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PDBsum entry 2c9a
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References listed in PDB file
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Key reference
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Title
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Molecular analysis of receptor protein tyrosine phosphatase mu-Mediated cell adhesion.
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Authors
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A.R.Aricescu,
W.C.Hon,
C.Siebold,
W.Lu,
P.A.Van der merwe,
E.Y.Jones.
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Ref.
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EMBO J, 2006,
25,
701-712.
[DOI no: ]
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PubMed id
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Abstract
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Type IIB receptor protein tyrosine phosphatases (RPTPs) are bi-functional cell
surface molecules. Their ectodomains mediate stable, homophilic, cell-adhesive
interactions, whereas the intracellular catalytic regions can modulate the
phosphorylation state of cadherin/catenin complexes. We describe a systematic
investigation of the cell-adhesive properties of the extracellular region of
RPTPmu, a prototypical type IIB RPTP. The crystal structure of a construct
comprising its N-terminal MAM (meprin/A5/mu) and Ig domains was determined at
2.7 A resolution; this assigns the MAM fold to the jelly-roll family and reveals
extensive interactions between the two domains, which form a rigid structural
unit. Structure-based site-directed mutagenesis, serial domain deletions and
cell-adhesion assays allowed us to identify the four N-terminal domains (MAM,
Ig, fibronectin type III (FNIII)-1 and FNIII-2) as a minimal functional unit.
Biophysical characterization revealed at least two independent types of
homophilic interaction which, taken together, suggest that there is the
potential for formation of a complex and possibly ordered array of receptor
molecules at cell contact sites.
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Figure 1.
Figure 1 The MIg crystal structure and analysis of the MAM
domain. (A) Ribbon diagram of MIg. The MAM-Ig linker in the MAM
domain is highlighted in purple. Disulphide bonds (orange) and
the N-glycosylation sites (CPK) are presented as stick models.
The N- and C-termini are labelled. Inset shows the L-shape of
the molecule. Arrowhead indicates the largest crystal contact
site. (B) Comparisons of the MAM domain to two closely related
 -sandwich
structures. Ribbon diagrams are shown for comparisons of
secondary structures and molecular surfaces are shown to display
surface features of binding sites (marked by arrowheads).
Structurally equivalent regions (inter-C  distances
<3.0 Å) are shown in green and structurally distinct
regions are highlighted (blue in front of the -sandwich
and red at the back). In 1GUI:A, the blue loops demarcate the
carbohydrate-binding groove. In 1KGY:A, the red loops surround
the hydrophobic ephrinB2-binding pocket, which constitutes the
primary dimerization site; the blue loop forms the second
ephrinB2-binding site. Regions that are not superposable are
coloured in grey. Disulphide bonds are shown as orange sticks.
All three structures are shown from the same view upon
superimposition on MAM. (C) Structural details of the MAM L1 and
L2 loops. The L1 and L2 loops are depicted in stick
representation whereas the remainder of the MAM domain is shown
as a solid surface. Residues selected for mutagenesis studies
are highlighted with yellow carbon atoms. Phe68 (shown in pink)
corresponds to the F74S cancer-linked mutation in RPTP  .
As in panel A, linker residues are coloured in purple and
cysteines in orange. Asparagine residues providing sites for
N-linked glycosylation are distinguished by standard atom
colouring (carbon: white, nitrogen: blue, oxygen: red). This
figure was produced using Pymol (http://pymol.sourceforge.net/).
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Figure 3.
Figure 3 Cell-adhesion assays. Insect Sf9 cells expressing
transmembrane RPTP  EGFP-fusion
constructs were observed by fluorescence microscopy. All RPTP
fusion
constructs (except for Ex -TM-EGFP)
have JM-D1-EGFP in the intracellular region, and are expressed
at the plasma membrane (indicated by white arrows); whereas EGFP
alone (control) is expressed uniformly in the cytosol. The
bright intracellular signal indicates overexpression of the
constructs in the endoplasmic reticulum and the Golgi apparatus.
Note the localization of MIF2, MIF3, Ex and
its mutants (L1m, L2m) at the cell–cell contact regions (also
see Figure 5B and C). IF14t and F14t fusion constructs (not
shown) did not induce cell aggregation. The Ex -TM-EGFP
construct contains only the first 10 intracellular residues of
RPTP .
Scale bar: 250 m
for the epifluorescence images; 20 m
(for constructs that form cell aggregates) and 10 m
(others) for the confocal images.
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The above figures are
reprinted
from an Open Access publication published by Macmillan Publishers Ltd:
EMBO J
(2006,
25,
701-712)
copyright 2006.
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