The Topographical Fate of Ligand-Receptor Complexes as Reflected by the Properties of Ligand Binding to Intact Cells

1996 ◽  
pp. 201-232 ◽  
Author(s):  
Lee E. Limbird
Keyword(s):  
Ligand Binding ◽  
Intact Cells ◽  
Receptor Complexes

Structural characteristics of growth hormone receptors on mouse 3T3 cells having different biological responses to growth hormone

1989 ◽  
Vol 3 (3) ◽  
pp. 239-245 ◽  
Author(s):  
E. Uchida ◽  
T. Hayakawa ◽  
S. Niimi ◽  
A. Tanaka ◽  
M. Morikawa
Keyword(s):  
Growth Hormone ◽  
Gel Electrophoresis ◽  
Structural Characteristics ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Type ◽  
3T3 Cells ◽  
Intact Cells ◽  
Receptor Complexes ◽  
Gh Receptor ◽  
Adipose Conversion

ABSTRACT Cultured 3T3-F442A preadipocytes are able to undergo GH-promoted differentiation into adipocytes. The relationship between the structure and function of GH receptors on 3T3 cells (3T3-F442A preadipocytes, differentiated adipocytes and 3T3-C2 cells, which vary in susceptibility to adipose conversion or with respect to carbohydrate and lipid metabolism) was studied by the covalent cross-linking of 125I-labelled human (h) GH to intact cells with the bifunctional reagent disuccinimidyl suberate. When preadipocytes were cross-linked and analysed using sodium dodecylsulphate-polyacrylamide gel electrophoresis, a prominent 125I-labelled hGH-receptor complex of Mr 130 000 was observed along with minor complexes (Mr 300 000, 230 000 and 60 000) on autoradiography. Non-reducing—reducing two-dimensional gel electrophoresis revealed that the higher molecular weight complexes also contained the Mr 130 000 complex. Neuraminidase and tunicamycin treatment demonstrated that the GH receptor on F442A preadipocytes is a sialo-glycoprotein with N-linked carbohydrate chains. When the differentiated 3T3-F442A adipocytes and 3T3-C2 cells (a sub-line with no susceptibility to adipose conversion with GH) were examined in the same way as 3T3-F442A preadipocytes, no differences were observed in the specificity of GH binding and in the molecular size of the 125I-labelled hGH-receptor complexes and their glycosylation characteristics. This suggests that the structural characteristics of the GH receptor are closely related in each cell type, but that the hormonal signals produced after GH binding to the receptor may cause different effects according to the cell type.

scholarly journals Dap10 and Dap12 Form Distinct, but Functionally Cooperative, Receptor Complexes in Natural Killer Cells

2000 ◽  
Vol 192 (7) ◽  
pp. 1059-1068 ◽  
Author(s):  
Jun Wu ◽  
Holly Cherwinski ◽  
Thomas Spies ◽  
Joseph H. Phillips ◽  
Lewis L. Lanier
Keyword(s):  
Ligand Binding ◽  
Nk Cells ◽  
Natural Killer ◽  
Tyrosine Kinases ◽  
Nk Cell ◽  
Adaptor Proteins ◽  
Transmembrane Segments ◽  
Receptor Complexes ◽  
Activating Receptors ◽  
Specific Association

Many of the activating receptors on natural killer (NK) cells are multisubunit complexes composed of ligand-binding receptors that are noncovalently associated with membrane-bound signaling adaptor proteins, including CD3ζ, FcεRIγ, DAP12, and DAP10. Because the DAP10 and DAP12 genes are closely linked, expressed in NK cells, and have remarkably similar transmembrane segments, it was of interest to determine the specificity of their interactions with ligand-binding receptors and to examine their signaling properties. Despite their similarities, DAP10, DAP12, FcεRIγ, and CD3ζ form specific receptor complexes with their ligand-binding partners in NK cells and transfectants. The transmembrane regions of DAP10 and DAP12 are sufficient to confer specific association with their partners. Although cross-linking of either DAP10- or DAP12-associated receptors has been shown to be sufficient to trigger NK cell–mediated cytotoxicity against Fc receptor–bearing cells, substantial synergy was observed in the induction of cytokine production when both receptors were engaged. Activation of the Syk/ZAP70 tyrosine kinases by the immunoreceptor tyrosine-based activation motif–containing DAP12 adaptor and of the phosphatidylinositol 3-kinase pathway by the YxNM-containing DAP10 adaptor may play an important role in the stimulation of NK cells and T cells.

