Ligand-stimulated beta 2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes

1995 ◽  
Vol 108 (9) ◽  
pp. 2983-2991 ◽  
Author(s):  
R.H. Moore ◽  
N. Sadovnikoff ◽  
S. Hoffenberg ◽  
S. Liu ◽  
P. Woodford ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Adrenergic Receptor ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Beta Agonist ◽  
Hek293 Cells ◽  
Fluid Phase Endocytosis ◽  
Beta 2

The small GTPase rab5 appears to be rate-limiting for the constitutive internalization of transferrin receptor and for fluid-phase endocytosis. However, it is unknown whether rab5 regulates receptors whose internalization is stimulated by the binding of ligand, and whether such receptors change the underlying rate of the endocytic pathways they utilize. As a model for ligand-stimulated endocytosis, we used transfected HEK293 cells expressing high levels of an epitope-tagged human beta 2-adrenergic receptor. Nearly all receptors were on the cell surface in the absence of agonist, but within ten minutes of agonist addition > 50% of receptors internalized and colocalized extensively with rab5. Hypertonic sucrose blocked beta 2-adrenergic receptor internalization, as well as that of transferrin receptor, suggesting a clathrin-mediated process. In contrast, an inhibitor of potocytosis had little effect upon beta 2-adrenergic receptor internalization, suggesting that this process did not require active caveolae. Consistent with this finding, caveolin was not detectable in the 12 beta 6 line, as assessed by western blotting with a polyclonal anti-caveolin antibody. Stimulated receptor internalization did not affect the rate or capacity of the constitutive endocytic pathway since there was no detectable increase in fluid-phase endocytosis after addition of beta-agonist, nor was there a significant change in the amount of surface transferrin receptor. Altogether, these data suggest that beta 2-adrenergic receptors internalize by a clathrin-mediated and rab5-regulated constitutive endocytic pathway. Further, agonist-stimulated receptor internalization has no detectable effect upon the function of this pathway.

scholarly journals Developmental Variation in Rab11-Dependent Trafficking in Trypanosoma brucei

2005 ◽  
Vol 4 (5) ◽  
pp. 971-980 ◽  
Author(s):  
Belinda S. Hall ◽  
Emma Smith ◽  
Wolfram Langer ◽  
Louisa A. Jacobs ◽  
David Goulding ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
Primary Role ◽  
Flagellar Pocket ◽  
Developmentally Regulated ◽  
Developmental Variation ◽  
Fluid Phase Endocytosis

ABSTRACT In Trypanosoma brucei, endocytosis is developmentally regulated and is substantially more active in the mammalian infective stage, where it likely plays a role in immune evasion. The small GTPase TbRAB11 is highly expressed in the mammalian stage and mediates recycling of glycosylphosphatidylinositol-anchored proteins, including the variant surface glycoprotein (VSG) and the transferrin receptor, plus trafficking of internalized anti-VSG antibody and transferrin. No function has been assigned to TbRAB11 in the procyclic (insect) stage trypanosome. The importance of TbRAB11 to both bloodstream and procyclic form viability was assessed by RNA interference (RNAi). Suppression of TbRAB11 in the bloodstream form was rapidly lethal and led to cells with round morphology and an enlarged flagellar pocket. TbRAB11 RNAi was also lethal in procyclic forms, which also became rounded, but progression to cell death was significantly slower and the flagellar pocket remained normal. In bloodstream forms, silencing of TbRAB11 had no effect on exocytosis of newly synthesized VSG, fluid-phase endocytosis, or transferrin uptake, but export of internalized transferrin was inhibited. Lectin endocytosis assays revealed a block to postendosomal transport mediated by suppressing TbRAB11. By contrast, in procyclic forms, depletion of TbRAB11 blocks both fluid-phase endocytosis and internalization of surface proteins. In normal bloodstream forms, most VSG is recycled, but in procyclics, internalized surface proteins accumulated in the lysosome. These data demonstrate that TbRAB11 controls recycling and is essential in both life stages of T. brucei but that its primary role is subject to developmental variation.

