Enhancement of endocytosis due to aminophospholipid transport across the plasma membrane of living cells.
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Formation of intracellular vesicles is initiated by membrane budding. Here we test the hypothesis that the plasma membrane surface area asymmetry could be a driving force for vesicle formation during endocytosis. The inner layer phospholipid number was therefore increased by adding exogenous aminophospholipids to living cells, which were then translocated from the outer to the inner layer of the membrane by the ubiquitous flippase. Addition of either phosphatidylserine or phosphatidylethanolamine led to an enhancement of endocytosis, showing that the observed acceleration does not depend on the lipid polar head group. Conversely, a closely related aminophospholipid that is not recognized by the flippase, lyso-alpha-phosphatidylserine, inhibited endocytosis, and similar results were obtained with a cholesterol derivative that also remains in the plasma membrane outer layer. Thus an increase of lipid concentration in the inner layer enhanced internalization, whereas an increase of the lipid concentration in the outer layer inhibited internalization. These experiments suggest that transient asymmetries in lipid concentration might contribute to the formation of endocytic vesicles. (+info)
Inhibition of clathrin-coated pit assembly by an Eps15 mutant.
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Recent data have shown that Eps15, a newly identified component of clathrin-coated pits constitutively associated with the AP-2 complex, is required for receptor-mediated endocytosis. However, its precise function remains unknown. Interestingly, Eps15 contains three EH (Eps15-Homology) domains also found in proteins required for the internalization step of endocytosis in yeast. Results presented here show that EH domains are required for correct coated pit targeting of Eps15. Furthermore, when cells expressed an Eps15 mutant lacking EH domains, the plasma membrane punctate distribution of both AP-2 and clathrin was lost, implying the absence of coated pits. This was further confirmed by the fact that dynamin, a GTPase found in coated pits, was homogeneously redistributed on the plasma membrane and that endocytosis of transferrin, a specific marker of clathrin-dependent endocytosis, was strongly inhibited. Altogether, these results strongly suggest a role for Eps15 in coated pit assembly and more precisely a role for Eps15 in the docking of AP-2 onto the plasma membrane. This hypothesis is supported by the fact that a GFP fusion protein encoding the ear domain of (alpha)-adaptin, the AP-2 binding site for Eps15, was efficiently targeted to plasma membrane coated pits. (+info)
Direct demonstration of the endocytic function of caveolae by a cell-free assay.
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The endocytic function of caveolae was challenged by taking advantage of a cell-free assay directly measuring the detachment of receptor-containing vesicles from isolated plasma membranes. Plasma membranes from cultured cells surface-labeled with 125I-cholera toxin (segregating in caveolae) were isolated as described previously. Following incubation of these labeled membranes in the presence of nucleotide(s) and cytosol, a significant proportion of the initially membrane-associated radioactivity was released into the incubation medium in sedimentable form (14*10(6 )g). Results of biochemical, morphological, and fractionation analysis of the material containing the released radioactivity directly demonstrated that caveolae are plasma membrane domains involved in an endocytic process and resulting in the formation of caveolae-derived vesicles. In addition, these studies allowed a direct comparison of caveolae- and clathrin-coated pit-mediated endocytosis and reveal that these two processes diverge in terms of kinetics, cytosol and nucleotide requirements as well as in terms of the density and size of the endocytic vesicles formed. (+info)
Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator.
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Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel give rise to the most common lethal genetic disease of Caucasian populations, CF. Although the function of CFTR is primarily related to the regulation of apical membrane chloride permeability, biochemical, immunocytochemical, and functional studies indicate that CFTR is also present in endosomal and trans Golgi compartments. The molecular pathways by which CFTR is internalized into intracellular compartments are not fully understood. To define the pathways for CFTR internalization, we investigated the association of CFTR with two specialized domains of the plasma membrane, clathrin-coated pits and caveolae. Internalization of CFTR was monitored after cell surface biotinylation and quantitation of cell surface CFTR levels after elution of cell lysates from a monomeric avidin column. Cell surface levels of CFTR were determined after disruption of caveolae or clathrin-coated vesicle formation. Biochemical assays revealed that disrupting the formation of clathrin-coated vesicles inhibited the internalization of CFTR from the plasma membrane, resulting in a threefold increase in the steady-state levels of cell surface CFTR. In contrast, the levels of cell surface CFTR after disruption of caveolae were not different from those in control cells. In addition, although our studies show the presence of caveolin at the apical membrane domain of human airway epithelial cells, we were unable to detect CFTR in purified caveolae. These results suggest that CFTR is constitutively internalized from the apical plasma membrane via clathrin-coated pits and that CFTR is excluded from caveolae. (+info)
Cutting edge: receptor-mediated endocytosis of heat shock proteins by professional antigen-presenting cells.
