Endothelial cell surface F ₁ -F _O ATP synthase is active in ATP synthesis and is inhibited by angiostatin

Abstract

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F 1-FO ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F 1 ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F 1 ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surfaceassociated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the ␣-and ␤-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelialsurface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

Angiostatin's molecular mechanism: Aspects of specificity and regulation elucidated

“…However, an exhaustive survey of cell types and native tissues has not been conducted. To some degree, these inner mitochondrial membrane domains appear to remain discrete even after fusion with the plasma membrane, as a stable pattern of discrete microfoci is evident in immunofluorescence and co-localization imaging studies [Moser et al, 2001]. It is of interest that only one mitochondrial inner membrane protein has yet been discovered to be absent on the EC plasma membrane.…”

“…A single previous publication had reported detection of F 1 F O ATP synthase on the surface of the tumor cell line A549 [Das et al, 1994]; however, this observation was also met with skepticism and was regarded to be the likely consequence of aberrant trafficking in a genetically unstable tumor cell line or perhaps an artifact of cell culture. Since Das [1998] and Moser and co-authors [Moser et al, 1999[Moser et al, , 2001] first presented data that mammalian cells may express certain mitochondrial proteins on the plasma membrane, other investigators have described a wide variety of mitochondrial matrix proteins that have been definitively localized to the plasma membrane surface of certain cells [Soltys and Gupta, 1999]. Moreover, in many cases such matrix proteins have been shown to play functional roles in their plasma membrane locations, including P32 protein, also known as the gC1q receptor for complement protein C1q [Soltys et al, 2000].…”

“…To further characterize the relevance of cell surface F 1 F O ATP synthase, Moser and co-authors [Moser et al, 1999[Moser et al, , 2001] tested two hypotheses. First, they asked whether non-inhibitory antibodies against F 1 F O ATP synthase could block angiostatin binding and activity.…”

Amyloid precursor protein and amyloid β-peptide bind to ATP synthase and regulate its activity at the surface of neural cells

“…In this assay system, ATP generated at the cell surface and diffusing into the extracellular space of cultured cells can be determined. 19 C6 astrocytoma cells were used to measure extracellular production of ATP in the presence and absence of soluble APP-Fc and Ab (Figure 7b). In the presence of either APP-Fc or Ab, extracellular production of ATP was reduced by approximately 25-30% compared to the control peptide derived from a different extracellular domain of APP than Ab.…”

“…18 Although first described as a predominantly mitochondrial protein, the entire F 1 F 0 -ATP synthase complex has also been localized to the cell surface of endothelial cells, fibroblasts and hepatocytes, where the complex produces ATP extracellularly. [19][20][21][22] Our observations demonstrate a novel function for APP and Ab in regulating ATP synthase activity through interaction with the a subunit of ATP synthase. ATP is thus not only the main energy source of mammalian cells, but also an important extracellular signaling molecule with multiple functions.…”