Characterization of the receptor for glucagon-like peptide-1(7–36)amide on plasma membranes from rat insulinoma-derived cells by covalent cross-linking

1989 ◽  
Vol 2 (2) ◽  
pp. 93-98 ◽  
Author(s):  
R. Göke ◽  
T. Cole ◽  
J. M. Conlon
Keyword(s):  
Ligand Binding ◽  
Protein Complex ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Binding Protein ◽  
Single Class ◽  
Intact Cells ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Rat Insulinoma

ABSTRACT 125I-Labelled glucagon-like peptide-1(7–36)amide was cross-linked to a specific binding protein in plasma membranes prepared from RINm5F rat insulinoma-derived cells using disuccinimidyl suberate. Consistent with the presence of a single class of binding site on the surface of intact cells, only a single radiolabelled band at Mr 63 000 was identified by SDS-PAGE after solubilization of the ligand—binding protein complex. The band was not observed when 10 nm glucagon-like peptide-1(7–36)amide was included in the binding assay, but 1 μm concentrations of glucagon-like peptide1(1–36)amide, glucagon-like peptide-2 and glucagon did not decrease the intensity of labelling. No change in the mobility of the band was observed under reducing conditions, suggesting that the binding protein in the receptor is not attached to other subunits via disulphide bonds. In control incubations using plasma membranes from pig intestinal epithelial cells, which do not contain specific binding sites for glucagon-like peptide-1(7–36)amide, no cross-linked ligand-binding protein complex was observed.

Regulation of interacting populations during endocytosis: models of growth factor-tumor promoter dynamics

1986 ◽  
Vol 250 (6) ◽  
pp. R1123-R1132
Author(s):  
M. Gex-Fabry ◽  
C. DeLisi
Keyword(s):  
Growth Factor ◽  
Ligand Binding ◽  
Phorbol Ester ◽  
Egf Receptor ◽  
Phorbol Esters ◽  
Time Lag ◽  
Tumor Promoter ◽  
Necessary Condition ◽  
Coated Pits ◽  
Receptor Complexes

A model of growth factor-cell receptor interactions, including internalization, sorting, recycling, and degradation and their modulation by tumor promoters, is developed, analyzed, and tested. In keeping with data and concepts based on a large number of systems, the main assumption is that after receptor-ligand binding the complex associates with a second membrane protein, localized in coated pits, and that this event is a necessary condition for receptor-mediated endocytosis and subsequent intracellular processes. As a consequence of the model, ligands having distinct receptors interfere at the cell surface through competition between their receptor complexes for a limited pool of coated pit proteins. The utility of the model is illustrated by a detailed analysis of binding, endocytosis, and degradation of epidermal growth factor (EGF) and their modulation by phorbol esters. The analysis permits quantitative characterization of the dynamics of the endocytic processes and leads to the following conclusions. The Scatchard plot changes from linear to nonlinear as the ratio of the number of coated pit proteins to the number of receptors decreases. Competition between phorbol ester and EGF-bound receptors for coated pit proteins predicts, in agreement with observation, conversion of nonlinear EGF Scatchard plots to linear plots subsequent to reincubation with phorbol esters. The postulated competition suggests a local homology between the phorbol ester receptor and the EGF receptor. Homologous and heterologous downregulations observed in numerous systems are natural consequences of the model. Preincubation with the heterologous ligand increases the time lag between ligand binding and lysosomal degradation and alters intracellular sorting.

scholarly journals Progesterone receptor structure and function altered by geldanamycin, an hsp90-binding agent.

