Adam Meets Eph: An ADAM Substrate Recognition Module Acts as a Molecular Switch for Ephrin Cleavage in trans (Janes et. al., 2005)

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Adam Meets Eph An ADAM Substrate Recognition
Module Acts as a Molecular Switch for Ephrin
Cleavage in trans(Janes et. al., 2005)
Temptation and Fall by Michelangelo http//www.ang
elo.edu/faculty/rprestia/1301/list_of_illustration
s6.htm
Johanna Kaiser and Anne McDonald
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Outline

• Background
• Study Question
• Summary of Conclusions
• Results and Methods
• Toolbox
• Results
• Discussion
• Future Directions
• Applications

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Background Ephrins and Eph Receptors

• Roles in regulating cell sorting (Wolpert et al.
  2002)
• Two subclasses (A and B)
• Ephrins membrane bound proteins that interact
  with Eph cell surface receptors on adjacent cells
• Eph a receptor tyrosine kinase
• Can send bidirectional signal, both in cell with
  Eph receptor and cell with ephrin ligand
• Eph receptors and ephrins are expressed
  separately in alternating cells
• Ephrin molecules mediate repulsion upon contact
  with Eph receptors

Figure 4.26 Wolpert et al. 2002
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• MEDIATES REPULSION UPON CONTACT????!!

Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
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Thats what a lot of scientists said too!

• ADHESION AND REPULSION
• Eph and ephrin mediate contact dependent cell
  interactions regulating repulsion and adhesion,
  mechanisms involved in cell guidance and
  assembly.
• Eph-ephrin tethers must break to allow repulsion.
• Two mechanisms for cleavage proposed
• -rapid endocytosis of the Eph/ephrin complex
  during retraction of interacting cells
• -regulated cleavage of ephrin ligands by
  transmembrane proteins -gt ADAM10

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Cleavage which direction?

• ephrin-A2 and A3
• Cleavage occurs in cis
• for all ADAM mediated protein shedding studied so
  far, cleavage has occurred in cis
• But, ADAM10 is different!

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ADAM10

• Multidomain, transmembrane protein
• Disintegrin (blocks integrin proteins)
• Metalloprotease - binds a metal ion in the active
  site which often serves to coordinate side chains
  and in necessary for action of protein
• ADAM10 cleaves ephrin-A2 and ephrin-A5
• Atypical cleavage sequence signature, atypical
  how substrate specificity is conveyed
• Was unclear how ADAM10 interacts with ephrins in
  a regulated way
• Therefore investigate interaction of ADAM10 with
  EphA3 and ephrin-A5 complex between cell surfaces

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THE STUDY

• How does ADAM10 interact with the EphA3/ephrin-A5
  complex between cell surfaces in a regulated way?

Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
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Summary of Finding

• Trans cleavage by ADAM10 and internalization of
  Ephrin-A5 occurs when in complex with EphA3

http//www.joemckeever.com/mt/cartoons/adam_eve.gi
f
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Overview of Mechanism


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Tool Box for study

• HEK293 cells
• Immunolocalization
• Co-immunoprecipitation
• Western Blot
• Fusion Proteins
• siRNA

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HEK293 cells

• HEK human embryonic kidney
• Dont express Eph receptors or ephrins, so
  creates a blank slate to use for experiments

http//www.olympusfluoview.com/gallery/cells/hek/h
eklarge.html
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Immunolocalization

• Create antibodies against protein of interested
  by injecting protein into another species (rat,
  goat, mouse)
• Create secondary antibodies with a detectable tag
  (Alexa beads)
• Shows location of protein of interest by forming
  complex of the protein and the primary and
  secondary (visible) antibodies.

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Co-immunoprecipitation

• one antibody used to immunoprecipitate a target
  protein and co-precipitate any bound, interacting
  proteins
• complex is detected by Western blot using a
  second antibody targeted against one of the
  bound, interacting proteins.

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Western Blot
(Alberts et. al., 2002)
http//www.bio.davidson.edu/courses/genomics/metho
d/Westernblot.html
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Fusion proteins

• Stop codon removed from sequence for one protein
  and start codon removed from sequence of another
• Two modified sequences attached together to form
  a fusion protein of the two original proteins
  attached together
• Sequence for bovine ADAM10 put into a virus with
  part of the sequence for human IgG for expression
  in HEK cells

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siRNA

• small piece of RNA complementary a section of an
  mRNA molecule
• siRNA binds to mRNA for protein of interest,
  causing section of mRNA to become double stranded
• Cell breaks down double stranded mRNA

Alberts et al. 2002
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RESULTSQuestions Study Asked

• What interactions are necessary between ADAM10,
  EphA3 and ephrin-A5 for cleavage to occur?
• Whats doing the cleavage, ADAM10 or EphA3?
• Where does the interaction on Eph-A3 and ADAM10
  take place?
• Does the recognition pocket of ADAM10 also play a
  role in ephrin-A5 cleavage and internalization?
• How does the cleavage happen? Cis or trans?

