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PubMed2005

TTP specifically regulates the internalization of the transferrin receptor.

Different plasma membrane receptors are internalized through saturable/noncompetitive pathways, suggesting cargo-specific regulation. Here, we report that TTP (SH3BP4), a SH3-containing protein, specifically regulates the internalization of the transferrin receptor (TfR). TTP interacts with endocytic proteins, including clathrin, dynamin, and the TfR, and localizes selectively to TfR-containing coated-pits (CCP) and -vesicles (CCV). Overexpression of TTP specifically inhibits TfR internalization, and causes the formation of morphologically aberrant CCP, which are probably fission impaired. This effect is mediated by the SH3 of TTP, which can bind to dynamin, and it is rescued by overexpression of dynamin. Functional ablation of TTP causes a reduction in TfR internalization, and reduced cargo loading and size of TfR-CCV. Tyrosine phosphorylation of either TTP or dynamin prevents their interaction, pointing to a possible mechanism of exclusion of TTP from some CCP. Thus, TTP might represent one of the long sought for molecules that allow cargo-specific control of clathrin endocytosis.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Different modes of internalization of apoptotic alkyl-lysophospholipid and cell-rescuing lysophosphatidylcholine.

The synthetic alkyl-lysophospholipid (ALP), Et-18-OCH3 (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), can induce apoptosis in tumour cells. Unlike conventional chemotherapeutic drugs, ALP acts at the cell-membrane level. We have reported previously that ALP is internalized, and interferes with phosphatidylcholine (PC) biosynthesis de novo, which appeared to be essential for survival in lymphoma cells [Van der Luit, Budde, Ruurs, Verheij and Van Blitterswijk (2002) J. Biol. Chem. 277, 39541-39547]. Here, we report that, in HeLa cells, ALP accumulates in lipid rafts, and that internalization is inhibited by low temperature, monensin, disruption of lipid rafts and expression of a dominant-negative mutant of dynamin bearing a replacement of Lys44 with alanine (K44A). Thus ALP is internalized via raft- and dynamin-mediated endocytosis. Dynamin-K44A alleviated the ALP-induced inhibition of PC synthesis and rescued the cells from apoptosis induction. Additional cell rescue was attained by exogenous lysoPC, which after internalization serves as an alternative substrate for PC synthesis (through acylation). Unlike ALP, and despite the high structural similarity to ALP, lysoPC uptake did not occur via lipid rafts and did not depend on functional dynamin, indicating no involvement of endocytosis. Albumin back-extraction experiments suggested that (radiolabelled) lysoPC undergoes transbilayer movement (flipping). We conclude that ALP is internalized by endocytosis via lipid rafts to cause apoptosis, while exogenous cell-rescuing lysoPC traverses the plasma membrane outside rafts by flipping. Additionally, our data imply the importance of ether bonds in lyso-phospholipids, such as in ALP, for partitioning in lipid rafts.

Antigens, CD↗
PubMed2005

Molecular and genetic characterization of the interactions between the Drosophila stoned-B protein and DAP-160 (intersectin).

The stoned locus of Drosophila produces a dicistronic transcript and encodes two proteins, stoned-A (STNA) and stoned-B (STNB). Both proteins are located at synaptic terminals. The STNB protein contains a domain that has homology with the mu-subunit of the AP (adaptor protein) complex, as well as a number of NPF (Asp-Pro-Phe) motifs known to bind EH (Eps15 homology) domains. Mutations at the stoned locus interact synergistically with mutations at the shibire (dynamin) locus and alter synaptic vesicle endocytosis. The STNB protein has also been shown to interact with synaptic vesicles via synaptogamin-I. We initiated an investigation of the possible interaction of DAP-160 (dynamin-associated protein of 160 kDa), a Drosophila member of the intersectin family, with the STNB protein. We show here that both of the viable stoned alleles interacted with a genetic construct that reduces DAP-160 levels to 25% of normal. One of these stoned alleles contains a substitution resulting in a stop codon in the open reading frame encoding STNB. This allele also shows markedly reduced levels of both DAP-160 and dynamin. As anticipated, the NPF motifs in STNB are found to be high-affinity binding motifs for the EH domains of DAP-160. One of the SH3 (Src homology 3) domains of DAP-160 also interacts with STNB. Finally, we show that immunoprecipitation of STNB from fly head extracts co-precipitates with DAP-160, and we conclude that the interaction of the STNB protein with both synaptotagmin I and DAP-160 may regulate synaptic vesicle recycling by recruiting dynamin to a pre-fission complex.

