scholarly journals Endocytic Clathrin-coated Pit Formation Is Independent of Receptor Internalization Signal Levels

1998 ◽  
Vol 9 (5) ◽  
pp. 1177-1194 ◽  
Author(s):  
Francesca Santini ◽  
Michael S. Marks ◽  
James H. Keen
Keyword(s):  
Plasma Membrane ◽  
Critical Role ◽  
Indirect Evidence ◽  
Receptor Internalization ◽  
Coat Proteins ◽  
Cytoplasmic Domains ◽  
Reporter Protein ◽  
Coated Pits ◽  
Pit Formation ◽  
In Cells

The mechanisms responsible for coated pit formation in cells remain unknown, but indirect evidence has argued both for and against a critical role of receptor cytoplasmic domains in the process. If the endocytic motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of constitutively internalized receptors at the membrane would be expected to govern the steady-state level of coated pits in cells. Here we directly test this hypothesis for broad classes of receptors containing three distinct constitutive internalization signals. Chimeric proteins consisting of an integral membrane reporter protein (Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/casein kinase II-based internalization signals were overexpressed to levels that saturated the internalization pathway. Quantitative confocal immunofluorescence microscopy indicated that the number of plasma membrane clathrin-coated pits and the concentration of their structural components were invariant when comparing cells expressing saturating levels of the chimeric receptors to nonexpressing cells or to cells expressing only the Tac reporter lacking cytoplasmic internalization signals. Biochemical analysis showed that the distribution of coat proteins between assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These results provide a clear indication that receptor endocytic signals do not determine coated pit levels by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated docking sites that act independently of endocytic signals.

scholarly journals Nef-mediated Clathrin-coated Pit Formation

1997 ◽  
Vol 139 (1) ◽  
pp. 37-47 ◽  
Author(s):  
Michelangelo Foti ◽  
Aram Mangasarian ◽  
Vincent Piguet ◽  
Daniel P. Lew ◽  
Karl-Heinz Krause ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cytoplasmic Tail ◽  
Fluid Phase ◽  
Receptor Internalization ◽  
Coat Proteins ◽  
Membrane Area ◽  
Sequence Of Events ◽  
Early Protein ◽  
Functional Consequences ◽  
Cluster Of Differentiation

The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.

Utilization of the indirect lysosome targeting pathway by lysosome-associated membrane proteins (LAMPs) is influenced largely by the C-terminal residue of their GYXXphi targeting signals

1999 ◽  
Vol 112 (23) ◽  
pp. 4257-4269 ◽  
Author(s):  
N.R. Gough ◽  
M.E. Zweifel ◽  
O. Martinez-Augustin ◽  
R.C. Aguilar ◽  
J.S. Bonifacino ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Optimal Size ◽  
Mutant Form ◽  
Binding Assays ◽  
Indirect Pathway ◽  
Reporter Protein ◽  
Terminal Residue ◽  
Targeting Signals ◽  
Mutant Forms ◽  
In Cells

A systematic study was conducted on the requirements at the C-terminal position for the targeting of LAMPs to lysosomes, examining the hypothesis that a bulky hydrophobic residue is required. Mutations deleting or replacing the C-terminal valine with G, A, C, L, I, M, K, F, Y, or W were constructed in a reporter protein consisting of the lumenal/extracellular domain of avian LAMP-1 fused to the transmembrane and cytoplasmic domains of LAMP-2b. The steady-state distribution of each mutant form in mouse L-cells was assessed by quantitative antibody binding assays and immunofluorescence microscopy; efficiency of internalization from the plasma membrane and delivery to the lysosome were also estimated. It is found that (a) only C-terminal V, L, I, M, and F mediated efficient targeting to lysosomes, demonstrating the importance hydrophobicity and an optimal size of the C-terminal residue in targeting; (b) efficiency of lysosomal targeting generally correlated with efficiency of internalization; and (c) mutant forms that did not target well to lysosomes showed unique distributions in cells rather than simply default accumulation in the plasma membrane. Interactions of the targeting signals with adaptor subunits were measured using a yeast two-hybrid assay. The results are consistent with the hypothesis that trafficking of LAMP forms in cells through the indirect pathway is determined by the affinities of their targeting signals, predominantly for the mu2 and mu3 adaptors involved at plasma membrane and endosomal cellular sorting sites, respectively.

