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 ␤ 1 integrin, affecting ␤ 1 integrin N-linked glycosylation and inhibiting its function as a matrix adhesion receptor. Because of this dramatic inhibition of ␤ 1 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 ␣ 5 ␤ 1 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 ␤ 1 integrin biosynthesis indicated that 16K expression down-regulated the level of the 110-kDa biosynthetic form of ␤ 1 integrin (premature form) and, consequently, the level of the 130-kDa form of ␤ 1 integrin (mature form). Further experiments showed that the normal levels of association between the premature ␤ 1 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 ␤ 1 integrin. Interestingly, both Western blotting and a pulse-chase experiment showed co-immunoprecipitation of calnexin and 16K. These results indicate that 16K expression inhibits ␤ 1 integrin surface expression and spreading on matrix by a novel mechanism that results in reduced levels of functional ␤ 1 integrin. Vacuolar Hϩ -ATPase (V-ATPase) 1 is a multi-subunit complex found in all of the eukaryotic cells and consists of the V1 domain, which contains the ATPase activity, and the V0 domain, which contains transmembrane proton channel. V-ATPase is ubiquitously expressed in the various intracellular compartments and is responsible for their acidification (1, 2). The 16-kDa subunit (16K) of the V0 domain is a highly hydrophobic protein, consisting of four transmembrane segments, and it forms a homohexamer that functions as the transmembrane proton channel. The 16K can associate with other proteins through interactions with its fourth transmembrane segment, TMD4. For example, the papillomavirus E5 oncogene product associates with the TMD4 of 16K, decoupling 16 kDa from the ATPase V1 and resulting in an elevated pH in the Golgi and facilitation of the oncogenic transformation process. In addition, the complex between bovine papillomavirus E5 and the 16K can associate with the platelet-derived growth factor receptor, leading to a ligand-independent activation of platelet-derived growth factor receptor signaling (3).␤ 1 integrin performs a critical function in the anchorage-dependent growth of many cell types and is transcriptionally regulated by a variety of factors during development and oncogenic transformation (4 -9). The TMD4 of 16 kDa has also bee...

Transmembrane BAX Inhibitor Motif Containing (TMBIM) Family Proteins Perturbs a trans-Golgi Network Enzyme, Gb3 Synthase, and Reduces Gb3 Biosynthesis

“…As a similar example, overexpression of the V-ATPase V0-c subunit has been known to reduce expression of the ␤1 integrin through their bindings. Interestingly, the reduced level of ␤1 integrin was not restored by treatment with MG132, a proteasome inhibitor (44), which is similar to the effect of GRINA-C on Gb3 synthase as shown in Fig. 7C.…”

Heme-binding Protein HRG-1 Is Induced by Insulin-like Growth Factor I and Associates with the Vacuolar H+-ATPase to Control Endosomal pH and Receptor Trafficking

“…4B). The c subunit is known to interact with proteins other than V-ATPase components (29,30), so we also tested whether HRG-1 associates with the V-ATPase holoenzyme. HA-HRG-1 could be coimmunoprecipitated with endogenous A subunit (cytosolic V 1 domain) (Fig.…”

ATP6V0C Competes with Von Hippel-Lindau Protein in Hypoxia-Inducible Factor 1α (HIF-1α) Binding and Mediates HIF-1α Expression by Bafilomycin A1

“…Of these subunits, subunit c in V0 (ATP6V0C), which is the target protein of bafilomycin, has been found to interact with other structures independently of V-ATPase, including gap junction complexes and mediatophores (Finbow et al, 1994). It also forms a complex with papillomavirus E5 oncoprotein plus plateletderived growth factor-␤ receptor (Goldstein et al, 1992) or with ␤1 integrin (Lee et al, 2004). Thus, ATP6V0C is likely to participate in diverse cell signaling via proteinprotein interactions.…”