Adaptor Proteins, Vesicular Transport↗
PubMed2004

Gbetagamma activation of Src induces caveolae-mediated endocytosis in endothelial cells.

Caveolae-mediated endocytosis in endothelial cells is stimulated by the binding of albumin to gp60, a specific albumin-binding protein localized in caveolae. The activation of gp60 induces its cell surface clustering and association with caveolin-1, the caveolar-scaffolding protein. This interaction leads to G(i)-induced Src kinase activation, which in turn signals dynamin-2-mediated fission and directed migration of caveolae-derived vesicles from apical to basal membrane. In this study, we investigated the possible role of the Gbetagamma heterodimer in signaling G(i)-induced Src activation and subsequent caveolae-mediated endocytosis. We observed using rat lung microvascular endothelial cells that expression of the C terminus of beta-adrenergic receptor kinase (ct-betaARK), an inhibitor Gbetagamma signaling, prevented gp60-dependent Src activation as well as caveolae-mediated endocytosis and transcellular transport of albumin and uptake of cholera toxin subunit B, a specific marker of caveolae internalization. Expression of ct-betaARK also prevented Src-mediated tyrosine phosphorylation of caveolin-1 and dynamin-2 and the resultant phosphorylation-dependent association of dynamin-2 and caveolin-1. Also, the direct activation of Gbetagamma using a specific cell-permeant activating peptide (myristoylated-SIRKALNILGYPDYD) simulated the effects of gp60 in inducing Src activation, caveolin-1, and dynamin-2 phosphorylation as well as caveolae-mediated endocytosis of cholera toxin subunit B. The myristoylated-SIRKALNILGYPDYD peptide-induced responses were inhibited by the expression of ct-betaARK. Taken together, our results demonstrate that Gbetagamma activation of Src signals caveolae-mediated endocytosis and transendothelial albumin transport via transcytosis.

Albumins↗
PubMed2003

Impairing actin filament or syndapin functions promotes accumulation of clathrin-coated vesicles at the apical plasma membrane of acinar epithelial cells.

In this article, we investigate the contributions of actin filaments and accessory proteins to apical clathrin-mediated endocytosis in primary rabbit lacrimal acini. Confocal fluorescence and electron microscopy revealed that cytochalasin D promoted apical accumulation of clathrin, alpha-adaptin, dynamin, and F-actin and increased the amounts of coated pits and vesicles at the apical plasma membrane. Sorbitol density gradient analysis of membrane compartments showed that cytochalasin D increased [14C]dextran association with apical membranes from stimulated acini, consistent with functional inhibition of apical endocytosis. Recombinant syndapin SH3 domains interacted with lacrimal acinar dynamin, neuronal Wiskott-Aldrich Syndrome protein (N-WASP), and synaptojanin; their introduction by electroporation elicited remarkable accumulation of clathrin, accessory proteins, and coated pits at the apical plasma membrane. These SH3 domains also significantly (p </= 0.05) increased F-actin, with substantial colocalization of dynamin and N-WASP with the additional filaments. Coelectroporation with the VCA domain of N-WASP blocked the increase in F-actin and reversed the morphological changes indicative of impaired apical endocytosis. We suggest that transient modulation of actin polymerization by syndapins through activation of the Arp2/3 complex via N-WASP coordinates dynamin-mediated vesicle fission at the apical plasma membrane of acinar epithelia. Trapping of assembled F-actin intermediates during this process by cytochalasin D or syndapin SH3 domains impairs endocytosis.

Actin Cytoskeleton↗
PubMed1999

Receptor-mediated Moloney murine leukemia virus entry can occur independently of the clathrin-coated-pit-mediated endocytic pathway.

