The Heme regulated gene (HRG)-1 encodes a 16kD transmembrane protein that is induced by IGF-1 in transformed cells and is associated with the Vacuolar - (H+) ATPase (V-ATPase) protein complex. V-ATPases pump protons across the membrane of endosomal compartments or into the extracellular environment to regulate acidification. V-ATPase expression and activity at the plasma membrane of cancer cells has been associated with an invasive phenotype. We previously showed that HRG-1 is essential for V-ATPase activity in endosomal acidification, so therefore we hypothesised that HRG-1 may also regulate extracellular pH to enhance cancer cell invasion. To test this we investigated HRG-1 expression in phenotypically distinct cancer cell lines and investigated whether it is required for V-ATPase- mediated extracellular acidification. We found that in highly invasive and migratory cell lines HRG-1 and the a1 subunit of the V-ATPase are both expressed at the plasma membrane. In less-aggressive cancer cells and in non transformed cells HRG-1 is not expressed at the cell membrane, and is restricted to the endocytic pathway. To investigate whether HRG-1 is required for V-ATPase function at the plasma membrane we stably over-expressed HRG-1 (HA-HRG-1) and empty vector (HA-EV) in MCF-7 cells and measured proton extrusion. MCF-7/HA-HRG-1 cells exhibited a greater effect on lowering the pH of the extracellular milieu compared to empty vector control cells and also exhibited a concomitant increase in cytosolic pH. The observed extracellular pH change in MCF-7/HRG-1 cells could be suppressed by the V-ATPase inhibitor Bafilomyocin A, demonstrating that this effect is V-ATPase dependent. We also observed that the expression level and activity of matrix metalloproteinase-2, a pH dependent degrading protease whose expression can be correlated with metastatic potential, was increased in MCF-7/HA-HRG-1 cells compared with control cells. Taken together, our findings suggest that HRG-1 expression at the plasma membrane of cancer cells enhances V-ATPase activity and this facilitates cell migration and invasion. Thus HRG-1 may be a suitable cell surface target for selectively disrupting V-ATPase activity and the invasive potential of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5244. doi:1538-7445.AM2012-5244