The mechanism of receptor‐mediated endocytosis

Smythe, Elizabeth; Warren, Graham

doi:10.1111/j.1432-1033.1991.tb16424.x

Cited by 153 publications

(51 citation statements)

References 149 publications

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“…Cargo receptors are continuously present in coated pits independent of their occupancy by ligand, while signaling receptors concentrate in coated pits only upon ligand binding (24,28,(34)(35)(36). Internalization of both types of receptors requires endocytic codes (14).…”

Section: Resultsmentioning

confidence: 99%

Ligand-induced endocytosis of epidermal growth factor receptors that are defective in binding adaptor proteins.

Nesterov

Wiley

Gill

1995

Proc. Natl. Acad. Sci. U.S.A.

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Ligand-activated epidermal growth factor receptors (EGFRs) associate with coated pit adaptor proteins (AP2) in vivo, implying a mechanism for receptor retention in coated pits during internalization. Using an in vitro binding assay, we localized the adaptor binding determinant to residues 970-991 of EGFRs and confirmed specificity by competition with a synthetic peptide corresponding to this sequence. A mutant EGFR lacking this AP2 binding determinant did not associate with AP2 in vivo but demonstrated internalization and down-regulation kinetics indistinguishable from its wildtype counterpart. Immunocytochemistry confirmed ligandinduced internalization of the mutant EGFR. These data suggest that endocytic determinants are distinct from AP2 binding determinants and that processes other than association with AP2 regulate endocytosis of EGFRs.Adaptor proteins anchor clathrin lattices to the cytosolic face of the plasma membrane and are an important structural component of coated pits (1, 2). The plasma membrane adaptor (AP2) is a heterotetramer consisting of two -100-kDa proteins (a-and 3-adaptins) and two smaller subunits of 50 and 16 kDa (3). The subunit composition distinguishes AP2 from the Golgi complex adaptor AP1. It has been proposed that, in addition to a well-established structural function involved in clathrin assembly and coated pit formation (1-5), AP2 complexes also participate in specific retention of receptors in coated pits and thus regulate their endocytosis (1, 6). In support of this hypothesis, in vitro binding studies have shown interaction of AP2 with the cytoplasmic domains of the low density lipoprotein receptor, the mannose 6-phosphate receptor, the asialoglycoprotein receptor, and lysosomal acid phosphatase (6-9). Binding in vitro appeared to require defined cytoplasmic sequences initially identified by their requirement for receptor internalization in vivo. These endocytic determinants or "codes" consist of 4-6 amino acids including an essential aromatic residue that adopt a tight turn configuration (10-14). Despite requisite specificity, the affinity of AP2 binding to receptor sequences in vitro is low and of unproven functional importance. A significant observation made by Sorkin and Carpenter (15) was that the epidermal growth factor receptor (EGFR) binds AP2 in vivo with high affinity in a temperature-and EGF-dependent fashion. It was subsequently shown that binding occurs through direct interaction between AP2 and EGFR molecules (16) and is regulated by tyrosine autophosphorylation of EGFR that introduces a conformational change in the receptor molecule, which exposes AP2 binding determinants (17).Two lines of evidence indicate that the AP2 binding region is found in the regulatory C terminus of EGFRs. Deletion of the C terminus abolishes AP2 binding in vivo, whereas highaffinity association between a recombinant EGFR C terminus and purified AP2 can be shown in vitro (16,17). Because the three regions responsible for EGFR endocytosis are also found in the same 228-amino aci...

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Section: Resultsmentioning

confidence: 99%

Ligand-induced endocytosis of epidermal growth factor receptors that are defective in binding adaptor proteins.

Nesterov

Wiley

Gill

1995

Proc. Natl. Acad. Sci. U.S.A.

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“…These distinct pathways include clathrin-mediated, caveolae/lipid raft-mediated, and clathrin-and caveolae-independent endocytosis. The classically described route for entry of proteins into cells is via clathrin-coated pits, whereby proteins bind to specific receptors on the cell surface, and these ligand⅐receptor complexes cluster and are internalized in an energy-dependent manner by clathrin-coated membrane vesicles (27). To examine the role of clathrin-coated pits in C105Y uptake, stable HuH7-GFP and HuH7-⌬Eps15 cell lines were created expressing either EGFP or EGFP-DIII, respectively (Fig.…”