To investigate receptor-mediated Moloney murine leukemia virus (MoMuLV) entry, the green fluorescent protein (GFP)-tagged ecotropic receptor designated murine cationic amino acid transporter (MCAT-1) (MCAT-1-GFP) was constructed and expressed in 293 cells (293/MCAT-1-GFP). 293/MCAT-1-GFP cells displayed green fluorescence primarily at the cell membrane and supported wild-type levels of MoMuLV vector binding and transduction. Using immunofluorescence labeling and confocal microscopy, it was demonstrated that the surface envelope protein (SU) gp70 of MoMuLV virions began to appear inside cells 5 min after virus binding and was colocalized with MCAT-1-GFP. However, clathrin was not colocalized with MCAT-1-GFP, suggesting that MoMuLV entry, mediated by MCAT-1, does not involve clathrin. Double immunofluorescence labeling of SU and clathrin in 293 cells expressing untagged receptor (293/MCAT-1) gave the same results, i.e., SU and clathrin did not colocalize. In addition, we examined the transduction ability of MoMuLV vector on HeLa cells overexpressing the dominant-negative GTPase mutant of dynamin (K44A). HeLa cells overexpressing mutant dynamin have a severe block in endocytosis by the clathrin-coated-pit pathway. No significant titer difference was observed when MoMuLV vector was tranduced into HeLa cells overexpressing either wild-type or mutant dynamin, while the transduction ability of vesicular stomatitis virus glycoprotein pseudotyped vector into HeLa cells overexpressing mutant dynamin was decreased significantly. Taken together, these data suggest that MoMuLV entry does not occur through the clathrin-coated-pit-mediated endocytic pathway.

3T3 Cells↗
PubMed2005

Involvement of clathrin-mediated endocytosis in human immunodeficiency virus type 1 entry.

Productive entry of human immunodeficiency virus (HIV) is believed to occur by direct fusion at the plasma membrane. Endocytic uptake of HIV particles has been observed in several studies but is considered to be nonproductive, leading to virus degradation in the lysosome. We show here that endocytosis contributes significantly to productive HIV entry in HeLa cells by using trans dominant-negative mutants of dynamin and Eps15. Inducible expression of a dominant-negative mutant of dynamin in a CD4-positive HeLa cell line reduced HIV infection by 40 to 80%. This effect was independent of the infectious dose and was observed for three different isolates. Analysis of reverse transcription products by real-time PCR and of virus entry by delivery of a virion-associated Vpr-beta-lactamase fusion protein revealed a similar reduction, indicating that the block occurred at the entry stage. A strong reduction of HIV entry was also observed upon transient transfection of a different trans dominant-negative variant of dynamin, and this reduction correlated with the relative inhibition of transferrin endocytosis. Expression of a dominant-negative variant of Eps15, which is specific for clathrin-dependent endocytosis, reduced HIV entry in HeLa cells by ca 95%, confirming the role of endocytosis for productive infection. In contrast, no effect was observed for a dominant-negative variant of caveolin. We conclude that dynamin-dependent, clathrin-mediated endocytosis can lead to productive entry of HIV in HeLa cells, suggesting this pathway as an alternative route of virus entry.

Caveolin 1↗
PubMed2003

Inhibition of GTP-dependent vesicle trafficking impairs internalization of plasmalemmal eNOS and cellular nitric oxide production.

The Ca2+ mobilizing peptide, bradykinin (BK), stimulates endothelial nitric oxide synthase (eNOS)-derived cellular nitric oxide (NO) production in association with altering the subcellular distribution of the enzyme. In the present study we examine the influence of cellular GTPases, particularly the large GTPase dynamin, on BK-mediated eNOS localization and cellular NO production. BK stimulation of ECV cells, which were stably transfected with eNOS-GFP (eNOS-GFP ECV304), increased NO production. This was associated with the mobilization of eNOS-GFP protein into Triton X-100-insoluble fractions of cell lysates, and an internalization of plasmalemmal eNOS-GFP in live and fixed ECV 304 cells. Incubation of digitonin-permeabilized ECV304 cells with the non-hydrolyzed GTP analog, GTP-gamma-S, abrogated the BK-mediated internalization of eNOS-GFP as assessed by confocal microscopy. Conversely, inhibition of clathrin-dependent endocytosis, via overexpression of AP 180 or pretreatment of cells with chlorpromazine, did not influence BK-mediated eNOS redistribution. Furthermore, specific inhibition of dynamin-2 GTPase function by overexpression of a dominant negative construct, K44A, prevented the BK-mediated enrichment of eNOS-GFP within low buoyant density, caveolin-enriched fractions of eNOS-GFP ECV304 cell lysates. Dynamin-2 K44A overexpression also markedly impaired BK-dependent, L-NAME-inhibited NO production as did incubation of permeabilized cells with GTP-gamma-s. These studies demonstrate that disruption of dynamin- and GTP-dependent, but clathrin-independent, vesicle trafficking pathways impairs BK-dependent cellular NO production, via inhibition of the internalization of eNOS-containing plasmalemmal vesicles.

