Autoradiographic analysis of formylpeptide chemoattractant binding, uptake and intracellular processing by neutrophils

1989 ◽  
Vol 94 (1) ◽  
pp. 155-168
Author(s):  
A.H. Janeczek ◽  
W.A. Marasco ◽  
P.J. van Alten ◽  
R.J. Walter
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Active Phase ◽  
Coated Pits ◽  
Surface Receptors ◽  
Dense Granules ◽  
Receptor Complexes ◽  
Intracellular Processing ◽  
Autoradiographic Analysis ◽  
Formylpeptide Receptor

Formylpeptide chemoattractant binds specifically to leukocyte cell surface receptors and during chemotactic activation, is internalized and processed by these cells. Using electron microscope autoradiographic techniques, we have examined the binding, uptake and disposition of fNle-Leu-Phe-Nle-[125I]Tyr-Lys by rabbit peritoneal neutrophils. Cells were incubated with ligand for 15 min at 4 degrees C, rinsed and then further incubated in buffer at 4 degrees C, 15 degrees C, 24 degrees C, or 37 degrees C for 0–40 min. For all cells incubated at 4 degrees C, grains were predominantly localized on the plasma membrane. Initially, this formylpeptide was seen in small clusters or microaggregates that were not associated with coated pits. Upon further incubation at 37 degrees C for 1 min, formylpeptide clustering on the plasma membrane increased and extensive internalization of formylpeptide was observed. Endocytosis of formylpeptide-receptor complexes clearly involved uncoated membrane pits and vesicles but did not appear to involve coated pits or coated vesicles. In the following 3 min, peptide proceeded in waves through compartments composed of small uncoated endocytic vesicles, then large vesicles, and then into dense granules. After 4 min at 37 degrees C, the most active phase of intracellular processing subsided. The percentage of grains in the cytoplasm, granule and small vesicle compartments very gradually increased during the remainder of the 40 min incubation. Formylpeptide neither bound at 4 degrees C nor accumulated at 37 degrees C on the cell surface in proximity to the underlying Golgi/centrosome region of the cell. At 24 degrees C, processing was slowed but formylpeptide-receptor complexes proceeded through a similar series of compartments. The t1/2 for formylpeptide uptake at 37 degrees C was 15–20 s, whereas at 24 degrees C the t 1/2 was approximately 10 min. No uptake was observed at 15 degrees C. The distinctive characteristics of formylpeptide binding and receptor-complex uptake seen here may be essential in initiating and maintaining continued chemotactic responsiveness.

scholarly journals alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions.

1979 ◽  
Vol 82 (3) ◽  
pp. 614-625 ◽  
Author(s):  
M C Willingham ◽  
F R Maxfield ◽  
I H Pastan
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Coated Vesicles ◽  
The Other ◽  
Con A ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Receptor Complexes ◽  
Transmission Electron ◽  
Epidermal Growth

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.

Experimental Dissection of the Cellular Machinery Involved in Receptor Mediated Endocytosis and Translocation

1981 ◽  
Vol 39 ◽  
pp. 528-531
Author(s):  
J.L. Salisbury
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Regulatory Protein ◽  
Fold Increase ◽  
Coated Pits ◽  
Surface Distribution ◽  
Receptor Mediated Endocytosis ◽  
Calcium Dependent ◽  
Receptor Complexes ◽  
Human Lymphoblastoid Cell

The cultured human lymphoblastoid cell line WiL2 is a model system of choice for studies on receptor mediated endocytosis (RME). These cells display antigen receptor immunoglobulin of the IgM class (rIgM) as integral plasma membrane proteins which are present in diffuse cell surface distribution in unstimulated cells. Initially, rIgM occurs over uncoated regions of the plasma membrane. Crosslinking rIgM with multivalent antibody (ligand) results in the entry of ferritin-labelled ligand-rIgM complexes into the RME pathway (Figure 1). Stimulation of RME by ligand challenge results in an approximately three-fold increase in cell surface area displaying clathrin coats on the cytoplasmic face of the membrane. The newly formed coated pits are located directly beneath ferritin-labelled ligand-receptor complexes and their appearance is sensitive to the calmodulin directed drug trifluoperazine dihydrochloride (TFP). Calmodulin is a calcium dependent regulatory protein which recognizes local transient fluxes of cytoplasmic Ca+2 and activates a wide variety of enzymes and other protein systems. In addition, antibodies raised against calf brain calmodulin were used in indirect immunofluorescence studies.

