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Department of Pharmacology (M.L.B., H.E.H.) and Department of Medicine, Division of Cardiovascular Medicine (M.L.B.), Vanderbilt University Medical Center, Nashville, Tennessee
Received December 22, 2005; accepted December 23, 2005
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Abstract |
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12 and endothelial nitric-oxide synthase (eNOS). Based on studies characterizing the interaction of G12 and the molecular chaperone Hsp90 and the interaction of eNOS and Hsp90, the group proposed an interaction between G12 and eNOS and sought to determine the regulatory mechanisms, including the inferred dependence on Hsp90. Their experiments using an overexpression model lead to the observation that the cotransfection of G12 and eNOS expression vectors increased overall eNOS expression. Additional studies in the overexpression model and in human umbilical vein endothelial cells (HUVEC) provide evidence for a mechanism that involves G12-dependent stabilization of eNOS protein and possibly mRNA. These data present yet another paradigm by which heterotrimeric G proteins, through stabilization of target proteins, can regulate the activity of downstream signaling pathways.
, 
, and 
subunits, are intracellular signal transducers for a large number of hormones, neurotransmitters, chemokines, and autocrine and paracrine factors. These stimuli exert their physiological effects by binding to G protein-coupled receptors on the surfaces of cells. G proteins become activated by a receptor-catalyzed guanine nucleotide exchange that results in the binding of GTP to G and dissociation of G-GTP from G and the receptor (Hamm, 2001
). G-GTP and G subsequently regulate multiple downstream effectors, and G protein signals are terminated by the intrinsic GTPase activity of G and reassociation of G-GTP with G (Hamm, 1998; Cabrera-Vera et al., 2003). The 16 known types of G subunits are divided into four families: Gs, which stimulates adenylyl cyclase; Gi, which inhibits adenylyl cyclase; Gq, which stimulates phospholipase C; and G12, which stimulates p115 RhoGEF, a guanine nucleotide exchange factor for the monomeric G protein RhoA (Neves et al., 2002). The G12 family was the last family of G proteins to be discovered and comprises G12 and G13 (Strathmann and Simon, 1991). Although the involvement of G12 in cellular growth (Fukuhara et al., 2001; Radhika and Dhanasekaran, 2001), development (Dettlaff-Swiercz et al., 2005; Lin et al., 2005; Ruppel et al., 2005), apoptosis (Althoefer et al., 1997; Berestetskaya et al., 1998), and migration (Parks and Wieschaus, 1991; Offermanns et al., 1997; Gu et al., 2002; Xu et al., 2003) is well established, the mechanisms by which G12 functions in these processes, including the identities of endogenous receptors coupled to G12 activation (Riobo and Manning, 2005) and the direct effectors of G12 signaling (Kurose, 2003; Zhu et al., 2004; Andreeva et al., 2005), are only just beginning to be discovered.
In this issue of Molecular Pharmacology, Andreeva et al. (2006) explore a proposed functional link between G12 and endothelial nitric-oxide synthase (eNOS). eNOS is one of three NOS isoforms in mammalian cells that generate nitric oxide (NO) from L-arginine. Because of the growing recognition of NO as an important regulator of physiologic processes such as vasodilation, vascular permeability, neurotransmission, and thrombosis, the regulation of eNOS in disease and in response to drugs as well as the cardiovascular protective effects of eNOS/NO signaling are areas of intense research (Li et al., 2002b; Hare and Stamler, 2005). Numerous physiologic stimuli regulate eNOS by transcriptional and posttranscriptional mechanisms (Li et al., 2002a; Fleming and Busse, 2003; Sessa, 2004). The eNOS promoter exhibits proximal elements characteristic of a constitutively expressed gene and regulatory cis elements for numerous transcription factors. eNOS mRNA stability is regulated, at least in part, by the binding of cytosolic proteins to a cytidine-rich region within the 3'-untranslated region. These proteins probably render the eNOS mRNA susceptible to RNase activity, and interference with their activity seems to be one mechanism for increasing eNOS expression. eNOS activity and protein stability are regulated by posttranslational mechanisms including addition of lipid moieties (e.g., myristoylation and palmitoylation facilitate membrane association) and phosphorylation (e.g., Ser1177 phosphorylation by Akt/protein kinase B and other kinases increases eNOS activity; Thr495 phosphorylation by protein kinase C decreases eNOS activity). Another important mechanism of eNOS regulation involves protein-protein interactions. For example, eNOS activity is stimulated by calmodulin and inhibited by caveolin-1 (Fulton et al., 2001; Ostrom et al., 2004) and eNOS activity is enhanced by an interaction involving the molecular chaperone Hsp90 (Garcia-Cardena et al., 1998). Subsequent studies suggest that Hsp90 functions as a scaffold for eNOS and regulatory enzymes such as calmodulin and Akt (Gratton et al., 2000; Fontana et al., 2002).