scholarly journals The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits

1992 ◽  
Vol 117 (2) ◽  
pp. 279-290 ◽  
Author(s):  
A Pelchen-Matthews ◽  
I Boulet ◽  
DR Littman ◽  
R Fagard ◽  
M Marsh
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Protein Tyrosine ◽  
Src Gene ◽  
Fluid Phase Endocytosis ◽  
Nih 3T3

The lymphocyte glycoprotein CD4 is constitutively internalized and recycled in nonlymphoid cells, but is excluded from the endocytic pathway in lymphocytic cells (Pelchen-Matthews, A., J. E. Armes, G. Griffiths, and M. Marsh. 1991. J. Exp. Med. 173: 575-587). Inhibition of CD4 endocytosis is dependent on CD4 expressing an intact cytoplasmic domain and is only observed in cells where CD4 can interact with the protein tyrosine kinase p56lck, a member of the src gene family. We have expressed p56lck, p60c-src, or chimeras of the two proteins in CD4-transfected NIH-3T3 or HeLa cells. Immunoprecipitation of CD4 and in vitro kinase assays showed that p56lck and the lck/src chimera, which contains the NH2 terminus of p56lck, can associate with CD4. In contrast, p60c-src and the src/lck chimera, which has the NH2 terminus of p60c-src, do not associate with CD4. Endocytosis assays using radioiodinated anti-CD4 monoclonal antibodies demonstrated that coexpression of CD4 with p56lck, but not with p60c-src, inhibited CD4 endocytosis, and that the extent of the inhibition depended directly on the relative levels of CD4 and p56lck expressed. The uptake of mutant CD4 molecules which cannot interact with p56lck was not affected. Measurement of the fluid-phase endocytosis of HRP or the internalization of transferrin indicated that the effect of p56lck was specific for CD4, and did not extend to other receptor-mediated or fluid-phase endocytic processes. Immunogold labeling of CD4 at the cell surface and observation by electron microscopy demonstrated directly that p56lck inhibits CD4 endocytosis by preventing its entry into coated pits.

scholarly journals Domains of the Rsp5 Ubiquitin-Protein Ligase Required for Receptor-mediated and Fluid-Phase Endocytosis

2001 ◽  
Vol 12 (2) ◽  
pp. 421-435 ◽  
Author(s):  
Rebecca Dunn ◽  
Linda Hicke
Keyword(s):  
Plasma Membrane ◽  
Point Mutations ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Temperature Sensitive ◽  
C2 Domain ◽  
Ww Domains ◽  
Amino Terminal ◽  
Regulatory Event

Yeast Rsp5p and its mammalian homologue, Nedd4, arehect domain ubiquitin-protein ligases (E3s) required for the ubiquitin-dependent endocytosis of plasma membrane proteins. Because ubiquitination is sufficient to induce internalization, E3-mediated ubiquitination is a key regulatory event in plasma membrane protein endocytosis. Rsp5p is an essential, multidomain protein containing an amino-terminal C2 domain, three WW protein-protein interaction domains, and a carboxy-terminal hect domain that carries E3 activity. In this study, we demonstrate that Rsp5p is peripherally associated with membranes and provide evidence that Rsp5p functions as part of a multimeric protein complex. We define the function of Rsp5p and its domains in the ubiquitin-dependent internalization of the yeast α-factor receptor, Ste2p. Temperature-sensitive rsp5 mutants were unable to ubiquitinate or to internalize Ste2p at the nonpermissive temperature. Deletion of the entire C2 domain had no effect on α-factor internalization; however, point mutations in any of the three WW domains impaired both receptor ubiquitination and internalization. These observations indicate that the WW domains play a role in the important regulatory event of selecting phosphorylated proteins as endocytic cargo. In addition, mutations in the C2 and WW1 domains had more severe defects on transport of fluid-phase markers to the vacuole than on receptor internalization, suggesting that Rsp5p functions at multiple steps in the endocytic pathway.

scholarly journals The conserved Pkh–Ypk kinase cascade is required for endocytosis in yeast

2002 ◽  
Vol 156 (2) ◽  
pp. 241-248 ◽  
Author(s):  
Amy K.A. deHart ◽  
Joshua D. Schnell ◽  
Damian A. Allen ◽  
Linda Hicke
Keyword(s):  
Fluid Phase ◽  
Receptor Internalization ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Extracellular Signals ◽  
Fluid Phase Endocytosis ◽  
Endocytic Machinery ◽  
Kinase Cascade ◽  
Signaling Receptors ◽  
Mutant Cells

Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast α-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base–regulated serine–threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum– and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in α-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in α-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.