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What interactions are necessary between ADAM10,
EphA3 and ephrin-A5 for cleavage to occur?
Question 1
Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
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ADAM10 associates with EphA3 on the cell surface
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not ephrin-A5
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Clustering ephrin-A5 increases cleavage
-Eph activation requires contact with
preclustered ligand (ephrin) -result suggests
Eph activation and ephrin cleavage are linked
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Whats doing the cleavage, ADAM10 or EphA3?
Question 2
Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
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Ephrin-A5 cleavage and internalization inhibited
by ADAM10 and metalloproteinase inhibitors
Control

• Exogenous expression of dominant negative
  ADAM10?MP blocked ephrin cleavage and
  internalization

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But what if theres another ADAM protein
responsible for ephrin cleavage?
-loss of expression resulted in decreased ephrin
shedding and undetectable internalized ephrins
with Alexa546 beads
-ADAM10 expression silenced with siRNA (RT-PCR,
depletion of ADAM10 mRNA)
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But what if theres another ADAM protein
responsible for ephrin cleavage?
-cells transfected with control siRNA showed
ADAM10 expression on cell surface and
internalized bead-labeled ephrin
CONCLUSION specific ADMA10 protein
down-regulation inhibits cleavage and
internalization of ephrin-A5 into Eph-A3
expressing cells.
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Question 3

• Where does the interaction on Eph-A3 take place?

Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
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Where does the interaction on Eph-A3 take place?

• EphA3 (ligand) interacts with ADAM10 via its
  ligand binding domain (LBD).

Legend ?LBD lacking ligand binding domain
(N-terminal) ?PDZb lacking the PDZ binding
domain (C-terminal) - untreated ephrin-A5
Fc crosslinked ephrin-A5 Fc a
anti/antibody
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Question 4

• Where does the interaction take place on ADAM10?

Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
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ADAM a metalloprotease
Adam by Albrecht Dürer http//en.wikipedia.org/wi
ki/Albrecht_DC3BCrer
Domains known to be important in
protein-protein interactions thereby controlling
cell adhesion and ADAM protease specificity
(Reddy et al., 2000 Smith et al., 2002 White,
2003).
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ADAM(DC)

• Recombinant construct containing the disintergrin
  and cysteine-rich domains

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Where does the interaction take place on ADAM10?

• The ADAM10 Cys-rich domain recognizes the
  EphA3/Ephrin-A5 Complex but Not the individual
  proteins.
• COMPLEX REQUIRED!

ADAM10(DC) does not associate with Ephrin A5
alone
ADAM10(DC) binds the EphA3/ephrin-A5 complex but
not the individual proteins
Binding to the EphA3/ephrin-A5 complex is
mediated by the cysteine-rich domain
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Question 5
Where is the substrate-recognition site of ADAM10?
Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
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Structure of the ADAM10 substrate-recognition
module
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Where is the substrate-recognition site of ADAM10?

• An acidic surface pocket in the ADAM10 Cys-rich
  domain???

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Where is the substrate-recognition site of ADAM10?

• An acidic surface pocket in the ADAM10 Cys-rich
  domain???

• Investigate by changing the local surface
  electrostatic potential
• Replace Glu acidic residues with neutral
  alanines
• Can ADAM still recognize its substrate?

Glu578
Glu573
Glu579
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Where is the substrate-recognition site of ADAM10?

• Mutate acidic surface pocket. Is binding
  maintained?

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Where is the substrate-recognition site of ADAM10?

• A second EphA3-interaction site outside the
  ADAM10 Cys-rich domain mediates the weaker,
  constitutive association with unligated EphA3

• Red circles show a significantly reduced
  interaction of ADAM10?MPEEE-A with the
  EphA3/ephrin-A5 complex.
• Blue circles indicate that the constitutive
  association with EphA3 remains unchanged.

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Question 6

• Does the recognition
• pocket of ADAM10 also play a role in ephrin-A5
  cleavage and internalization?

Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
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Does the recognition pocket of ADAM10 also play a
role in ephrin-A5 cleavage?

• Ephrin-A5 cleavage was substantially reduced or
  not present in mutant ADAM10

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Does the recognition pocket of ADAM10 also play a
role in ephrin-A5 cleavage and internalization?

• Green is ephrin
• Red is EphA3
• Blue is ADAM

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How does the cleavage happen? Cis or trans?
Question 7
Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
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How does the cleavage happen cis or trans?

• Experiments so far have suggested that cleavage
  happens in trans
• Expression of ADAM10 and EphA3 in the same cell
  promotes cleavage of ephrin-A5, but expression of
  ADAM10 with ephrin-A5 on the same cell does not.
• Unlike every other ADAM mediated protein
  ectodomain shedding!

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Cleavage happens in trans!
Red EphA3 Green ephrin
-ephrin-A5 gets internalized into EphA3
expressing cells -ADAM10 inhibition in EphA3
expressing cells has effect, inhibition in ephrin
expressing cells does not.
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Confirmation cleavage in trans
(and making sure those big fluorescent tags
werent messing things up)
-HA-ADAM10?MP transfected EphA3/HEK293 cells
(green) dont internalize ephrin-A5,
untransfected cells did (blue cells, white,
filled arrow heads)
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Confirmation cleavage in trans
(and making sure those big fluorescent tags
werent messing things up)
-HA-ADAM10?MP transfected ephrin-A5/HEK293 cells
(arrows) -All EphA3 cells internalize ephrin-A5
cleaved from surface of interacting cell
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• Conclusion dominant-negative ADAM10?MP inhibits
  ephrin cleavage when expressed in EphA3 cells, in
  trans of the ephrin substrate. The ADAM10
  responsible for the ephrin-A5 cleavage is located
  on Eph-A3 expressing cells

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Discussion/Conclusions

• A substrate-recognition model has been presented
  ADAM10 associates with the ephrin-A5/EphA3
  complex to reposition the proteinase domain and
  allow effective cleavage in trans
• Mechanism involves the internalization of ephrin
  into the Eph-expressing cell
• ADAM must be presented on the juxtaposed cell (in
  trans) for cleavage of ephrin to occur

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Future Directions of Research

• How general is this mechanism?
• Do other ephrin/receptor pairs share the same
  fate?
• Do other ADAM proteases act on other ephrin
  pairs?
• Do other ADAMs use a similar substrate
  recognition and activation mechanism like the one
  proposed?

(Mancia and Shapiro, 2005)
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Applications

• Cancer ? general progression and metastasis
• Cancer ? mutations in kinase domain of ephA3 have
  been detected in colorectal cancer.
  
  
• Spinal/Brain injuries ? Eph signaling roles in
  axon regeneration
• Stem cells ? migration and cell fate
  determination
• Mental illness
• Drug discovery? efforts to interfere (/-) with
  Eph receptor-ephrin signaling pathways
• MUCH, MUCH MORE!!!

(Pasquale, 2005).
http//www.family.org.au/bioethics/cloning/
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References

• Janes, Peter W., Nayanendu Saha, William
  A. Barton, Momchil V. Kolev, Sabine
  H. Wimmer-Kleikamp, Eva Nievergall, Carl
  P. Blobel, Juha-Pekka Himanen, Martin Lackmann,
  and Dimitar B. Nikolov, 2005. Adam Meets Eph An
  ADAM Substrate Recognition Module Acts as a
  Molecular Switch for Ephrin Cleavage In trans.
  Cell. 123(2) 291-304.
• Mancia, F., and L. Shapiro. 2005. ADAM and Eph
  How ephrin-signaling becomes detached. Cell.
  123(2) 185-187.
• Mellitzer, G., Q. Xu, and D. Wilkinson, 2000.
  Control of cell behaviour by signalling through
  Eph receptors and ephrins. Current Opinion in
  Neurobiology. 10 400-408.
• Pasquale, E. B. Eph receptor signalling casts a
  wide net on cell behaviour. 2005. Nature Reviews
  Molecular Cell Biology. 6 462-475.
• Wolpert, L., R. Beddington, T. Jessel, P.
  Lawrence, E. Meyerowitz, and J. Smith, 2002.
  Principles of Development, Second Edition.
  Oxford, New York Oxford University Press.
• Alberts, B., A. Johnson, J. Lewis, M. raff, K.
  Roberts, and P. Walter, 2002. Molecular Biology
  of the Cell, Fourth Edition. New York Garland
  Science.