Investigating Ephrin B3 Ligand-Induced Clustering of EphB3 and EphA4 Receptors

1. Eph B3
Investigating Ephrin B3 Ligand-Induced Clustering of
EphB3 and EphA4 Receptors
Drexel University, Department of BiologyArjun Ganesh, Dr. Jerome Ricard
Acknowledgements
References
Introduction
Methods
Data/Results Conclusion
Future Work
• Dependence receptors are transmembrane proteins
which, when not bound to their ligand, prompt
apoptosis in the cell (Goldschneider and Mehlen,
2010).
• EphA4 and EphB3 are receptor tyrosine kinases
(RTK) that have been identified as dependence
receptors.
• Both receptors have a common ligand: EphrinB3
• We hypothesize that EphrinB3 could induce
clustering of EphA4 and EphB3 and that it could be
crucial for the regulation of the apoptotic function.
• The HEK cells utilized in this study have
endogenous EphA4 and EphB3 receptors, so the
next step would be to repeat this experiment with
murine embryonic fibroblasts, which do not have
endogenous receptors.
• EphA4 and EphB3 will be overexpressed in MEFs to
see whether apoptosis is dependent upon the
clustering of these two receptors.
• While ephrin is a common ligand to these receptors
and can prevent apoptosis upon binding, EphB3 and
EphA4 play a role in mediation of cell repulsion,
leading to cytoskeletal re-organization and functional
morbidities upon recovery.
• A ligand must be found that can prevent the
apoptotic propensities of these dependence
receptors while not contributing to detrimental
neuronal circuit re-organization
Fig. 1. EphrinB3 induces the clustering of EphA4 and EphB3
The first two lanes in each treatment groups are the total protein extracts, while the third lane is
the co-immunoprecipitation. The first lane of each treatment group was treated with the anti-V5
antibody, probing for EphB3. The second lane of each group was treated with anti-GFP, probing
for EphA4. The third lane was treated with anti-GFP, and the appearance of bands around 150
kDA reveal the presence of EphA4 in the co-immunoprecipitation, thus signifying an interaction
between the two receptors.
• The treatment groups lacking EphrinB3 did not
show any signs of clustering
• Addition of EphrinB3 induced the association of
EphA4 and EphB3
• The presence of EphA4 after immunoprecipitation
indicates that EphB3 and EphA4 belong to the
same complex
• It does not support any claims of EphA4 and
EphB3 interacting directly with one another
• The absence of interaction in the cells treated with
EB3 in the absence of serum was not expected.
• HEK293 cells were maintained in a DMEM medium
containing ten percent Fetal Bovine Serum (FBS).
• 250,000 cells were transfected in 60mm plates with five
micro-grams of pcDNA3.1-EphB3-V5 and pBiFC-EphA4-
CrNplasmids, using Lipofectamine (Life Technologies) to
manufacturer’s instructions.
• EphrinB3 was pre-clustered in order to elicit proper
signaling from the receptors
• The four different media were prepared and pipetted into
appropriate plates:
• DMEM
• DMEM (10% FBS)
• DMEM + pre-clustered EphrinB3
• DMEM (10%FBS) + pre-clustered EphrinB3
• Proteins were extracted with RIPA buffer after a 30
minute incubation
• The proteins were immunoprecipitated with one micro-
gram of anti-V5 antibody per 100 micro-gram of proteins.
• The immunoprecipitated proteins were washed
systematically with buffers containing decreasing
concentrations of salt.
• The proteins were resolved on an SDS PAGE gel and
probed by western blotting using anti-GFP and anti-V5
antibodies.
• The detection was performed using a chemiluminescent
substrate and then exposing the membrane to an
autoradiography film.
We would like to thank the Office of Undergraduate
Research and the Department of Biology at Drexel
University.
EphrinB3
EphrinB3
Eph A4 Eph A4 Eph B3Eph B3
Eph B3
Apoptosis Survival
Fig. 2. The binding of EphrinB3 to EphA4 and EphB3 induces clustering of the two
receptors and inhibits apoptotic tendencies.
Goldschneider, D., and P. Mehlen. "Dependence
Receptors: A New Paradigm in Cell Signaling and
Cancer Therapy." Oncogene 29.13 (2010): 1865-
882. Web.