Animals↗
PubMed2004

Differences in endosomal targeting of human (beta)1- and (beta)2-adrenergic receptors following clathrin-mediated endocytosis.

The beta(2)-adrenergic receptor (beta(2)AR) undergoes agonist-mediated endocytosis via clathrin-coated pits by a process dependent on both arrestins and dynamin. Internalization of some G protein-coupled receptors, however, is independent of arrestins and/or dynamin and through other membrane microdomains such as caveolae or lipid rafts. The human beta(1)AR is less susceptible to agonist-mediated internalization than the beta(2)-subtype, and its endocytic route, which is unknown, may be different. We have found that (i) co-expression of arrestin-2 or -3 enhanced the internalization of both subtypes whereas co-expression of dominant-negative mutants of arrestin-2 or dynamin impaired their internalization, as did inhibitors of clathrin-mediated endocytosis. (ii) Agonist stimulation increased the phosphorylation of beta(2)AR but not beta(1)AR. (iii) In response to agonist, each subtype redistributed from the cell surface to a distinct population of cytoplasmic vesicles; those containing beta(1)AR were smaller and closer to the plasma membrane whereas those containing beta(2)AR were larger and more perinuclear. (iv) When subcellular fractions from agonist-treated cells were separated by sucrose density gradient centrifugation, all of the internalized beta(2)AR appeared in the lighter endosomal-containing fractions whereas some of the internalized beta(1)AR remained in the denser plasma membrane-containing fractions. (v) Both subtypes recycled with similar kinetics back to the cell surface upon removal of agonist; however, recycling of beta(2)AR but not beta(1)AR was inhibited by monensin. Based on these results, we propose that the internalization of beta(1)AR is both arrestin- and dynamin-dependent and follows the same clathrin-mediated endocytic pathway as beta(2)AR. But during or after endocytosis, beta(1)AR and beta(2)AR are sorted into different endosomal compartments.

Adrenergic beta-1 Receptor Agonists↗
PubMed2005

NOSTRIN functions as a homotrimeric adaptor protein facilitating internalization of eNOS.

Intracellular trafficking of endothelial nitric oxide synthase (eNOS) between different compartments is incompletely understood. Recently, we described a novel eNOS-interacting protein, NOSTRIN, which upon overexpression drives eNOS away from the plasma membrane towards intracellular compartments. Sequence similarity of NOSTRIN and pacsins/syndapins suggested a role for NOSTRIN in endocytosis. Accordingly, we show here that NOSTRIN interacts with the large GTPase dynamin and the actin nucleation promoting factor N-WASP by means of its SH3 domain, which also represents the docking site for eNOS. Via a coiled-coil region in the C-terminal portion of the protein, NOSTRIN oligomerizes, mainly forming trimers, which would allow simultaneous interaction with multiple binding partners of the SH3 domain. Consistent with this notion, expression of dynamin-2-GFP in CHO cells stably expressing eNOS (CHO-eNOS) results in recruitment of eNOS to dynamin-positive structures, only when NOSTRIN is present as well. Similarly, when N-WASP-GFP and NOSTRIN are co-expressed in CHO-eNOS cells, both proteins strongly co-localize with eNOS and are recruited to structures running along actin filaments. If, however, the actin cytoskeleton is depolymerized by cytochalasin D, NOSTRIN and eNOS are associated with extended structures in the cell periphery, possibly being unable to leave the plasma membrane. Together, these results indicate that NOSTRIN may facilitate endocytosis of eNOS by coordinating the function of dynamin and N-WASP.