Section: Table 1 Primary Structures Of Peptides Used In This Studymentioning

confidence: 99%

Mechanism of Uptake of C105Y, a Novel Cell-penetrating Peptide

Rhee

Davis

2006

Journal of Biological Chemistry

143

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C105Y, a synthetic peptide (CSIPPEVKFNKPFVYLI) based on the amino acid sequence corresponding to residues 359 -374 of ␣1-antitrypsin, enhances gene expression from DNA nanoparticles. To investigate how this enhancement occurs, C105Y was fluorescently labeled to study its uptake and intracellular trafficking. When human hepatoma cells (HuH7) were incubated with fluorescently labeled C105Y for as little as 3 min, C105Y displayed nuclear and cytoplasmic staining with enrichment of fluorescent signal in the nucleus and nucleolus. Uptake and nucleolar localization were observed with the short sequence PFVYLI, but not with SIPPEVK-FNK, and the D-isomer was readily taken up into cells but not into the nucleus. We found that the C105Y peptide is routed to the nucleolus very rapidly in an energy-dependent fashion, whereas membrane translocation and nuclear localization are energy-independent. When we tested the involvement of known endocytosis pathways in uptake and trafficking of this peptide, we demonstrated that C105Y peptide is internalized by a clathrin-and caveolin-independent pathway, although lipid raft-mediated endocytosis may play a role in peptide intracellular trafficking. Efficient energy-independent cell entry with rapid nuclear localization probably accounts for enhancement of gene expression from inclusion of C105Y into DNA nanoparticles.

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“…In A431 cells, the mitotic block appears to arrest clathrin assembly at various stages of invagination (13), suggesting that the continuous activity of an enzyme (or enzymes) is required for vesicle formation (reviewed in Ref. 14). Using in vitro reconstitution assays, mitotic cytosol has been shown to inhibit invagination of clathrin-coated pits and one of the factors responsible is cdc2 kinase (15).…”

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confidence: 99%

Casein Kinase II Activity Is Required for Transferrin Receptor Endocytosis

Cotlin

Siddiqui

Simpson

et al. 1999

Journal of Biological Chemistry

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The effect of protein kinase inhibitors on transferrin receptor (TR) internalization was examined in HeLa, A431, 3T3-L1 cells, and primary chicken embryo fibroblasts. We show that TR endocytosis is not affected by tyrosine kinase or protein kinase C inhibitors, but is inhibited by one serine/threonine kinase inhibitor, H-89. Inhibition occurred within 15 min, was completely reversible after H-89 withdrawal, and was specific for endocytosis rather than pinocytosis since a TR mutant lacking an internalization signal was not affected. Interestingly, H-89 also inhibited the internalization of a TR chimera containing the major histocompatibility complex class II invariant chain cytoplasmic tail, indicating that the effect was not specific for the TR. Since H-89 inhibits a number of kinases, we employed a permeabilized cell endocytosis assay to further characterize the kinase. In permeabilized 3T3-L1 cells, addition of pseudosubstrate inhibitor peptides of casein kinase II (CKII) blocked TR internalization by more than 50%, whereas pseudosubstrates of cyclic AMP-dependent kinase A, protein kinase C, and casein kinase I had no effect. Furthermore, addition of purified CKII to the cell-free reactions containing CKII pseudosubstrates reversed the endocytosis block, suggesting that CKII or a CKII-like activity is required for constitutive endocytosis. The transferrin receptor (TR)1 binds the serum iron transport protein transferrin (Tf), internalizes through clathrincoated pits, and facilitates Tf iron release in the sorting endosome. Efficient TR internalization requires a cytoplasmic tail tyrosine-containing motif, Tyr-Thr-Arg-Phe (1, 2). Studies by Ohno et al. (3) using yeast two-hybrid analysis demonstrate that the TR cytoplasmic tail signal interacts with one of the four subunits of the AP-2 adaptor complex, the 2 chain. This interaction provides a mechanism for promoting TR clustering in clathrin-coated pits and subsequent internalization. The AP-2 adaptor complex is also required for clathrin recruitment (4 -6) and lattice assembly (7) and, together with its direct interaction with receptor cytoplasmic tails, links the cell surface receptors to the clathrin-based endocytic machinery (reviewed in Refs. 8 and 9).Despite the extensive characterization of many of the proteins involved in endocytosis, little is known about how the clathrin-based endocytic machinery is regulated. What is evident, however, is endocytosis via clathrin-coated pits is blocked during mitosis (10), starting at prophase and continuing through telophase (11,12). In A431 cells, the mitotic block appears to arrest clathrin assembly at various stages of invagination (13), suggesting that the continuous activity of an enzyme (or enzymes) is required for vesicle formation (reviewed in Ref. 14). Using in vitro reconstitution assays, mitotic cytosol has been shown to inhibit invagination of clathrin-coated pits and one of the factors responsible is cdc2 kinase (15).The role of kinases in receptor trafficking has been demonstrated in studies on the asia...

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The mechanism of receptor‐mediated endocytosis

Cited by 153 publications

References 149 publications

Ligand-induced endocytosis of epidermal growth factor receptors that are defective in binding adaptor proteins.

Ligand-induced endocytosis of epidermal growth factor receptors that are defective in binding adaptor proteins.

Mechanism of Uptake of C105Y, a Novel Cell-penetrating Peptide

Casein Kinase II Activity Is Required for Transferrin Receptor Endocytosis

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