Signalosomes: delivering cardioprotective signals from GPCRs to mitochondria.

ISCHEMIC PRECONDITIONING (PC) was originally described by Murry et al. (6) in 1986. In the ensuing years, much has been learned about the signaling pathways that are activated by PC, but we still know little about how these signals reduce cell death (5). Brief intermittent periods of ischemia and reperfusion are thought to lead to the release of agonists such as adenosine, bradykinin (BK), and opioids from the heart. These agonists are thought to bind to G protein-coupled receptors (GPCRs), where they initiate a signaling cascade that leads to cardioprotection. Gi appears to be involved as the Gi inhibitor pertussis toxin blocks the protection afforded by PC. A number of kinases have been shown to be activated by PC and inhibitors of these kinases attenuate protection, suggesting a role for these kinases in PC. Among the kinases that are thought to be involved downstream of Gi protein-coupled receptors is phosphatidylinositol 3-kinase (PI3K), which phosphorylates and activates Akt. Downstream signaling from Akt activates nitric oxide (NO) synthase (NOS) isoforms and inactivates glycogen synthase kinase (GSK) through phosphorylation. The NO generated by NOS is reported to activate PKG, which, in turn, leads to the stimulation of mitochondrial PKC-ε and activation of the mitochondrial ATP-sensitive K (mitoKATP) channel, which has been reported to be an important component of the protection afforded by PC. As discussed previously, activation of these signaling pathways is thought to converge on the mitochondria, resulting in inhibition of the mitochondrial permeability transition pore (MPT). Although previous studies have identified many signaling pathways involved in PC, the challenges for the future are to understand how these signals generated by GPCRs are targeted to the mitochondria, establish the identity of the mitochondrial targets, and determine how these mitochondrial targets result in reduced cell death. The study by Quinlan et al. (8) provides some novel insights into these issues. Quinlan et al. (8), studying pharmacological PC mediated by BK, showed that after BK stimulation, the BK receptor localizes with caveolin, endothelial NOS (eNOS), and PKG in a light layer of mitochondria that they have termed the signalosome. PKG was present in this light mitochondrial layer in the absence of BK stimulation, but the BK receptor, caveolin, and eNOS were all recruited to the signalosome after stimulation of the receptor. The addition of this signalosome to mitochondria from a non-PC heart resulted in the activation of the mitoKATP channel in the non-PC mitochondria. BK-mediated activation of the mitoKATP channel was blocked by bafilomycin or methyl-cyclodextrin, suggesting a role for endosomal and caveolin signaling, respectively. These finding fit with recent data suggesting that GPCR localization in endosomal vesicles can result in unique signaling (4, 10, 11). Endosomal localization of GPCRs was originally described as part of receptor desensitization. For example, after agonist stimulation of the -adrenergic receptor, the receptor is phosphorylated by G protein receptor kinase 2 (GRK2). Phosphorylation by GRK2 recruits -arrestin, which decouples the receptor from G protein signaling. PI3K is also recruited to this complex, and the receptor complex is internalized in endosomes. This mechanism was originally thought to function solely for receptor desensitization. However, a recent study (4) has shown that the receptor-arrestin complex recruits a number of signaling kinases, such as ERK and GSK, which can result in novel signaling. For example, activation of the angiotensin receptor results in an increase in ERK in the nuclear compartment; however, overexpression of -arrestin results in increased endosomal localization of the angiotensin receptor and increased cytosolic ERK (10). Interestingly, inhibition of GRK2 signaling, by overexpression of a peptide that blocks GRK2 binding to the receptor and receptor phosphorylation, inhibits the protective effects of PC and the PCmediated increase in ERK (11). It is thus tempting to speculate that perhaps PC (or pharmacological PC) results in spatial localization of kinase signaling through the assembly of endosomal vesicles, which traffic to mitochondria and coordinate the movement of kinases to the mitochondria (see Fig. 1). These endosomal vesicles may be the source of the light mitochondrial fraction. This mechanism would allow the selective transfer of signals from the GPCR to the mitochondria. The presence of the BK receptor, caveolin, and eNOS, along with kinases such as PKG, in the light mitochondrial fraction would be consistent with BK-mediated assembly and transfer of vesicles containing these components from the plasma membrane to the mitochondria. Regarding a role for caveolin, it is also interesting to note that for the -adrenergic receptor, phosphorylation with GRK2 resulted in internalization of the receptor via clathrin-coated vesicles, whereas phosphorylation with PKA resulted in internalization of the receptor into caveolae (9). There is mounting evidence that signaling in cells does not occur by free diffusion in the cytosol. The cytosolic or whole cell levels of signals such as cAMP (1) or ERK (10) tell us little about signaling in the different regions of the cell. Rather, there is considerable evidence that adapter proteins such as -arrestin, 14-3-3, A-kinase anchoring proteins, and others assemble signaling complexes that are recruited to the appropriate compartments, likely by mechanisms that involve movement by microtubules (3) or by similar mechanisms. It is noteworthy Address for reprint requests and other correspondence: E. Murphy, Pulmonary Vascular Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bldg. 10, Rm. 7N112, 10 Center Dr., Bethesda, MD 20892 (e-mail: [email protected]). Am J Physiol Heart Circ Physiol 295: H920–H922, 2008; doi:10.1152/ajpheart.00738.2008.