scholarly journals Postendocytotic Trafficking of the Follicle-Stimulating Hormone (FSH)-FSH Receptor Complex

2003 ◽  
Vol 17 (11) ◽  
pp. 2162-2176 ◽  
Author(s):  
Hanumanthappa Krishnamurthy ◽  
Hiroshi Kishi ◽  
Mei Shi ◽  
Colette Galet ◽  
Ravi Sankar Bhaskaran ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Human Kidney ◽  
Degradation Pathway ◽  
Fsh Receptor ◽  
Amino Acid Residues ◽  
Down Regulation ◽  
Receptor Complexes ◽  
C Terminus ◽  
Recycling Pathway

Abstract Although the fates of the internalized hormone-receptor complexes formed by the lutropin/choriogonadotropin and the TSH receptors have been examined in some detail, much less is known about the fate of the internalized FSH-FSH receptor (FSHR) complex. Using biochemical and imaging approaches we show here that the majority of the internalized FSH-FSHR complex accumulates in endosomes and subsequently recycles back to the cell surface where the bound, intact hormone dissociates back into the medium. Only small amounts of FSH and the FSHR are routed to a lysosomal degradation pathway, and the extent of FSH-induced down-regulation of the cell surface and total FSHR is minimal. This pathway was detected in heterologous (human kidney 293T) cells transfected with the rat (r) or human (h) FSHR as well as in a mouse Sertoli cell line (MSC-1) or a mouse granulosa cell line (KK-1) transfected with the rFSHR. Additional experiments using a series of C-terminal deletions of the rFSHR and the hFSHR showed that the recycling of the internalized FSH-FSHR complex and the extent of hFSH-induced down-regulation is dictated by a short stretch of amino acids present at the extreme C-terminal end of the receptor. We conclude that most of the internalized FSH-FSHR complex is recycled back to the cell surface, that this recycling pathway is highly dependent on amino acid residues present near the C terminus of the FSHR, and that it is an important determinant of the extent of down-regulation of the FSHR.

Both proteasomes and lysosomes degrade the activated erythropoietin receptor

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 600-608 ◽  
Author(s):  
Pierre Walrafen ◽  
Frédérique Verdier ◽  
Zahra Kadri ◽  
Stany Chrétien ◽  
Catherine Lacombe ◽  
...  
Keyword(s):  
Cell Surface ◽  
Intracellular Signaling ◽  
Erythropoietin Receptor ◽  
Janus Kinase ◽  
Receptor Internalization ◽  
Down Regulation ◽  
Epo Receptor ◽  
Receptor Complexes ◽  
Regulation Mechanisms ◽  
Rapid Activation

AbstractActivation of the erythropoietin receptor (EpoR) after Epo binding is very transient because of the rapid activation of strong down-regulation mechanisms that quickly decrease Epo sensitivity of the cells. Among these down-regulation mechanisms, receptor internalization and degradation are probably the most efficient. Here, we show that the Epo receptor was rapidly ubiquitinated after ligand stimulation and that the C-terminal part of the Epo receptor was degraded by the proteasomes. Both ubiquitination and receptor degradation by the proteasomes occurred at the cell surface and required Janus kinase 2 (Jak2) activation. Moreover, Epo-EpoR complexes were rapidly internalized and targeted to the lysosomes for degradation. Neither Jak2 nor proteasome activities were required for internalization. In contrast, Jak2 activation was necessary for lysosome targeting of the Epo-EpoR complexes. Blocking Jak2 with the tyrphostin AG490 led to some recycling of internalized Epo-Epo receptor complexes to the cell surface. Thus, activated Epo receptors appear to be quickly degraded after ubiquitination by 2 proteolytic systems that proceed successively: the proteasomes remove part of the intracellular domain at the cell surface, and the lysosomes degrade the remaining part of the receptor-hormone complex. The efficiency of these processes probably explains the short duration of intracellular signaling activated by Epo.

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 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.

Direct visualization of binding, aggregation and internalization of human growth hormone in cultured human lymphocytes

1984 ◽  
Vol 107 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Rina Eshet ◽  
Shulamit Peleg ◽  
Zvi Laron
Keyword(s):  
Growth Hormone ◽  
Cell Surface ◽  
Human Growth Hormone ◽  
Direct Observation ◽  
Hormone Receptor ◽  
Human Lymphocytes ◽  
Human Growth ◽  
Direct Visualization ◽  
Down Regulation ◽  
Receptor Complexes