scholarly journals Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

2015 ◽  
Vol 26 (19) ◽  
pp. 3504-3519 ◽  
Author(s):  
Camilo Garay ◽  
Gurjeet Judge ◽  
Stefanie Lucarelli ◽  
Stephen Bautista ◽  
Rohan Pandey ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Gene Silencing ◽  
Receptor Internalization ◽  
Membrane Structures ◽  
Coated Pits ◽  
Akt Phosphorylation ◽  
Erbb2 Expression ◽  
Epidermal Growth

Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation.

μ1A deficiency induces a profound increase in MPR300/IGF-II receptor internalization rate

2001 ◽  
Vol 114 (24) ◽  
pp. 4469-4476 ◽  
Author(s):  
Christoph Meyer ◽  
Eeva-Liisa Eskelinen ◽  
Medigeshi Ramarao Guruprasad ◽  
Kurt von Figura ◽  
Peter Schu
Keyword(s):  
Plasma Membrane ◽  
Fold Increase ◽  
Retrograde Transport ◽  
Receptor Internalization ◽  
Recycling Rate ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
Internalization Rate ◽  
Clathrin Adaptor ◽  
Golgi Network

The mannose-6-phosphate/IGF-II receptor MPR300 mediates sorting of lysosomal enzymes from the trans-Golgi network to endosomes and endocytosis of hormones, for example, of IGF-II. We analyzed transport of MPR300 in μ1A-adaptin-deficient fibroblasts, which lack a functional AP-1 clathrin adaptor complex. In μ1A-adaptin-deficient fibroblasts, the homologous MPR46 accumulates in endosomes due to a block in retrograde transport to the trans-Golgi network. The MPR300-mediated endocytosis is markedly enhanced. We demonstrate that the seven-fold increase in endocytosis is not associated with an increased steady-state concentration of receptors at the plasma membrane, but with an increased internalization rate of MPR300. Internalization of other receptors that are also endocytosed by AP-2 is not affected. More MPR300 receptors are found in clathrin-coated pits of the plasma membrane, whereas outside coated-areas, more MPR300 are concentrated in clusters and all intracellular receptors reside in endosomes, which are in equilibrium with the plasma membrane. Thus AP-1-mediated transport of MPR300 from endosomes to the TGN controls indirectly the recycling rate of the receptor between the plasma membrane and endosomes.

Regulation of clathrin-coated vesicle formation

2001 ◽  
Vol 29 (4) ◽  
pp. 375-377 ◽  
Author(s):  
E. Hill ◽  
O. Olusanya ◽  
J. van der Kaay ◽  
C. P. Downes ◽  
P. D. Andrews ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Coat Proteins ◽  
Concerted Action ◽  
Coated Pits ◽  
The Real ◽  
Permeabilized Cells ◽  
Real Challenge

The formation of clathrin-coated pits at the plasma membrane requires the concerted action of many different molecules. The real challenge lies in determining the hierarchy of these interactions. We are using assays in both intact and permeabilized cells to dissect the temporal requirements for clathrin-coated vesicle formation, and also to examine the role of phosphorylation of the coat proteins.

Quantitative measurement of alpha 6 beta 1 and alpha 6 beta 4 integrin internalization under cross-linking conditions: a possible role for alpha 6 cytoplasmic domains

1994 ◽  
Vol 107 (12) ◽  
pp. 3339-3349 ◽  
Author(s):  
G. Gaietta ◽  
T.E. Redelmeier ◽  
M.R. Jackson ◽  
R.N. Tamura ◽  
V. Quaranta
Keyword(s):  
Cell Surface ◽  
Human Keratinocytes ◽  
Receptor Internalization ◽  
Cross Linking ◽  
Cytoplasmic Domains ◽  
Reporter Protein ◽  
Teratocarcinoma Cells ◽  
Significant Difference ◽  
Epithelial Cell Surface ◽  
Discrete Domains