scholarly journals Down-Regulation of Cell Surface Receptors Is Modulated by Polar Residues within the Transmembrane Domain

2000 ◽  
Vol 11 (8) ◽  
pp. 2643-2655 ◽  
Author(s):  
Lolita Zaliauskiene ◽  
Sunghyun Kang ◽  
Christie G. Brouillette ◽  
Jacob Lebowitz ◽  
Ramin B. Arani ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transmembrane Domain ◽  
Gradient Centrifugation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Surface Proteins ◽  
Down Regulation ◽  
Surface Receptors ◽  
Receptor Complexes ◽  
Polar Residues

How recycling receptors are segregated from down-regulated receptors in the endosome is unknown. In previous studies, we demonstrated that substitutions in the transferrin receptor (TR) transmembrane domain (TM) convert the protein from an efficiently recycling receptor to one that is rapidly down regulated. In this study, we demonstrate that the “signal” within the TM necessary and sufficient for down-regulation is Thr11Gln17Thr19 (numbering in TM). Transplantation of these polar residues into the wild-type TR promotes receptor down-regulation that can be demonstrated by changes in protein half-life and in receptor recycling. Surprisingly, this modification dramatically increases the TR internalization rate as well (∼79% increase). Sucrose gradient centrifugation and cross-linking studies reveal that propensity of the receptors to self-associate correlates with down-regulation. Interestingly, a number of cell surface proteins that contain TM polar residues are known to be efficiently down-regulated, whereas recycling receptors for low-density lipoprotein and transferrin conspicuously lack these residues. Our data, therefore, suggest a simple model in which specific residues within the TM sequences dramatically influence the fate of membrane proteins after endocytosis, providing an alternative signal for down-regulation of receptor complexes to the well-characterized cytoplasmic tail targeting signals.

scholarly journals A conserved regulatory module regulates receptor kinase signaling in immunity and development

2021 ◽  
Author(s):  
Thomas A. DeFalco ◽  
Pauline Anne ◽  
Sean R. James ◽  
Andrew Willoughby ◽  
Oliver Johanndrees ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cellular Responses ◽  
Receptor Signaling ◽  
Receptor Kinase ◽  
Surface Receptors ◽  
Receptor Complexes ◽  
Regulatory Circuit ◽  
Regulatory Module ◽  
Molecular Patterns ◽  
Receptor Kinase Signaling

ABSTRACTLigand recognition by cell-surface receptors underlies development and immunity in both animals and plants. Modulating receptor signaling is critical for appropriate cellular responses but the mechanisms ensuring this are poorly understood. Here, we show that signaling by plant receptors for pathogen-associated molecular patterns (PAMPs) in immunity and CLAVATA3/EMBRYO SURROUNDING REGION-RELATED peptides (CLEp) in development employ a similar regulatory module. In the absence of ligand, signaling is dampened through association with specific type-2C protein phosphatases (PP2Cs). Upon activation, PAMP and CLEp receptors phosphorylate divergent cytosolic kinases, which, in turn, phosphorylate the phosphatases, thereby promoting their release from the receptor complexes. Our work reveals a regulatory circuit shared between immune and developmental receptor signaling, which may have broader important implications for plant receptor kinase-mediated signaling in general.

Intracellular signalling

2010 ◽  
pp. 169-176
Author(s):  
R. Andres Floto
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Intracellular Signalling ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Transmission Of Information ◽  
Effective Transmission

This section outlines the general principles of intracellular signalling. Focusing on cell surface receptors, the requirements for effective transmission of information across the plasma membrane are outlined. The principal mechanisms utilized in mammalian signal transduction are described. For each, the pathological consequences of aberrant signalling and means by which pathways can be pharmacologically targeted are described in molecular terms....