Abstract. The distribution of human growth hormone (hGH) receptor complexes on IM-9 cultured lymphocytes was studied using fluorescein and 125I-labelled hGH (F-hGH and [125I]hGH). The cells labelled with F-hGH were visualized with a sensitive video intensification microscopic system which permitted direct observation of the location of the fluorescent hormone on the surface of the living lymphocytes. At 4°C F-hGH bound diffusely to the cell surface and remained dispersed but following incubation for 30 min at 37°C the hormone receptor complexes aggregated into patches on the cell surface and formed a single cap on one pole of the cell. Progressive internalization into the cell was demonstrated at 37°C with [125I]hGH. It is hypothesized that the aggregation and internalization of the hGH receptor complexes are associated with the action and degradation of the hormone and probably also with the mechanism of down-regulation of the receptors.

scholarly journals The post-endocytotic fate of the gonadotropin receptors is an important determinant of the desensitization of gonadotropin responses

2005 ◽  
Vol 34 (2) ◽  
pp. 447-457 ◽  
Author(s):  
R S Bhaskaran ◽  
M Ascoli
Keyword(s):  
Cell Surface ◽  
Leydig Cells ◽  
Degradation Pathway ◽  
Cell Surface Receptors ◽  
Wild Type ◽  
Gonadotropin Receptors ◽  
Surface Receptors ◽  
Receptor Complexes ◽  
Mutant Receptors ◽  
Stimulation Of

Internalization of the ligand/receptor complexes is a consequence of the activation of the gonadotropin receptors. Since the recycling or degradation of the internalized receptors results in the maintenance or loss of cell surface receptors respectively and this contributes to the loss of responsiveness, we hypothesized that the fate of the internalized receptors could be an important component of desensitization. We examined this hypothesis using the wild-type and mutants of the human LH (hLHR) receptors and follitropin receptors expressed in MA-10 and KK-1 cells respectively. The receptor mutants were chosen because they are routed mostly to a lysosomal degradation pathway whereas the wild-type receptors are recycled back to the surface. We have shown that agonist stimulation of cells expressing the mutant receptors results in a more pronounced loss of cell surface receptors and agonist responses than stimulation of cells expressing the wild-type receptors. We concluded that receptor recycling promotes the maintenance of cell surface receptors and preserves hormonal responsiveness. This property of the hLHR is likely to be physiologically important because there at least two hLHR-expressing tissues in pregnant women, the maternal corpus luteum and the fetal Leydig cells, where a loss of hormonal responsiveness induced by the elevated levels of human chorionic gonadotropin that occur during pregnancy is not desirable.

scholarly journals Major Reservoir for Heparin-Releasable TFPIα (Tissue Factor Pathway Inhibitor α) Is Extracellular Matrix

2021 ◽  
Author(s):  
Julie A. Peterson ◽  
Susan A. Maroney ◽  
Nicholas D. Martinez ◽  
Alan E. Mast
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Surface ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
Human Kidney ◽  
Laminin 5 ◽  
C Terminus ◽  
Tissue Factor Pathway

Objective: Human endothelial cells produce 2 alternatively spliced TFPI (tissue factor pathway inhibitor) isoforms that maintain anticoagulant properties of the vasculature. TFPIβ is glycosylphosphatidylinositol anchored on the cell surface. TFPIα has a basic C terminus sharing homology with VEGF (vascular endothelial growth factor) and is a heparin-releasable protein, suggesting it binds glycosaminoglycans on the endothelium surface. However, this is unclear because TFPIα is not on the surface of cultured endothelial cells. This study identifies the source of heparin-releasable TFPIα. Approach and Results: ELISA assays localized heparin-releasable TFPIα to the extracellular matrix (ECM) of Ea.hy926 cells and human umbilical vein endothelial cells. Immunofluorescence microscopy for TFPIα showed punctate intracytoplasmic staining and ECM staining beneath individual cells. Flow cytometry identified TFPIβ but not TFPIα on the cell surface. TFPIα localization to ECM was confirmed with ELISA and immunohistochemistry studies of umbilical cord veins. The TFPIα C terminus interacted with Ea.hy926 ECM glycosaminoglycans, and a homologous VEGF peptide competed for this binding, suggesting these interactions modulate VEGF responses. Immobilized TFPIα C-terminal peptide bound to several ECM proteoglycans in Ea.hy926 conditioned media. Immunofluorescence studies of human kidney colocalized TFPIα with 4 of these proteoglycans surrounding the microvasculature: glypican-1, syndecan-4, thrombospondin, and laminin-5. The absence of TFPIα on the surface of endothelial cells and its co-localization with specific ECM proteins suggests TFPIα binds to unique proteoglycan structures. Conclusions: ECM contained the primary vascular pool of heparin-releasable TFPIα. By localizing to ECM, TFPIα is positioned to inhibit the procoagulant activity of tissue factor surrounding the vasculature.

scholarly journals Erk1/2 Inactivation-Induced c-Jun Degradation Is Regulated by Protein Phosphatases, UBE2d3, and the C-Terminus of c-Jun