Actins↗
PubMed2003

Actin microfilaments et al.--the many components, effectors and regulators of epithelial cell endocytosis.

The aim of this review is to introduce the advances made over the past several years regarding the participation of actin and actin-associated proteins in clathrin-mediated endocytosis in simple cell models, and then to consider the evidence for the involvement of these effectors in apical clathrin-mediated endocytosis in epithelial cells. Basic mechanisms of clathrin-mediated endocytosis are initially addressed, followed by a detailed description of the actin cytoskeleton: its organization, function and, most importantly, the essential role played by proteins and signaling pathways responsible for the regulation of actin filament dynamics. Our focus then shifts to the GTPase, dynamin and its pivotal role as a bridge between various components of the clathrin endocytic machinery and the actin cytoskeleton. Mechanisms and effectors of dynamin-dependent endocytosis are then described, with a particular emphasis on novel proteins, which link dynamin to actin filaments. We consider additional effectors proposed to interact with actin to facilitate clathrin-mediated endocytosis in a dynamin-independent manner. The multiple roles which actin filaments are thought to play in endocytosis are addressed followed by a more detailed characterization of actin filament participation specifically in apical endocytosis. We conclude by discussing how these concepts may be integrated to improve drug internalization at the apical plasma membrane of epithelial cells.

Actin Cytoskeleton↗
PubMed1999

Role of arrestins in endocytosis and signaling of alpha2-adrenergic receptor subtypes.

We investigated the role of arrestins in the trafficking of human alpha2-adrenergic receptors (alpha2-ARs) and the effect of receptor trafficking on p42/p44 MAP kinase activation. alpha2-ARs expressed in COS-1 cells demonstrated a modest level of agonist-mediated internalization, with alpha2c > alpha2b > alpha2a. However, upon coexpression of arrestin-2 (beta-arrestin-1) or arrestin-3 (beta-arrestin-2), internalization of the alpha2b AR was dramatically enhanced and redistribution of receptors to clathrin coated vesicles and endosomes was observed. Internalization of the alpha2c AR was selectively promoted by coexpression of arrestin-3, while alpha2a AR internalization was only slightly stimulated by coexpression of either arrestin. Coexpression of GRK2 had no effect on the internalization of any alpha2-AR subtype, either in the presence or absence of arrestins. Internalization of the alpha2b and alpha2c ARs was inhibited by coexpression of dominant negative dynamin-K44A. However, alpha2-AR-mediated activation of either endogenous or cotransfected p42/p44 mitogen-activated protein (MAP) kinase was not affected by either dynamin-K44A or arrestin-3. Moreover, activation of p42/p44 MAP kinase by endogenous epidermal growth factor, lysophosphatidic acid, and beta2-adrenergic receptors was also unaltered by dynamin-K44A. In summary, our data suggest that internalization of the alpha2b, alpha2c, and to a lesser extent alpha2a ARs, is both arrestin- and dynamin-dependent. However, endocytosis does not appear to be required for alpha2-adrenergic, epidermal growth factor, lysophosphatidic acid, or beta2-adrenergic receptor-mediated p42/p44 MAP kinase activation in COS-1 cells.

Amino Acid Sequence↗
PubMed1999

Regulation of muscarinic acetylcholine receptor sequestration and function by beta-arrestin.

After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic beta-arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the beta2-adrenergic receptor have demonstrated that beta-arrestin also targets the receptors to clathrin-coated pits for subsequent internalization and activation of mitogen-activated protein kinases. We and others have previously shown that muscarinic acetylcholine receptors (mAChRs) of the m1, m3, and m4 subtype require functional dynamin to sequester into HEK-293 tsA201 cells, whereas m2 mAChRs sequester in a dynamin-independent manner. To investigate the role of beta-arrestin in mAChR sequestration, we determined the effect of overexpressing beta-arrestin-1 and the dominant-negative inhibitor of beta-arrestin-mediated receptor sequestration, beta-arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60-75% in cells overexpressing beta-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of beta-arrestin-1 V53D as well as dynamin K44A significantly suppressed m1 mAChR-mediated activation of mitogen-activated protein kinases. Finally, we investigated whether mAChRs sequester into clathrin-coated vesicles by overexpressing Hub, a dominant-negative clathrin mutant. Although sequestration of m1, m3, and m4 mAChRs was inhibited by 50-70%, m2 mAChR sequestration was suppressed by less than 10%. We conclude that m1, m3, and m4 mAChRs expressed in HEK-293 tsA201 cells sequester into clathrin-coated vesicles in a beta-arrestin- and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.

