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

Loading...

PPT – Adam Meets Eph: An ADAM Substrate Recognition Module Acts as a Molecular Switch for Ephrin Cleavage in trans (Janes et. al., 2005) PowerPoint presentation | free to view - id: 34917-OTVlN



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

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

Description:

Eve by Albrecht D rer ... Eve by Albrecht D rer. http://en.wikipedia. ... Wolpert, L., R. Beddington, T. Jessel, P. Lawrence, E. Meyerowitz, and J. Smith, 2002. ... – PowerPoint PPT presentation

Number of Views:385
Avg rating:3.0/5.0
Slides: 53
Provided by: joha194
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

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


1
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
2
Outline
  • Background
  • Study Question
  • Summary of Conclusions
  • Results and Methods
  • Toolbox
  • Results
  • Discussion
  • Future Directions
  • Applications

3
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
4
  • MEDIATES REPULSION UPON CONTACT????!!

Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
5
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

6
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!

7
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

8
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
9
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
10
Overview of Mechanism


11
Tool Box for study
  • HEK293 cells
  • Immunolocalization
  • Co-immunoprecipitation
  • Western Blot
  • Fusion Proteins
  • siRNA

12
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
13
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.

14
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.

15
Western Blot
(Alberts et. al., 2002)
http//www.bio.davidson.edu/courses/genomics/metho
d/Westernblot.html
16
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

17
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
18
RESULTS Questions 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?

19
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
20
ADAM10 associates with EphA3 on the cell surface
21
not ephrin-A5
22
Clustering ephrin-A5 increases cleavage
-Eph activation requires contact with
preclustered ligand (ephrin) -result suggests
Eph activation and ephrin cleavage are linked
23
Whats doing the cleavage, ADAM10 or EphA3?
Question 2
Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
24
Ephrin-A5 cleavage and internalization inhibited
by ADAM10 and metalloproteinase inhibitors
Control
  • Exogenous expression of dominant negative
    ADAM10?MP blocked ephrin cleavage and
    internalization

25
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)
26
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.
27
Question 3
  • Where does the interaction on Eph-A3 take place?

Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
28
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
29
Question 4
  • Where does the interaction take place on ADAM10?

Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
30
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).
31
ADAM(DC)
  • Recombinant construct containing the disintergrin
    and cysteine-rich domains

32
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
33
Question 5
Where is the substrate-recognition site of ADAM10?
Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
34
Structure of the ADAM10 substrate-recognition
module
35
Where is the substrate-recognition site of ADAM10?
  • An acidic surface pocket in the ADAM10 Cys-rich
    domain???

36
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
37
Where is the substrate-recognition site of ADAM10?
  • Mutate acidic surface pocket. Is binding
    maintained?

38
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.

39
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
40
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

41
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

42
How does the cleavage happen? Cis or trans?
Question 7
Eve by Albrecht Dürer http//en.wikipedia.org/wik
i/Albrecht_DC3BCrer
43
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!

44
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.
45
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)
46
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
47
  • 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

48
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

49
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)
50
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/
51
(No Transcript)
52
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.
About PowerShow.com