Expression of the Vacuolar H+-ATPase 16-kDa Subunit Results in the Triton X-100-insoluble Aggregation of β1 Integrin and Reduction of Its Cell Surface Expression

Abstract Vacuolar H+-ATPase functions as a vacuolar proton pump and is responsible for acidification of intracellular compartments such as the endoplasmic reticulum, Golgi, lysosomes, and endosomes. Previous reports have demonstrated that a 16-kDa subunit (16K) of vacuolar H+-ATPase via one of its transmembrane domains, TMD4, strongly associates with β1 integrin, affecting β1 integrin N-linked glycosylation and inhibiting its function as a matrix adhesion receptor. Because of this dramatic inhibition of β1 integrin-mediated HEK-293 cell motility by 16K expression, we investigated the mechanism by which 16 kDa was having this effect. Using HT1080 cells whose α5β1 integrin-mediated adhesion to fibronectin has been extensively studied, the expression of 16 kDa also resulted in reduced cell spreading on fibronectin-coated substrates. A pulse-chase study of β1 integrin biosynthesis indicated that 16K expression down-regulated the level of the 110-kDa biosynthetic form of β1 integrin (premature form) and, consequently, the level of the 130-kDa form of β1 integrin (mature form). Further experiments showed that the normal levels of association between the premature β1 integrin form and calnexin were significantly decreased by the expression of either 16 kDa or TMD4. Expression of 16 kDa also resulted in a Triton X-100-insoluble aggregation of an unusual 87-kDa form of β1 integrin. Interestingly, both Western blotting and a pulse-chase experiment showed co-immunoprecipitation of calnexin and 16K. These results indicate that 16K expression inhibits β1 integrin surface expression and spreading on matrix by a novel mechanism that results in reduced levels of functional β1 integrin.