title: Alterations of EHD1/EHD4 protein levels interfere with L1/NgCAM endocytosis in neurons and disrupt axonal targeting.
abstract: Axon growth is regulated by many proteins, including adhesion molecules, which need to be trafficked correctly to axons. The adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM) travels to axons via an endocytosis-dependent pathway (transcytosis), traversing somatodendritic endosomes. The Eps15 homology domain (EHD) family proteins (EHD1-EHD4) play important roles in endosomal recycling and possibly in endocytosis. We investigated whether EHD1 regulates L1/NgCAM trafficking in neurons. Both short hairpin-mediated downregulation and overexpression of EHD1 led to dendritic mistargeting of NgCAM. Downregulation of EHD1 showed increased endosomal accumulation of NgCAM, whereas, surprisingly, overexpression of EHD1 led to impairment of L1/NgCAM internalization in neurons but not in fibroblasts. Transferrin internalization, however, was unaffected. At longer overexpression times of EHD1, NgCAM endocytosis returned to normal, suggesting rapid upregulation of compensatory endocytic pathways. EHD1 is capable of hetero-oligomerization, and an endogenous complex of EHD1 and EHD4 was identified previously. We therefore tested whether short-term overexpression of other EHD family members showed a similar endocytosis defect. Expression of EHD4, but not of EHD3, also caused a defect in L1/NgCAM endocytosis. Oligomerization of EHD1 was required to cause NgCAM endocytosis defects, and simultaneous expression of EHD1 and EHD4 rescued NgCAM endocytosis. Therefore, balanced levels of EHD1-EHD4 are important for NgCAM endocytosis in neurons. Our data suggest that EHD1 plays roles in both endosomal recycling and a specialized endocytosis pathway in neurons used by NgCAM. We propose that EHD1 and EHD4 act as hetero-oligomeric complexes in this pathway.
authors.affiliation: Department of Neuroscience, University of Virginia Medical School, Charlottesville, Virginia 22936, USA.
authors.forename: Chan Choo C
authors.initials: CC
authors.lastname: Yap
authors.forename: Zofia M
authors.initials: ZM
authors.lastname: Lasiecka
authors.forename: Steven
authors.initials: S
authors.lastname: Caplan
authors.forename: Bettina
authors.initials: B
authors.lastname: Winckler
mesh_terms: D000818:Animals; D001369:Axons / Q000502:physiology*; D019556:COS Cells; D049108:Cell Enlargement; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000502:physiology; D005347:Fibroblasts / Q000502:physiology; D006624:Hippocampus / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D039842:Neural Cell Adhesion Molecule L1 / Q000378:metabolism*; D009474:Neurons / Q000502:physiology*; D016716:PC12 Cells; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors; D014168:Transferrin / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: C549917:EHD1 protein, rat; C456611:Ehd4 protein, rat; D009419:Nerve Tissue Proteins; D039842:Neural Cell Adhesion Molecule L1; D014168:Transferrin; D033921:Vesicular Transport Proteins
references.citation: Int Rev Cell Mol Biol. 2009;272:303-89
references.pmid: 19121821
references.citation: J Cell Sci. 2009 Feb 1;122(Pt 3):389-400
references.pmid: 19139087
references.citation: Traffic. 2002 Aug;3(8):575-89
references.pmid: 12121420
references.citation: Mol Biol Cell. 2006 Jan;17(1):163-77
references.pmid: 16251358
references.citation: Exp Cell Res. 2007 Jan 15;313(2):219-31
references.pmid: 17097635
references.citation: BMC Syst Biol. 2008 Jan 02;2:1
references.pmid: 18171472
references.citation: EMBO J. 2002 Jun 3;21(11):2557-67
references.pmid: 12032069
references.citation: Dev Biol. 2009 Mar 1;327(1):4-11
references.pmid: 19121301
references.citation: Traffic. 2006 Jan;7(1):52-60
references.pmid: 16445686
references.citation: Nat Neurosci. 2007 Jan;10(1):19-26
references.pmid: 17189949
references.citation: J Biol Chem. 2007 Jun 1;282(22):16612-22
references.pmid: 17412695
references.citation: J Cell Sci. 2008 May 1;121(Pt 9):1514-25
references.pmid: 18411247
references.citation: Eur J Neurosci. 2000 Jun;12(6):1913-23
references.pmid: 10886332
references.citation: Mol Biol Cell. 2009 Jun;20(11):2731-43
references.pmid: 19369419
references.citation: J Cell Biol. 1995 Oct;131(1):69-80
references.pmid: 7559787
references.citation: Annu Rev Neurosci. 1998;21:97-125
references.pmid: 9530493
references.citation: Gene. 1999 Nov 15;240(1):227-32
references.pmid: 10564830
references.citation: Trends Biochem Sci. 2006 Oct;31(10):544-6
references.pmid: 16904324
references.citation: J Cell Biol. 2003 Sep 29;162(7):1317-28
references.pmid: 14517209
references.citation: J Biol Chem. 2001 Jan 12;276(2):1285-90
references.pmid: 11035015
references.citation: J Cell Biol. 2008 Feb 25;180(4):827-42
references.pmid: 18299352
references.citation: J Cell Biol. 2002 May 13;157(4):679-91
references.pmid: 12011113
references.citation: J Neurosci. 2009 Sep 16;29(37):11674-85
references.pmid: 19759314
references.citation: J Neurosci. 2009 Mar 25;29(12):3865-74
references.pmid: 19321783
references.citation: Biol Cell. 2003 Oct;95(7):419-24
references.pmid: 14597259
references.citation: J Biol Chem. 2009 Jul 24;284(30):20121-9
references.pmid: 19487694
references.citation: Proc Natl Acad Sci U S A. 2007 Jul 24;104(30):12270-5
references.pmid: 17640889
references.citation: Mol Cell. 2006 Feb 3;21(3):319-30
references.pmid: 16455487
references.citation: J Cell Sci. 2008 Nov 15;121(Pt 22):3757-69
references.pmid: 18940911
references.citation: Nat Cell Biol. 2001 Jun;3(6):567-72
references.pmid: 11389441
references.citation: Traffic. 2008 Dec;9(12):2043-52
references.pmid: 18801062
references.citation: J Cell Biol. 2010 Jan 25;188(2):271-85
references.pmid: 20083601
references.citation: Mol Biol Cell. 2005 Aug;16(8):3642-58
references.pmid: 15930129
references.citation: Traffic. 2008 Jun;9(6):995-1018
references.pmid: 18331452
references.citation: J Biol Chem. 2003 Apr 4;278(14):12222-30
references.pmid: 12529370
references.citation: J Biol Chem. 2001 Aug 31;276(35):33054-60
references.pmid: 11423532
references.citation: Nature. 2003 Jul 10;424(6945):194-7
references.pmid: 12853957
references.citation: J Neurosci. 2001 Dec 1;21(23):9194-203
references.pmid: 11717353
references.citation: J Cell Biol. 2002 Jun 24;157(7):1105-12
references.pmid: 12070130
references.citation: J Neurosci. 2005 May 25;25(21):5236-47
references.pmid: 15917464
references.citation: Science. 2004 Sep 24;305(5692):1972-5
references.pmid: 15448273
references.citation: J Biol Chem. 2004 Mar 12;279(11):10593-605
references.pmid: 14676205
references.citation: Trends Cell Biol. 2009 Jul;19(7):317-24
references.pmid: 19540123
references.citation: BMC Cell Biol. 2007 Jan 18;8:3
references.pmid: 17233914
references.citation: Nature. 2007 Oct 18;449(7164):923-7
references.pmid: 17914359