2021 ◽  
Vol 22 (8) ◽  
pp. 3889
Author(s):  
Weiming Ouyang ◽  
David M. Frucht
Keyword(s):  
Protein Degradation ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Degradation Pathway ◽  
Protein Phosphatase 1 ◽  
Amino Acid Residues ◽  
Ubiquitin E3 Ligase ◽  
C Terminus ◽  
Or Gene ◽  
E2 Enzymes

Constitutive photomorphogenic 1 (COP1) is the ubiquitin E3 ligase that mediates degradation of c-Jun protein upon Erk1/2 inactivation. It remains unknown how this protein degradation pathway is regulated. In this study, we investigated the roles of protein phosphatases, ubiquitin-conjugating E2 enzymes (UBE2), and an intrinsic motif of c-Jun in regulating this degradation pathway. By using pharmacological inhibitors and/or gene knockdown techniques, we identified protein phosphatase 1 (PP1) and PP2A as the phosphatases and UBE23d as the UBE2 promoting c-Jun degradation, triggered by Erk1/2 inactivation. In addition, we report that the C-terminus of c-Jun protein facilitates its degradation. The addition of a C-terminal tag or deletion of the last four amino acid residues from the C-terminus of c-Jun protects it from degradation under Erk1/2-inactivating conditions. Taken together, this study reveals that the Erk1/2 inactivation-triggered and COP1-mediated c-Jun degradation is extrinsically and intrinsically regulated, providing a new understanding of the mechanisms underlying this protein degradation pathway.

scholarly journals Epidermal growth factor and transforming growth factor-alpha: differential intracellular routing and processing of ligand-receptor complexes.

1991 ◽  
Vol 2 (8) ◽  
pp. 599-612 ◽  
Author(s):  
R Ebner ◽  
R Derynck
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Surface ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Alpha ◽  
Down Regulation ◽  
Surface Receptors ◽  
Receptor Complexes ◽  
Epidermal Growth ◽  
Factor Alpha

Two structurally related but different polypeptide growth factors, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), exert their activities after interaction with a common cell-surface EGF/TGF-alpha-receptor. Comparative studies of the effects of both ligands have established that TGF-alpha is more potent than EGF in a variety of biological systems. This observation is not explained by differences in affinities of the ligands for the receptor, because the affinity-constants of both factors are very similar. We have compared the intracellular processing of ligand-receptor complexes using either EGF or TGF-alpha in two different cell systems. We found that TGF-alpha dissociates from the EGF/TGF-alpha-receptor at much higher pH than EGF, which may reflect the substantial difference in the calculated isoelectric points. After internalization, the intracellular TGF-alpha is more rapidly cleared than EGF, and a substantial portion of the released TGF-alpha represents undegraded TGF-alpha in contrast to the mostly degraded EGF. In addition, TGF-alpha did not induce a complete down-regulation of cell surface receptors, as observed with EGF, which is at least in part responsible for a much sooner recovery of the ligand-binding ability after down-regulation, in the case of TGF-alpha. These differences in processing of the ligand-receptor complexes may explain why TGF-alpha exerts quantitatively higher activities than EGF.

The distribution of antigen on the surface of RBL-2H3 rat basophilic leukemia cells observed by back-scattered electron imaging

1986 ◽  
Vol 44 ◽  
pp. 274-275
Author(s):  
R.F. Stump ◽  
J.R. Pfeiffer ◽  
JC. Seagrave ◽  
D. Huskisson ◽  
J.M. Oliver
Keyword(s):  
Cell Surface ◽  
Dynamic Properties ◽  
Leukemia Cells ◽  
Antigen Binding ◽  
Scattered Electron ◽  
Ige Receptor ◽  
Receptor Complexes ◽  
Rat Basophilic Leukemia Cells ◽  
Electron Imaging ◽  
Basophilic Leukemia

In RBL-2H3 rat basophilic leukemia cells, antigen binding to cell surface IgE-receptor complexes stimulates the release of inflammatory mediators and initiates a series of membrane and cytoskeletal events including a transformation of the cell surface from a microvillous to a lamellar topography. It is likely that dynamic properties of the IgE receptor contribute to the activation of these responses. Fewtrell and Metzger have established that limited crosslinking of IgE-receptor complexes is essential to trigger secretion. In addition, Baird and colleagues have reported that antigen binding causes a rapid immobilization of IgE-receptor complexes, and we have demonstrated an apparent increase with time in the affinity of IgE-receptor complexes for antigen.

scholarly journals From cell surface to signalling and back: the life of the mammalian FSH receptor

FEBS Journal ◽  
2020 ◽  
Author(s):  
Antara A. Banerjee ◽  
Shaini Joseph ◽  
Smita D. Mahale
Keyword(s):  
Cell Surface ◽  
Fsh Receptor
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