimeric hammerhead ribozyme was constructed and analyzed for specificity toward the targeted ␥ 7 subunit. In vitro cleavage analysis of this ribozyme revealed a highly efficient cleavage activity directed exclusively toward the ␥ 7 RNA transcript. In particular, this ribozyme did not result in cleavage of the ␥ 12 RNA transcript, which is 75% identical to the ␥ 7 RNA transcript. Using a transient transfection assay, in vivo analysis of this ribozyme showed a specific reduction in both the mRNA and protein expression of the ␥ 7 subunit in HEK 293 cells. Coincident with this loss in ␥ 7 subunit, there was a specific reduction in the protein expression of the ␤ 1 subunit, suggesting that the ␤ 1 and ␥ 7 subunits may functionally interact to form a ␤␥ dimer in vivo. Functional analysis of the consequences of ribozyme-mediated suppression of the ␥ 7 subunit expression indicated that it was associated with significant attenuation of isoproterenol-, but not prostaglandin E 1 -, stimulated adenylylcyclase activity. Suppression of the ␥ 7 subunit expression had no effect on carbachol-and ATP-mediated stimulation of phosphatidylinositol turnover. Taken together, these results not only indicate the feasibility of using the ribozyme technology to determine the roles of individual ␥ subunits in receptor-G protein-effector pathways in vivo, but they point to a specific role of the ␥ 7 subunit in the regulation of adenylylcyclase activity in response to isoproterenol.While the role of heterotrimeric G proteins in signal transduction is well established, a central question that remains to be resolved is how the specificity of signal transduction from receptor to G protein to effector is encoded in the proteinprotein interactions between an expanding number of signaling partners. Perhaps the simplest way to encode the specificity of signal transduction would be for each type of receptor to interact with a specific G protein ␣ and ␤␥ subunit combination to converge on one or more types of effectors. In this scenario, the specific combination of G protein ␣ and ␤␥ subunits would provide the level of selectivity that is needed to interact with the different types of receptors. That ␤␥ subunits, as well as ␣ subunits, contribute to the selectivity of interaction with receptor is supported by several studies. Reconstitution of receptors with G proteins consisting of common ␣ subunits but distinct ␤␥ subunits shows a range of differences in coupling (1-3). Moreover, the differences in coupling are attributable to the ␥ component, with both the prenyl group and the structure of the ␥ subunit tail being specific determinants of receptor-G protein intera...

Oxygen tensions in the major venous inputs to the systemic and portal-vein hearts of normoxic Atlantic hagfish (12.3 +/- 1.7 and 11.0 +/- 1.6 mmHg, respectively) are low compared with typical vertebrate values. Anoxia and poisoning with cyanide and azide do not significantly affect in situ performance of the systemic heart. Idoacetate poisoning, however, results in a significant decrease in cardiac performance of the systemic heart to 12% of the initial value after 3 h. Activities of mitochondrial enzymes of hagfish ventricle suggest a small potential for aerobic metabolism compared with those in the aerobic ventricle of Atlantic cod. Activities of enzymes of carbohydrate metabolism indicate similar anaerobic capacity in hagfish and cod ventricle. The ratio of pyruvate kinase to cytochrome c oxidase, an index of anaerobic to aerobic capacity, is 5.6 times greater in hagfish than cod ventricle. Metabolite concentrations in freeze-clamped ventricles of normoxic and hypoxic hagfish indicate hypoxia-induced activation of glycogenolysis, enhanced substrate flow across 6-phosphofructokinase, and an apparent secondary constriction of glycolysis at the level of glyceraldehyde-phosphate dehydrogenase. Carbohydrate utilization via the glycolytic pathway appears essential for maintenance of cardiac performance in both normoxic and anoxic hagfish. Under conditions of severe hypoxia, ATP provision is probably met by anaerobic glycolysis.

Abstract. Signal transducing heterotrimeric G proteins are responsible for coupling a large number of cell surface receptors to the appropriate effector(s). Of the three subunits, 16 o~, 4/~, and 5 3' subunits have been characterized, indicating a potential for over 300 unique combinations of heterotrimeric G proteins. To begin deciphering the unique G protein combinations that couple specific receptors with effectors, we examined the subcellular localization of the 3' subunits. Using anti-peptide antibodies specific for each of the known 3, subunits, neonatal cardiac fibroblasts were screened by standard immunocytochemistry. The anti-3"5 subunit antibody yielded a highly distinctive pattern of intensely fluorescent regions near the periphery of the cell that tended to protrude into the cell in a fibrous pattern. Dual staining with anti-vinculin antibody showed co-localization of the 3'5 subunit with vinculin. In addition, the 3's subunit staining extended a short distance out from the vinculin pattern along the protruding stress fiber, as revealed by double staining with phalloidin. These data indicated that the 3'5 subunit was localized to areas of focal adhesion. Dual staining of rat aortic smooth muscle cells and Schwann cells also indicated co-localization of the 3'5 subunit and vinculin, suggesting that the association of the 3"s subunit with areas of focal adhesion was widespread.H ETEROTRIMERIC G proteins are responsible for transmitting signals from a large variety of ligandbound receptors to specific effector(s), which, in turn, modulate the concentrations of intracellular signals, including cyclic-AMP, inositol 1,4,5-trisphosphate, diacylglycerol, and ions such as K ÷ and Ca 2+ (reviewed in references 15, 39). Given its interposition between receptor and effector, much of the fidelity of signal transmission must reside in the ot/sy subunit structure of the G protein. While it has generally been assumed that structural heterogeneity of the ot subunits provides the specificity for receptor-G protein and G protein-etfector interactions (reviewed in reference 46), a growing body of evidence indicates that a similar structural heterogeneity of the/5 and 3' subunits exists, and that this heterogeneity, particularly in the 3' subunits, contributes to the specificities of these interactions (24,25,35,37,45). While the functional significance of the individual /5 and 3' subunits identified thus far has yet to be elucidated, evidence indicates that/5~ subunits directly regulate the activities of several effectors. The rapidly expanding family of /5"r regulated effectors include the type II and IV adenylyl cyclase (11,22,48), phospholipase A2 (23), the/5 family of phospholipase (3,6,47), muscarinic and /5-adrenergic receptor kinases (16,36), and a plasma membrane Ca 2+

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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 ...