PMID- 10491193
OWN - NLM
STAT- MEDLINE
DCOM- 19991122
LR  - 20190620
IS  - 0014-2956 (Print)
IS  - 0014-2956 (Linking)
VI  - 265
IP  - 1
DP  - 1999 Oct 1
TI  - Direct interaction of EEA1 with Rab5b.
PG  - 361-6
AB  - The early endosomal autoantigen EEA1 is essential for early endosomal membrane
      fusion. It binds to endosomes via a C-terminal domain (EEA1-CT). To identify
      proteins interacting with EEA1-CT, we screened a human brain library in the yeast
      two-hybrid system. Fourteen clones reacted strongly with EEA1-CT. Sequencing of
      these clones revealed that they all contained the ORF of the small GTPase, Rab5b.
      Further two-hybrid analysis suggested that Rab5b also interacts with the
      N-terminus of EEA1 (EEA1-NT). The interaction of both EEA1-CT and EEA1-NT with
      Rab5b was confirmed biochemically, and was found to be GTP dependent. Confocal
      immunofluorescence microscopy indicated that EEA1 colocalizes with Rab5b on early
      endosomes. Although EEA1-CT and EEA1-NT interacted strongly with wild-type Rab5b 
      in the two-hybrid system, we detected no interaction with wild-type Rab5a, even
      though GTPase-deficient mutants of both Rab5a and Rab5b interacted equally well
      with EEA1. This difference could not be explained by differences in intrinsic
      GTPase activities, as these were found to be very similar. Instead, we speculate 
      that yeast may contain a GTPase-activating protein (GAP) activity that stimulates
      Rab5a but not Rab5b. In contrast, pig brain cytosol was found to contain a GAP
      activity that stimulates the GTPase activity of Rab5b in preference to that of
      Rab5a. These data provide evidence that EEA1 interacts with both Rab5a and Rab5b,
      and that the GTPase activities of the two proteins are differentially regulated
      in vivo.
FAU - Callaghan, J
AU  - Callaghan J
AD  - Department of Biochemistry, The Norwegian Radium Hospital, Oslo.
FAU - Nixon, S
AU  - Nixon S
FAU - Bucci, C
AU  - Bucci C
FAU - Toh, B H
AU  - Toh BH
FAU - Stenmark, H
AU  - Stenmark H
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Eur J Biochem
JT  - European journal of biochemistry
JID - 0107600
RN  - 0 (Autoantigens)
RN  - 0 (Membrane Proteins)
RN  - 0 (Nerve Tissue Proteins)
RN  - 0 (Peptide Fragments)
RN  - 0 (Protein Isoforms)
RN  - 0 (Vesicular Transport Proteins)
RN  - 0 (early endosome antigen 1)
RN  - 86-01-1 (Guanosine Triphosphate)
RN  - EC 3.6.5.2 (rab5 GTP-Binding Proteins)
SB  - IM
MH  - Animals
MH  - Autoantigens/genetics/*metabolism
MH  - Brain
MH  - Cytosol/metabolism
MH  - Endosomes
MH  - Fluorescent Antibody Technique
MH  - Gene Library
MH  - Guanosine Triphosphate/metabolism
MH  - Humans
MH  - Hydrolysis
MH  - Membrane Proteins/genetics/*metabolism
MH  - Nerve Tissue Proteins/genetics/*metabolism
MH  - Peptide Fragments/genetics/metabolism
MH  - Protein Binding
MH  - Protein Isoforms/genetics/metabolism
MH  - Swine
MH  - Two-Hybrid System Techniques
MH  - Vesicular Transport Proteins
MH  - rab5 GTP-Binding Proteins/genetics/*metabolism
EDAT- 1999/09/22 00:00
MHDA- 1999/09/22 00:01
CRDT- 1999/09/22 00:00
PHST- 1999/09/22 00:00 [pubmed]
PHST- 1999/09/22 00:01 [medline]
PHST- 1999/09/22 00:00 [entrez]
AID - ejb743 [pii]
AID - 10.1046/j.1432-1327.1999.00743.x [doi]
PST - ppublish
SO  - Eur J Biochem. 1999 Oct 1;265(1):361-6. doi: 10.1046/j.1432-1327.1999.00743.x.