Arrestins↗
PubMed1998

Involvement of growth factor receptor-bound protein-2 in rat hepatocyte growth.

Growth factor receptor-bound protein-2 (GRB-2) is a protein linking receptor tyrosine kinase and Sos (Son of Sevenless gene; Ras GDP/GTP exchange protein), leading to activation of the Ras-mitogen-activated protein kinase (MAPK) cascade. So far, it remains unclear how GRB-2 plays a role in signal transduction pathways evoked by hepatotrophic factors. This study was attempted to evaluate the involvement of GRB-2 in signalling in rat hepatocyte growth. Using rat cultured hepatocytes stimulated by hepatotrophic factors and regenerating livers after partial hepatectomy (PH) we examined GRB-2-mediated linkage of hepatotrophic factor receptors to signal transducing molecules such as Sos or dynamin-II by immunoprecipitation and western blot analysis. In primary cultured hepatocytes stimulated with hepatocyte growth factor (HGF) or epidermal growth factor (EGF), GRB-2 linked HGF receptor or EGF receptor, respectively, to Sos which activated the mitogen-activated protein kinase (MAPK) cascade. In contrast, in primary cultured hepatocytes stimulated with insulin, GRB-2 linked insulin receptor substrate-1 (IRS-1) to dynamin-II as well as Sos. In the early phase after PH, GRB-2 activated the Ras-MAPK cascade by linking HGF receptor, IRS-1, or EGF receptor to Sos. In the late phase after PH, a complex of IRS-1-GRB-2 associated with dynamin-II, indicating that GRB-2 may transduce signals from IRS-1 to dynamin-II. We conclude that GRB-2 may play a role in transmitting signals from hepatotrophic factors to not only MAPK but also to other signalling pathways in hepatocyte growth.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Roles of cortactin, an actin polymerization mediator, in cell endocytosis.

Cortactin, an actin-binding protein and a substrate of Src, is encoded by the EMS1 oncogene. Cortactin is known to activate Arp2/3 complex-mediated actin polymerization and interact with dynamin, a large GTPase and proline rich domain-containing protein. Transferrin endocytosis was significantly reduced in cells by knock-down of cortactin expression as well as in vivo introduction of cortactin immunoreagents. Cortactin-dynamin interaction displayed morphologically dynamic co-distribution with a change in the endocytosis level in cells treated with an actin depolymerization reagent, cytochalasin D. In an in vitro beads assay, a branched actin network was recruited onto dynamin-coated beads in a cortactin Src homology domain 3 (SH3)-dependent manner. In addition, cortactin was found to function in the late stage of clathrin coated vesicle formation. Taken together, cortactin is required for optimal clathrin mediated endocytosis in a dynamin directed manner.

Actin-Related Protein 2↗
PubMed1999

beta-arrestins regulate interleukin-8-induced CXCR1 internalization.

The functional role of neutrophils during acute inflammatory responses is regulated by two high affinity interleukin-8 receptors (CXCR1 and CXCR2) that are rapidly desensitized and internalized upon binding their cognate chemokine ligands. The efficient re-expression of CXCR1 on the surface of neutrophils following agonist-induced internalization suggests that CXCR1 surface receptor turnover may involve regulatory pathways and intracellular factors similar to those regulating beta2-adrenergic receptor internalization and re-expression. To examine the internalization pathway utilized by ligand-activated CXCR1, a CXCR1-GFP construct was transiently expressed in two different cell lines, HEK 293 and RBL-2H3 cells. While interleukin-8 stimulation promoted CXCR1 sequestration in RBL-2H3 cells, receptor internalization in HEK 293 cells required co-expression of G protein-coupled receptor kinase 2 and beta-arrestin proteins. The importance of beta-arrestins in CXCR1 internalization was confirmed by the ability of a dominant negative beta-arrestin 1-V53D mutant to block internalization of CXCR1 in RBL-2H3 cells. A role for dynamin was also demonstrated by the lack of CXCR1 internalization in dynamin I-K44A dominant negative mutant-transfected RBL-2H3 cells. Agonist-promoted co-localization of transferrin and CXCR1-GFP in endosomes of RBL-2H3 cells confirmed that receptor internalization occurs via clathrin-coated vesicles. Our data provides a direct link between agonist-induced internalization of CXCR1 and a requirement for G protein-coupled receptor kinase 2, beta-arrestins, and dynamin during this process.

