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G Raposo

Publications and source records attributed to G Raposo.

43 records · Page 3Linked to original sources
PubMed1993

The invariant chain induces compact forms of class II molecules localized in late endosomal compartments.

The invariant chain (Ii) binds to newly synthesized major histocompatibility complex (MHC) class II molecules and is targeted to an acidic compartment where it is degraded. To evaluate its role on the conformation and the subcellular distribution of murine MHC class II molecules we have established stable L cell transfectants expressing class II IAk heterodimers alone or in conjunction with p31 and p41 Ii chains. In these cells, class II molecules were present under three forms: alpha beta heterodimers bearing high mannose carbohydrate moieties, and fully glycosylated alpha beta heterodimers that are sensitive or resistant to sodium dodecyl sulfate dissociation at 20 degrees C. The latter class II molecules called compact heterodimers, were here highly induced in Ii-positive cells. Using in situ iodination of endosomal compartments, class II heterodimers were detected in late endosomal compartments essentially as compact forms in Ii-positive cells, and as non-compact forms in Ii-negative cells. Using confocal microscopy, IAk molecules were located in compartments distinct from early endosomes labeled with transferrin, but partially coincident with vesicles containing fluid-phase markers, and highly coincident with compartments containing large amounts of cathepsins B, D, H, and L in Ii-positive and Ii-negative cells. At the ultrastructural level, class II molecules were mostly present in multivesicular bodies, even without Ii expression. But Ii chains were needed to induce an efficient presentation of the hen egg lysozyme antigen and were sufficient to promote a major conformational change of the late endosomal, and/or lysosomal resident, class II molecules. Ii molecules are presumably playing a chaperoning function favoring the association of peptides with class II molecules in endosomal compartments.

Antigen Presentation↗
PubMed1993

Caveolae and sorting in the trans-Golgi network of epithelial cells.

VIP21 is a 21 kDa membrane protein present in TGN-derived transport vesicles isolated from the epithelial MDCK cell line. The membrane topology and subcellular localization of VIP21 were studied using antibodies against the N- and C-terminal domains. The protein was found to have a structure with little or no exposure to the exoplasmic side of the membrane. VIP21 was localized to the TGN, consistent with its presence in TGN-derived transport vesicles. Unexpectedly, it was also very abundant in the non-clathrin-coated plasma membrane invaginations called caveolae. We have previously proposed that VIP21 is associated with glycosphingolipid-enriched membrane domains in the TGN which may be involved in the sorting of proteins into vesicles directed to the apical plasma membrane. Caveolae are specialized lipid structures with similarities to the glycolipid microdomains in the TGN. The presence of VIP21 in both locations suggests that the mechanisms governing inclusion of proteins into caveolar plasma membrane domains are related to the processes of protein and lipid sorting at the TGN. This connection is confirmed by the recent finding that the amino acid sequence of VIP21 is almost identical to that of caveolin, a protein previously localized to caveolae.

Animals↗
PubMed1989

Internalization of beta-adrenergic receptor in A431 cells involves non-coated vesicles.

To characterize the mechanism of internalization of beta-adrenergic catecholamine receptors on human epidermoid A431 carcinoma cells, their distribution was analyzed by immunocytochemistry using the monoclonal anti-receptor antibody BRK2. In preconfluent cultures, the receptors appeared to be randomly distributed on the cell surface. Exposure to the agonist isoproterenol induced an overall decrease in the number of cell surface receptors as determined by binding experiments and visualized by immunofluorescence. When cells were incubated at 4 degrees C with BRK2 and anti-mouse IgG-gold and then transferred at 37 degrees C, non-coated invaginations and vesicles were labeled. The addition of isoproterenol resulted in an increased rate of internalization of the receptor-BRK2-anti-IgG-gold complex. When incubation with the two antibody reagents was prolonged (with or without isoproterenol), non-coated vesicles fused in the endosomal compartment, and receptors were transferred to multivesicular bodies and lysosomes. At no stage in this process was there any indication that clathrin-coated pits or vesicles participated. Furthermore, we found that an intracellular potassium depletion treatment known to inhibit endocytosis, did not affect the normal pattern of desensitization of beta-adrenergic receptors.

Antibodies, Monoclonal↗
PubMed1987

Redistribution of muscarinic acetylcholine receptors on human fibroblasts induced by regulatory ligands.

Distribution of the muscarinic acetylcholine receptor on human fibroblasts was determined by immunofluorescence and immunoperoxidase staining using the anti-muscarinic receptor antibody M-35b. The receptor appeared to be randomly distributed on the cell surface in 1- or 2-day nonconfluent cultures. Brief exposure to 0.1 mM carbamylcholine (15 min to 1 hr) induced receptor accumulation in several restricted domains of the cell surface. This process was associated with sequestration into uncoated vesicles. Random receptor distribution was restored by incubation in ligand-free medium for 4 hr after carbamylcholine treatment, and vesicular profiles were no longer detectable. When incubation with the agonist was prolonged (3 hr at 37 degrees C), endocytotic 'smooth vesicles' fused and formed multivesicular structures presumably implicated in receptor down-regulation. Conversely, when nonconfluent cells were exposed to the muscarinic antagonist atropine, receptor redistribution was revealed, leading to the formation of clusters where receptor accumulated. Muscarinic receptor redistribution induced by atropine therefore does not involve the sequestration process seen in carbamylcholine-treated cells.

Antibodies, Monoclonal↗