Production and characterization of monoclonal antibodies against amphiphysins

Jin, YuLian; Kim, Kyung-Yong; Soung, Nak-Kyun; Shin, Eun‐Young; Kim, Eung‐Gook; Kim, Seung Ryul

doi:10.1038/emm.2001.13

Cited by 8 publications

(7 citation statements)

References 19 publications

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“…We demonstrate here that dynamin II, amphiphysin II, and intersectin 1 are present in both COS-7 and HEK 293 cell lines and are expressed at comparable levels between the two cell types (COS-7 being of fibroblastic origin, HEK 293 of epithelial origin). This finding is congruent with the reported expression of these proteins in virtually all peripheral cell types (Herskovits et al 1993;Tsutsui et al 1997;Wigge et al 1997a,b;Guipponi et al 1998;Okamoto et al 1999;Sengar et al 1999;Pucharcos et al 2000Pucharcos et al , 2001Jin et al 2001;Predescu et al 2003;Sarret et al 2004), and with their broad involvement in ligand-induced receptor endocytosis (for reviews, see Wigge and McMahon 1998;Marsh and McMahon 1999;Santolini et al 1999;McPherson et al 2001;McPherson 2002). It also indicates that the present overexpression experiments are physiologically relevant.…”

Section: Discussionsupporting

confidence: 92%

Cell-type-specific pathways of neurotensin endocytosis

et al. 2005

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The neurotensin receptor subtype 1 (NTS1) is a G-protein-coupled receptor (GPCR) mediating a large number of central and peripheral effects of neurotensin. Upon stimulation, NTS1 is rapidly internalized and targeted to lysosomes. This process depends on the interaction of the phosphorylated receptor with beta-arrestin. Little is known about other accessory endocytic proteins potentially involved. Here, we investigated the involvement of dynamin, amphiphysin, and intersectin in the internalization of NTS1 receptor-ligand complexes in transfected COS-7 and HEK 293 cells, by using the transferrin receptor as an internal control for the constitutive endocytic pathway. We found that NTS1 endocytosis was not only arrestin-dependent, but also dynamin-dependent in both COS-7 and HEK 293 cells, whereas internalization of the transferrin receptor was independent of arrestin but required dynamin. Overexpression of the SH3 domain of amphiphysin II had no effect on receptor internalization in either cell type. By contrast, overexpression of full-length intersectin or of its SH3 domain (but not of its EH domain) inhibited NTS1 internalization in COS-7 but not in HEK 293 cells. This difference between COS-7 and HEK 293 cells was not attributable to differences in endogenous intersectin levels between the two cell lines. Indeed, the same constructs inhibited transferrin endocytosis equally well in COS-7 and HEK 293 cells. However, immunogold electron microscopy revealed that internalized NTS1 receptors were associated with clathrin-coated pits in COS-7 cells but with smooth vesicles in HEK 293 cells, suggesting that NTS1 internalization proceeds via different endocytic pathways in these two cell types.

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Section: Discussionsupporting

confidence: 92%

Cell-type-specific pathways of neurotensin endocytosis

et al. 2005

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“…We determined the anti-HVEM activity of supernatants of the hybridoma cells by ELISA (Jin et al, 2001). ninety-six-well polypropylene microplates (Falcon, Oxnard, CA) were coated with 5 mg/ml HVEM-Fc in NaHCO3 overnight at 4 o C, blocked with 3% BSA, and incubated with the supernatants.…”

Section: Elisa Screeningmentioning

confidence: 99%

High levels of soluble herpes virus entry mediator in sera of patients with allergic and autoimmune diseases

Jung

La²,

Kim³

et al. 2003

Exp Mol Med

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“…Extracts were immunoprecipitated with anti-GFP antibodies (Sigma) or mouse monoclonal anti-amphiphysin1 antibodies (N8-2) (12). In vitro phosphorylated samples and immunocomplexes were subjected to SDS-PAGE and autoradiography.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Amphiphysin1 by Mitogen-activated Protein Kinase

Wang

Adachi

Nakamura

et al. 2004

Journal of Biological Chemistry

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Amphiphysin1, which can simultaneously bind to dynamin1 and the clathrin adaptor AP-2, is essential for dynamin1 recruitment during receptor-mediated endocytosis, but little is known about its regulatory mechanism. Here, we purified a 120-kDa mitogen-activated protein kinase (MAPK) substrate protein from porcine brains and identified the protein as amphiphysin1. Serine phosphorylation of amphiphysin1 was rapidly induced by nerve growth factor (NGF) in PC12 cells, and the induction was blocked by a MAPK inhibitor. Furthermore, when phosphorylated by MAPK in vitro or by NGF treatment in vivo, amphiphysin1 failed to bind to AP-2, but its association with dynamin1 was unaffected. Consistent with this, mutation of consensus MAPK phosphorylation sites increased amphiphysin1 binding to AP-2 and their intracellular colocalization. Thus, we propose that MAPK phosphorylation of amphiphysin1 controls NGF receptor/TrkA-mediated endocytosis by terminating the amphiphysin1-AP-2 interaction. This perhaps helps to regulate the availability of amphiphysin1-dynamin1 complexes for binding to the endocytic vesicle.Clathrin-mediated endocytosis, which involves the orchestration of several molecular components, is crucial for various intracellular communications including the control of the levels of transmembrane receptors and their ligands, the recycling of synaptic vesicles in nerve terminals, and signal transduction (1). Among endocytic proteins, amphiphysin1 has recently been postulated to perform an essential function in endocytosis (2). Amphiphysin1 interacts with dynamin1 through its SH3 (Srchomology 3) domain (3) and thereby recruits dynamin1 to the site of clathrin-dependent endocytosis (4) by binding to the clathrin adaptor AP-2 (␣-adaptin subunit), which is associated with plasma membrane receptors. Phosphorylation and dephosphorylation events play an important role in the regulation of the assembly of endocytic protein complexes; phosphorylation of amphiphysin1 negatively regulates its association with AP-2, and phosphorylation of dynamin1 results in its dissociation from amphiphysin1 (5). These modifications may contribute to the availability of amphiphysin1 and dynamin1 for binding to the coated vesicle. Recently, cylin-dependent kinase Cdk5 was identified as a protein kinase responsible for this phosphorylation reaction (6 -8). Cdk5 phosphorylated amphiphysin1 and thereby promoted the dissociation of amphiphysin1 from AP-2. Likewise, Cdk5-phosphorylation of dynamin1 inhibited its binding to amphiphysin1. The results indicate that Cdk5 plays an important physiological role in endocytosis at nerve terminals by altering protein-protein interaction. However, the exact molecular mechanism underlying the regulation of endocytosis through phosphorylation reaction has not fully been elucidated.The 42/45-kDa MAPK 1 is activated by extracellular signals. Activation of MAPK requires its dual phosphorylation on threonine and tyrosine residues catalyzed by MAPK kinase (MAPKK), and MAPKK is activated by phosphorylation by Raf-1 (9)...

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Production and characterization of monoclonal antibodies against amphiphysins

Cited by 8 publications

References 19 publications

Cell-type-specific pathways of neurotensin endocytosis

Cell-type-specific pathways of neurotensin endocytosis

High levels of soluble herpes virus entry mediator in sera of patients with allergic and autoimmune diseases

Regulation of Amphiphysin1 by Mitogen-activated Protein Kinase

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