1995 ◽  
Vol 15 (12) ◽  
pp. 6804-6812 ◽  
Author(s):  
D F Smith ◽  
L Whitesell ◽  
S C Nair ◽  
S Chen ◽  
V Prapapanich ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Binding Capacity ◽  
Biological Significance ◽  
Binding Agent ◽  
Complex Dynamic ◽  
Intact Cells ◽  
Receptor Complexes ◽  
Assembly Pathway ◽  
And Function

The assembly of progesterone receptor (PR) heterocomplexes in vitro involves at least eight components of the molecular chaperone machinery, and as earlier reports have shown, these proteins exhibit complex, dynamic, but ordered, interactions with one another and PR. Using the selective hsp90 binding agent geldanamycin (GA), we have found that PR assembly in vitro can be arrested at a previously observed intermediate assembly step. Like mature PR complexes, the intermediate complexes contain hsp90, but they differ from mature complexes by the presence of hsp70, p60, and p48 and the absence of immunophilins and p23. Arrest of PR assembly is likely due to GA's ability to directly block binding of p23 to hsp90. An important functional consequence of GA-mediated assembly arrest in vitro is the inability of the resulting PR complexes to bind progesterone, despite the presence of hsp90 in the receptor complexes. The biological significance of the in vitro observations is demonstrated by GA's ability to (i) rapidly block PR's hormone binding capacity in intact cells and (ii) alter the composition of COS cell PR complexes in a manner similar to that observed during in vitro reconstitutions. An updated model for the cyclic assembly pathway of PR complexes that incorporates the present findings with earlier results is presented.

scholarly journals Internalization and recycling of insulin receptors in hepatoma cells. Absence of regulation by receptor occupancy

1984 ◽  
Vol 222 (1) ◽  
pp. 111-117 ◽  
Author(s):  
Y Chvatchko ◽  
E Van Obberghen ◽  
M Fehlmann
Keyword(s):  
Insulin Receptor ◽  
Cellular Localization ◽  
Insulin Analogue ◽  
Insulin Receptors ◽  
Receptor Occupancy ◽  
Hepatoma Cells ◽  
Down Regulation ◽  
Intact Cells ◽  
Receptor Complexes ◽  
Before And After

Insulin receptors of Fao hepatoma cells were labelled with a 125I-labelled photoreactive insulin analogue or by surface iodination catalysed by lactoperoxidase. Cells were then incubated at 37 degrees C, and the cellular localization of the labelled receptors was assessed by limited exposure of intact cells to trypsin. The results show that: (1) photolabelled insulin-receptor complexes are internalized and recycled in Fao hepatoma cells; (2) the dynamics of photolabelled insulin receptors (internalization and recycling) is similar before and after down-regulation; (3) the unoccupied receptors labelled by surface iodination are internalized and recycled similarly to covalent insulin-receptor complexes; (4) insulin does not induce internalization of surface-iodinated insulin receptors. We conclude that internalization and recycling of insulin receptors are independent of receptor occupancy by insulin in Fao hepatoma cells.

scholarly journals Architecture and subunit arrangement of native AMPA receptors elucidated by cryo-EM

Science ◽  
2019 ◽  
Vol 364 (6438) ◽  
pp. 355-362 ◽  
Author(s):  
Yan Zhao ◽  
Shanshuang Chen ◽  
Adam C. Swensen ◽  
Wei-Jun Qian ◽  
Eric Gouaux
Keyword(s):  
Molecular Structure ◽  
Electron Microscopy ◽  
Signal Transduction ◽  
Ligand Binding ◽  
Ampa Receptor ◽  
Ampa Receptors ◽  
Mammalian Brain ◽  
Receptor Complexes ◽  
Cryo Electron Microscopy ◽  
Chemical Synapses

Glutamate-gated AMPA receptors mediate the fast component of excitatory signal transduction at chemical synapses throughout all regions of the mammalian brain. AMPA receptors are tetrameric assemblies composed of four subunits, GluA1–GluA4. Despite decades of study, the subunit composition, subunit arrangement, and molecular structure of native AMPA receptors remain unknown. Here we elucidate the structures of 10 distinct native AMPA receptor complexes by single-particle cryo–electron microscopy (cryo-EM). We find that receptor subunits are arranged nonstochastically, with the GluA2 subunit preferentially occupying the B and D positions of the tetramer and with triheteromeric assemblies comprising a major population of native AMPA receptors. Cryo-EM maps define the structure for S2-M4 linkers between the ligand-binding and transmembrane domains, suggesting how neurotransmitter binding is coupled to ion channel gating.

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