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Immunization with heat shock proteins (HSPs) induces Ag-specific CTL responses. The specificity of the immune response is based on peptides associated with HSPs. To investigate how exogenous HSP/peptide complexes gain access to the MHC class I-restricted Ag presentation pathway, we incubated the monocytic cell line P388D1 and the dendritic cell line D2SC/1 with gold-labeled HSPs gp96 and HSC70. We show that HSPs bind specifically to the surface of these APCs and are internalized spontaneously by receptor-mediated endocytosis, demonstrating the existence of specific receptors for HSPs on these cells. In addition, we observe colocalization of internalized HSPs and surface MHC class I molecules in early and late endosomal structures. These findings provide possible explanations for the immunogenicity of HSP/peptide complexes and for the transfer of HSP-associated peptides onto MHC class I molecules. (+info)
Bradykinin-induced internalization of the human B2 receptor requires phosphorylation of three serine and two threonine residues at its carboxyl tail.
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The binding of bradykinin (BK) to B2 receptor triggers the internalization of the agonist-receptor complex. To investigate the mechanisms and the receptor structures involved in this fundamental process of receptor regulation, the human B2 receptor was mutated within its cytoplasmic tail by complementary strategies of truncation, deletion, and amino acid substitution. Ligand binding, signal transduction, internalization as well as phosphorylation were studied for the mutated receptors expressed in COS, CHO, and HEK 293 cells. Truncation of 44 out of 55 amino acid residues of the receptor's cytoplasmic tail corresponding to positions 321-364 did not alter the kinetics of BK binding and the receptor coupling to phospholipase C and phospholipase A2. By contrast, truncations after positions 320 and 334, deletions within the segment covering positions 335-351, as well as alanine substitution of serine and threonine residues within segment 335-351 diminished the internalization capacity of the mutant receptors. Mutants with a markedly reduced internalization potential failed to produce BK-induced receptor phosphorylation suggesting that phosphorylation may be involved in receptor internalization. The mutagenesis approaches converged at the conclusion that three serines in positions 339, 346, and 348 and two threonines in positions 342 and 345, contained in a sequence segment that is highly conserved between species, have a critical role in the ligand-dependent internalization and phosphorylation of kinin receptors and can intervene in these processes in an alternative manner. However, mutants lacking these residues were still sensitive to dominant-negative forms of beta-arrestin and dynamin, suggesting the existence of additional receptor structure(s) involved in the receptor sequestration through clathrin-coated vesicles. (+info)
Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis.
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Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner. (+info)
Functional properties of leptin receptor isoforms: internalization and degradation of leptin and ligand-induced receptor downregulation.
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Long (ObRb) and short (ObRa) leptin receptor isoforms are thought to play essential roles in mediating leptin signaling and the transport and degradation of leptin, respectively. Although the capacity of these cloned receptor species to mediate signal transduction has been reported, there is no information on the ability of individual receptor species to mediate leptin internalization and degradation or to undergo ligand-induced downregulation. We therefore studied these parameters in Chinese hamster ovary (CHO) cells stably expressing either ObRa or ObRb isoforms of the leptin receptor. We determined that both ObRa and ObRb mediated internalization of 125I-labeled leptin by a temperature- and coated pit-dependent mechanism. Both ObRa and ObRb also mediated degradation of 125I-leptin by a lysosomal mechanism, and this was more efficiently mediated by ObRa in these cells. Neither leptin internalization nor degradation by ObRa was affected by mutation of the conserved Box 1 motif. By studying deletion mutants of ObRa, we found that efficient internalization was dependent on a motif located between amino acids 8 and 29 of the intracellular domain of ObRa. Exposure of cells expressing ObRa or ObRb to unlabeled leptin for 90 min at 37 degrees C produced downregulation of available surface receptors, and this effect was of greater magnitude in cells expressing ObRb. Whereas CHO cells expressing the growth hormone receptor showed marked downregulation of ligand binding after exposure to dexamethasone (DEX) or phorbol myristic acid (PMA), PMA had no effect on expression of ObRa or ObRb, and DEX reduced binding to cells expressing ObRb by 15%. Thus, the two leptin receptor isoforms, ObRa and ObRb, mediate leptin internalization by a coated pit-dependent mechanism, leptin degradation by a lysosomal pathway, and ligand-induced receptor downregulation. The differential capacity of the two receptor isoforms may relate to the different roles of the receptor isoforms in the biology of leptin. (+info)