Abstract
Ectodomain shedding is a posttranslational modification mechanism, which liberates extracellular domains of membrane proteins through juxtamembrane processing executed mainly by the ADAM (a disintegrin and metalloprotease) family of metalloproteases. Shedding is a unique and effective mechanism for inducing multifaceted effects through the soluble extracellular domains released and/or the remaining membrane-bound portions; however, the physiological functions of shedding are not yet fully understood. In this study, we performed unbiased proteomic screening for shedding targets in a lipopolysaccharide (LPS)-stimulated macrophage cell line to elucidate a new immunological function of shedding. We identified VIP36 (36-kDa vesicular integral membrane protein), a lectin domain-containing transmembrane protein postulated as a cargo receptor for Golgi-to-endoplasmic reticulum transport, as a new target for shedding and found that the shedding of VIP36 occurs mainly on the cell surface. In addition, we demonstrate that the amount of VIP36 precisely regulates phagocytosis in macrophages and that the shedding of VIP36 is required for this regulation. These results substantially expand our knowledge of the immunological and cell biological functions of both the shedding process and VIP36 itself.
Highlights
Shedding is a processing mechanism for membrane proteins inducing multifaceted effects
VIP36 localizes mainly to the ER and Golgi [14] and is postulated to be a cargo receptor for Golgi-to-ER transport [17, 18], we show here that only the Endo H-resistant glycoform of VIP36, which should be present in the late Golgi or on the cell surface, is susceptible to shedding (Fig. 2D) and that VIP36 significantly accumulates on the cell surface upon inhibition of shedding (Fig. 2F)
These results clearly demonstrate that cell surface VIP36 is subject to shedding
Summary
Shedding is a processing mechanism for membrane proteins inducing multifaceted effects. Ectodomain shedding is a posttranslational modification mechanism, which liberates extracellular domains of membrane proteins through juxtamembrane processing executed mainly by the ADAM (a disintegrin and metalloprotease) family of metalloproteases. We performed unbiased proteomic screening for shedding targets in a lipopolysaccharide (LPS)-stimulated macrophage cell line to elucidate a new immunological function of shedding. We demonstrate that the amount of VIP36 precisely regulates phagocytosis in macrophages and that the shedding of VIP36 is required for this regulation These results substantially expand our knowledge of the immunological and cell biological functions of both the shedding process and VIP36 itself. Shedding of VIP36 Regulates Phagocytosis loproteases distantly related to ADAMs, we employed a proteomic method called two-dimensional difference gel electrophoresis (DIGE) to screen for shedding targets on a lipopolysaccharide (LPS)-stimulated macrophage cell line, Raw 264.7. We report here that VIP36 (36-kDa vesicular integral membrane protein) [9] is a shedding target and plays a role in phagocytosis in a shedding-dependent manner
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