Animals↗
PubMed2000

Syndapin isoforms participate in receptor-mediated endocytosis and actin organization.

Syndapin I (SdpI) interacts with proteins involved in endocytosis and actin dynamics and was therefore proposed to be a molecular link between the machineries for synaptic vesicle recycling and cytoskeletal organization. We here report the identification and characterization of SdpII, a ubiquitously expressed isoform of the brain-specific SdpI. Certain splice variants of rat SdpII in other species were named FAP52 and PACSIN 2. SdpII binds dynamin I, synaptojanin, synapsin I, and the neural Wiskott-Aldrich syndrome protein (N-WASP), a stimulator of Arp2/3 induced actin filament nucleation. In neuroendocrine cells, SdpII colocalizes with dynamin, consistent with a role for syndapin in dynamin-mediated endocytic processes. The src homology 3 (SH3) domain of SdpI and -II inhibited receptor-mediated internalization of transferrin, demonstrating syndapin involvement in endocytosis in vivo. Overexpression of full-length syndapins, but not the NH(2)-terminal part or the SH3 domains alone, had a strong effect on cortical actin organization and induced filopodia. This syndapin overexpression phenotype appears to be mediated by the Arp2/3 complex at the cell periphery because it was completely suppressed by coexpression of a cytosolic COOH-terminal fragment of N-WASP. Consistent with a role in actin dynamics, syndapins localized to sites of high actin turnover, such as filopodia tips and lamellipodia. Our results strongly suggest that syndapins link endocytosis and actin dynamics.

Actins↗
PubMed2000

Mechanisms of agonist-induced down-regulation of the human kappa-opioid receptor: internalization is required for down-regulation.

Previously, we showed that the human kappa-opioid receptor (hkor) stably expressed in Chinese hamster ovary (CHO) cells underwent down-regulation after prolonged U50,488H treatment. In the present study, we determined the mechanisms underlying this process. U50, 488H caused a significant down-regulation of the hkor, although etorphine did not. Neither U50,488H nor etorphine caused down-regulation of the rat kappa-opioid receptor. Thus, similar to internalization, there are agonist and species differences in down-regulation of kappa-opioid receptors. Expression of the dominant negative mutants arrestin-2(319-418) or dynamin I-K44A significantly reduced U50,488H-induced down-regulation of the hkor. Coexpression of GRK2 or GRK2 and arrestin-2 permitted etorphine to induce down-regulation of the hkor, although expression of arrestin-2 or dynamin I alone did not. Expression of the dominant negative mutants rab5A-N133I or rab7-N125I blunted U50,488H-induced down-regulation. Pretreatment with lysosomal enzyme inhibitors [(2S, 3S)trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester or chloroquine] or proteasome inhibitors (proteasome inhibitor I, MG-132, or lactacystin) decreased the extent of U50,488H-induced down-regulation. A combination of chloroquine and proteasome inhibitor I abolished U50,488H-induced down-regulation. These results indicate that U50,488H-induced down-regulation of the hkor involves GRK-, arrestin-2-, dynamin-, rab5-, and rab7-dependent mechanisms and receptors seem to be trafficked to lysosomes and proteasomes for degradation. Thus, U50,488H-induced internalization and down-regulation of the hkor share initial common mechanisms. To the best of our knowledge, these results represent the first report on the involvement of both rab5 and rab7 in agonist-induced down-regulation of a G protein-coupled receptor. In addition, this study is among the first to show the involvement of proteasomes in agonist-induced down-regulation of a G protein-coupled receptor.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