In epithelial cells integrins are segregated on discrete domains of the plasma membrane. Redistribution may also occur during migration or differentiation. However, little is known about the mechanisms that control such redistribution. Receptor internalization may be a part of one such mechanism. We developed a quantitative assay and measured internalization of two epithelial integrin heterodimers, alpha 6 beta 1 and alpha 6 beta 4, induced by cross-linking with specific antibodies. alpha 6 beta 1 is a receptor for EHS laminin, while alpha 6 beta 4 is a receptor for a component of the basement membrane. alpha 6 beta 4 plays an important role in the establishment of hemidesmosomes, and becomes redistributed on the epithelial cell surface when cells are in a migratory phase. We report that alpha 6 beta 4 is efficiently internalized in human keratinocytes. More than 25% of cell surface alpha 6 beta 4 was internalized at 30 minutes, after cross-linking with A9, an anti-beta 4 monoclonal antibody. alpha 6 beta 1 is also internalized, in melanoma and teratocarcinoma cells, with maximum values of 20% of total receptors expressed at the cell surface. No significant difference was observed between the alpha 6 isoforms A and B in these assays. To determine whether alpha 6 cytoplasmic domains could influence integrin endocytosis, we prepared chimeric constructs with the extracellular domain of a reporter protein (CD8), and the cytoplasmic domains of either alpha 6 A or alpha 6 B. Both alpha 6 cytoplasmic domains but not a control cytoplasmic domain promoted internalization of the chimeric proteins, after cross-linking with antibody. Internalization of alpha 6 integrins may have a role in redistributing these receptors at the cell surface. Furthermore, the cytoplasmic domains of alpha 6 may be involved in regulating integrin internalization.

scholarly journals Endosome acidification and receptor trafficking: bafilomycin A1 slows receptor externalization by a mechanism involving the receptor's internalization motif.

1993 ◽  
Vol 4 (12) ◽  
pp. 1251-1266 ◽  
Author(s):  
L S Johnson ◽  
K W Dunn ◽  
B Pytowski ◽  
T E McGraw
Keyword(s):  
Plasma Membrane ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Specific Inhibitor ◽  
Aromatic Amino Acids ◽  
Receptor Trafficking ◽  
Receptor Internalization ◽  
General Mechanism ◽  
Bafilomycin A1 ◽  
Coated Pits

To examine the relationship between endosome acidification and receptor trafficking, transferrin receptor trafficking was characterized in Chinese hamster ovary cells in which endosome acidification was blocked by treatment with the specific inhibitor of the vacuolar H(+)-ATPase, bafilomycin A1. Elevating endosome pH slowed the receptor externalization rate to approximately one-half of control but did not affect receptor internalization kinetics. The slowed receptor externalization required the receptor's cytoplasmic domain and was largely eliminated by substitutions replacing either of two aromatic amino acids within the receptor's cytoplasmic YTRF internalization motif. These results confirm, using a specific inhibitor of the vacuolar proton pump, that proper endosome acidification is necessary to maintain rapid recycling of intracellular receptors back to the plasma membrane. Moreover, receptor return to the plasma membrane is slowed in the absence of proper endosome acidification by a signal-dependent mechanism involving the receptor's cytoplasmic tyrosine-containing internalization motif. These results, in conjunction with results from other studies, suggest that the mechanism for clustering receptors in plasma membrane clathrin-coated pits may be an example of a more general mechanism that determines the dynamic distribution of membrane proteins among various compartments with luminal acidification playing a crucial role in this process.

Changes Occur in Plasma Membrane Turnover when K562 Leukemia Cells are Induced to Differentiate

1982 ◽  
Vol 40 ◽  
pp. 286-287
Author(s):  
L. M. Marshall
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Intracellular Transport ◽  
Parent Cell ◽  
Membrane Turnover ◽  
Coated Pits ◽  
Surface Distribution ◽  
Specific Proteins ◽  
Erythroleukemic Cell ◽  
Specific Membrane

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.

Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

2015 ◽  
Vol 57 ◽  
pp. 189-201 ◽  
Author(s):  
Jay Shankar ◽  
Cecile Boscher ◽  
Ivan R. Nabi
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Cancer Cell ◽  
Cellular Response ◽  
Spatial Organization ◽  
Essential Feature ◽  
Cell Signalling ◽  
Coated Pits ◽  
Signalling Molecules ◽  
Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

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