Regulation of mannose receptor synthesis and turnover in mouse J774 macrophages.

The mannose receptor, present on the plasma membrane of macrophages, promotes the internalization of glycoproteins and glycoconjugates via both endocytic and phagocytic pathways. The expression of this receptor is tightly modulated during monocyte/Mphi differentiation and cellular activation. We isolated clonal populations from murine J774 macrophage tumor cells, which differ in their surface expression of functional mannose receptors. To examine the potential mechanisms regulating receptor function in these cell lines, the interaction of receptor with ligand as well as receptor synthesis and degradation was analyzed. J774 clones with both high and low levels of mannose receptor activity were found to synthesize significant amounts of receptor protein, suggesting that the protein may be regulated at the level of synthesis and degradation. In J774 clones expressing very low receptor activity and protein, the half-life of mannose receptor molecules was substantially decreased. The evolution of multiple mechanisms modulating mannose receptor function may be critical in fine-tuning the role of this receptor in antigen processing and in scavenger and host defense functions.

Detection of biotinylated cell surface receptors and MHC molecules in a capture ELISA: a rapid assay to measure endocytosis.

Cell surface receptors and antigens, such as TfR and MHC molecules, are endocytosed and subsequently redisplayed on the plasma membrane. The internalization and recycling of MHC molecules is thought to play an important role in antigen presentation, but studying this process has been hindered due to the lack of a rapid and easily quantitated assay. The combination of a cleavable biotin reagent to label surface molecules and a capture ELISA to detect these molecules of interest, allows for the quantitation of their cell surface expression, endocytosis and recycling. The endocytosis of TfR and MHC II molecules was readily quantitated in B cell lines using this procedure with results nearly identical to previously published data using more laborious radioactive methods. Evidence for the recycling of class II antigens and TfR back to the plasma membrane was obtained by monitoring the exit of these molecules from endosomes. Exposing cells to hypertonic media blocks clathrin-dependent endocytosis and was found to inhibit the internalization of MHC class II proteins on B cells. This flexible assay to capture and quantitate the cell surface expression and endocytosis of MHC molecules and other surface antigens offers a sensitive and non-radioactive alternative to study the intracellular trafficking of diverse membrane proteins.

Biotin labeling and quantitation of cell-surface proteins.

Cell surface receptors, such as transferrin receptors and MHC molecules, are internalized into the endocytic pathway and recycled to the plasma membrane. Previous assays used to measure endocytosis and recycling were cumbersome and often required radioactive reagents. This unit describes protocols that employ the combination of a cleavable biotin reagent to label surface molecules and a capture ELISA to detect these molecules allowing for rapid and safe quantitation of cell surface protein expression, endocytosis, and recycling.