Phorbol ester promotes endocytosis by activating a factor involved in endosome fusion

1999 ◽  
Vol 112 (15) ◽  
pp. 2549-2557 ◽  
Author(s):  
A. Aballay ◽  
P.D. Stahl ◽  
L.S. Mayorga
Keyword(s):  
Phorbol Ester ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Early Endosomes ◽  
Zinc Depletion ◽  
Fluid Phase Endocytosis ◽  
Endocytic Vesicles

Previous studies indicate that a zinc- and phorbol ester-binding factor is necessary for in vitro endosome fusion and for the effect of Rab5 on endosome fusion. Rab5 is a small GTPase that regulates membrane fusion between early endosomes derived from either receptor-mediated endocytosis or fluid-phase endocytosis. In its GTP-bound form, Rab5 promotes endocytosis and enhances fusion among early endosomes. To determine if PMA stimulates endocytosis by activating a factor required for endosome fusion, we overexpressed wild-type Rab5, a dominant negative mutant (Rab5:S34N), and a GTPase deficient mutant (Rab5:Q79L) in BHK-21 cells. The phorbol ester PMA stimulates endocytosis and increases the number and the size of endocytic vesicles, even in the presence of Rab5:S34N. Zinc depletion with N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and addition of calphostin C (CPC), an inhibitor of PKC that interacts with zinc and phorbol ester binding motifs, inhibited both basal and Rab5-stimulated fluid phase endocytosis. These two reagents also inhibited the size and number of endocytic vesicles promoted by Rab5. These results suggest that PMA stimulates endocytosis by regulating the dynamics of the early endosome compartment.

scholarly journals Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

1996 ◽  
Vol 7 (3) ◽  
pp. 355-367 ◽  
Author(s):  
D J Spiro ◽  
W Boll ◽  
T Kirchhausen ◽  
M Wessling-Resnick
Keyword(s):  
Transferrin Receptor ◽  
Kinase Inhibitor ◽  
Morphological Changes ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Phosphatidylinositol 3 Kinase ◽  
Vitro Assay ◽  
Phosphatidylinositol 3

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

scholarly journals Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells.

1991 ◽  
Vol 115 (6) ◽  
pp. 1561-1572 ◽  
Author(s):  
T Ludwig ◽  
G Griffiths ◽  
B Hoflack
Keyword(s):  
Lysosomal Enzymes ◽  
Rat Kidney ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis ◽  
Normal Rat ◽  
Endocytic Compartments ◽  
Endocytic Pathways

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.

Characterisation of insulin and [beta]2-adrenergic receptor heteromers: experimental and bioinformatics approach

2015 ◽  
Author(s):  
Maja Mandic ◽  
Sanja Glisic ◽  
Christina Pedersen ◽  
Nevena Veljkovic ◽  
Jane Nohr ◽  
...  
Keyword(s):  
Adrenergic Receptor ◽  
Receptor Heteromers ◽  
Beta 2

Ligand and Structure Based Models for the Identification of Beta 2 Adrenergic Receptor Antagonists

2015 ◽  
Vol 11 (3) ◽  
pp. 222-236 ◽  
Author(s):  
Jayadev Joshi ◽  
Manali Dimri ◽  
Subhajit Ghosh ◽  
Nitisha Shrivastava ◽  
Rina Chakraborti ◽  
...  
Keyword(s):  
Adrenergic Receptor ◽  
Receptor Antagonists ◽  
Beta 2
Sign in / Sign up

Export Citation Format

Share Document