Earlier work by Vaiskunaite et al. (2001) demonstrated an interaction between G12 and Hsp90 that is required for G12 activation of serum response element, cytoskeletal changes, and mitogenic responses. Based on this study and other published data, Andreeva et al. (2006) hypothesized a macromolecular interaction between G12 and eNOS and sought to characterize how this might impact eNOS activity and how formation of such a complex might be regulated. Using coimmunoprecipitation of overexpressed G12 and eNOS, the authors provide evidence for the existence of a complex containing the two proteins. The authors found, through two lines of evidence, that the G12-eNOS interaction in the overexpression model is independent of the activation state of the exogenous G12. The requirement of Hsp90 was explored using geldanamycin as a disruptor of Hsp90-substrate interactions. In contrast to the interactions of endogenous Hsp90 with overexpressed G12 and eNOS individually, the G12-eNOS complex is surprisingly unaffected by geldanamycin, suggesting that G12 and eNOS interact independent of Hsp90. During the course of the study, the authors observed that cotransfection of G12 and eNOS increases the overall expression of eNOS, an effect also independent of the G12 activation state. Based on the known promoter and cDNA sequences within the eNOS expression vector, the authors excluded the possibility that G12 mediates transcriptional control of eNOS. Therefore, they sought to define the effects of G12 on eNOS mRNA and protein stability using kinetic analyses in the overexpression model following treatments with the RNA polymerase inhibitor actinomycin D and the ribosome inhibitor cycloheximide. These experiments led the authors to conclude that the mechanism by which G12 increases eNOS levels involves stabilizing eNOS mRNA and protein. Using human umbilical vein endothelial cells to extend these findings to a physiologically relevant cellular model, the authors provide two lines of evidence for a functional link between endogenous G12 and eNOS proteins. The first experiment used small interfering RNA knockdown of endogenous G12 to demonstrate a parallel decrease in endogenous eNOS protein expression. The second experiment used the knowledge that eNOS expression in human umbilical vein endothelial cells is decreased by long-term treatment with thrombin (Eto et al., 2001) to correlate this finding with a parallel decrease in the expression of endogenous G12 protein.
A number of important biochemical and physiologic questions remain to be explored. First, what are the specificities of eNOS for G12 versus other G and, conversely, of G12 for eNOS versus other NOS isoforms? Andreeva et al. (2006) reveal that in addition to G12, G13 and possibly Gs can affect eNOS expression and that Gq, Gz, and G lack effects on eNOS expression (A. V. Andreeva R. Vaiskunaite, M. A. Kutuzov, J. Profirovic, R. A. Skidgel, T. Voyno-Yasenetskaya, unpublished observations). Others have shown that G13 increases inducible NOS expression in a renal epithelial cell line by transcriptional and possibly posttranscriptional mechanisms (G13 effects on inducible NOS mRNA and protein stability were not measured; Kitamura et al., 1996). Together with the current data, such findings imply that regulation of NOS isoforms may be a generalized property of the G12 family. Second, how are Hsp90 and other regulatory proteins involved in the likely macromolecular G12-eNOS complex? Although the data confirm that the individual interactions of G12 and eNOS with Hsp90 are disrupted by geldanamycin, it remains possible that persistence of the G12-eNOS interaction is an artifact of the overexpression model. However, the unpublished data of Andreeva et al. (A. V. Andreeva, R. Vaiskunaite, M. A. Kutuzov, J. Profirovic, R. A. Skidgel, T. Voyno-Yasenetskaya, unpublished observations) on the existence of a G13-eNOS interaction and the group's earlier report that Hsp90 interacts with G12 and not G13 (Vaiskunate et al., 2001) suggest that Hsp90 is not required for the interactions between eNOS and the G12 family. Whether G12 modulates the association of eNOS with other regulatory proteins (e.g., calmodulin, Akt, and caveolin-1) has yet to be tested. Third, what is the molecular basis of G protein-dependent stabilization of eNOS mRNA and protein? The effects of G12 on eNOS expression reported by Andreeva et al. (2006) are paradoxical given the cumulative evidence that signaling through Rho, a canonical G12 effector, negatively regulates eNOS expression in endothelial cells. Laufs and Liao (1998) provided the first evidence that Rho negatively regulates eNOS mRNA stability. Later studies demonstrated that the effects of thrombin on eNOS mRNA stability are dependent on Rho kinase (ROCK) and independent of Akt (Eto et al., 2001; Ming et al., 2002). Thus, it could be predicted that G12 links thrombin to Rho/ROCK via p115 RhoGEF and negatively regulates eNOS expression (Fig. 1, right arrow). Fourth, where and/or when is a G12-eNOS interaction relevant in vivo? Based on their finding that the eNOS stabilizing effects of G12 are independent of its activation state, Andreeva et al. (2006) reason that unstimulated cells may represent a physiologic context in which G12, among other mechanisms, could maintain eNOS levels (Fig. 1, left arrow). Down-regulation of G12 by long-term thrombin stimulation might then represent "negative feedback" by Rho/ROCK. Whether the thrombin-induced decrease in eNOS expression is a direct consequence of Rho/ROCK activation or is more closely linked to the G12 down-regulation observed in the current study is an open question.
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In summary, Andreeva et al. (2006) characterize a novel functional link between G12 and eNOS that involves G12-dependent stabilization of eNOS protein and possibly mRNA. This intriguing discovery may represent a newly observed mechanism by which heterotrimeric G proteins regulate the activity of downstream signaling pathways. Further studies to determine the molecular basis of the G12-dependent stabilization of eNOS and to identify other target proteins regulated by G proteins in this manner are required to validate this mechanism.
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Acknowledgements |
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Footnotes |
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Please see the related article on page 975.
ABBREVIATIONS: eNOS, endothelial nitric-oxide synthase; NOS, nitric-oxide synthase; Hsp90, 90-kDa heat shock protein; ROCK, Rho kinase.
Address correspondence to: Heidi E. Hamm, Vanderbilt University Medical Center, 2200 Pierce Ave., 442 Robinson Research Building, Nashville, TN 37232-6600. E-mail: heidi.hamm{at}vanderbilt.edu.
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