Intracellular signalling

2020 ◽  
pp. 256-265
Author(s):  
R. Andres Floto
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Intracellular Signalling ◽  
Signalling Pathways ◽  
Molecular Processes ◽  
Surface Receptors ◽  
Intracellular Signalling Pathways ◽  
Transmission Of Information ◽  
Effective Transmission

This chapter outlines the general principles of intracellular signalling. Focusing on cell surface receptors, the requirements for effective transmission of information across the plasma membrane are outlined. The principal mechanisms utilized in mammalian signal transduction are described. For each, the pathological consequences of aberrant signalling and means by which pathways can be pharmacologically targeted are described in molecular terms. Intracellular signalling pathways permit the transmission and integration of information within cells. Mammalian receptor signalling relies on only a small number of distinct molecular processes which interact to determine cellular responses. Rapid advances in our knowledge of the mechanisms of intracellular signalling has greatly increased understanding of how cells function physiologically, how they malfunction pathologically, and how their behaviour might be manipulated therapeutically.

scholarly journals Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway.

1986 ◽  
Vol 102 (4) ◽  
pp. 1271-1283 ◽  
Author(s):  
M G Roth ◽  
C Doyle ◽  
J Sambrook ◽  
M J Gething
Keyword(s):  
Influenza Virus ◽  
Cell Surface ◽  
Dna Sequences ◽  
Acquired Resistance ◽  
Endocytic Pathway ◽  
Wild Type ◽  
Cytoplasmic Domains ◽  
Coated Pits ◽  
Herpes Simplex Virus 1 ◽  
Surface Receptors

Chimeric genes were created by fusing DNA sequences encoding the ectodomain of the influenza virus hemagglutinin (HA) to DNA coding for the transmembrane and cytoplasmic domains of either the G glycoprotein of vesicular stomatitis virus or the gC glycoprotein of Herpes simplex virus 1. CV-1 cells infected with SV40 vectors carrying the recombinant genes expressed large amounts of the chimeric proteins, HAG or HAgC on their surfaces. Although the ectodomains of HAG and HAgC differed in their immunological properties from that of HA, the chimeras displayed the biological functions characteristic of the wild-type protein. Both HAG and HAgC bound erythrocytes as efficiently as HA did and, after brief exposure to an acidic environment, induced the fusion of erythrocyte and CV-1 cell membranes. However, the behavior of HAG and HAgC at the cell surface differed from that of HA in several important respects. HAG and HAgC were observed to collect in coated pits whereas wild-type HA was excluded from those structures. In the presence of chloroquine, which inhibits the exit of receptors from endosomes, HAG and HAgC accumulated in intracellular vesicles. By contrast, chloroquine had no effect on the location of wild-type HA. HAG and HAgC labeled at the cell surface exhibited a temperature-dependent acquisition of resistance to extracellular protease at a rate similar to the rates of internalization observed for many cell surface receptors. HA acquired resistance to protease at a rate at least 20-fold slower. We conclude that HAG and HAgC are efficiently routed into the endocytic pathway and HA is not. However, like HA, HAG was degraded slowly, raising the possibility that HAG recycles to the plasma membrane.

scholarly journals Rapid endocytosis of the transferrin receptor in the absence of bound transferrin.

1985 ◽  
Vol 100 (2) ◽  
pp. 633-637 ◽  
Author(s):  
C Watts
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Cell Line ◽  
Transferrin Receptor ◽  
Protein A ◽  
Transferrin Receptors ◽  
Coated Pits ◽  
Prolonged Incubation ◽  
Surface Receptors ◽  
Recycling Pathway

The rate of endocytosis of transferrin receptors, occupied or unoccupied with transferrin, was measured on the cell line K562. At 37 degrees C, receptors, radioiodinated on the cell surface at 4 degrees C, were internalized equally rapidly in the presence or absence of transferrin. In both cases, 50% of the labeled receptors became resistant to externally added trypsin in 5 min. An antitransferrin antibody was used to show directly that the receptors had entered the cells without bound transferrin. The distribution of the receptors on the cell surface was revealed by antibody and protein A-gold staining after prolonged incubation in the presence or absence of transferrin. The receptors were concentrated in coated pits under both conditions. The data suggest that endocytosis of transferrin receptors is not "triggered" by ligand binding and raise the possibility that ligand-induced down-regulation of surface receptors may not occur by this mechanism. Instead receptors may be recognized as being ligand-occupied, not at the cell surface, but at some other site in the recycling pathway such as the endosome.

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