PT  - JOURNAL ARTICLE
AU  - Katherine M. Stefanski
AU  - Charles M. Russell
AU  - Justin M. Westerfield
AU  - Rajan Lamichhane
AU  - Francisco N. Barrera
TI  - PIP<sub>2</sub> promotes conformation-specific dimerization of the EphA2 membrane region
AID  - 10.1101/2020.10.14.338293
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.10.14.338293
4099  - http://biorxiv.org/content/early/2020/10/14/2020.10.14.338293.short
4100  - http://biorxiv.org/content/early/2020/10/14/2020.10.14.338293.full
AB  - The impact of the EphA2 receptor on cancer malignancy hinges on the two different ways it can be activated. EphA2 induces anti-oncogenic signaling after ligand binding, but ligand-independent activation of EphA2 is pro-oncogenic. It is believed that the transmembrane (TM) domain of EphA2 adopts two alternate conformations in the ligand-dependent and the ligand-independent states. However, it is poorly understood how the difference in TM helical crossing angles found in the two conformations impacts the activity and regulation of EphA2. We devised a method that uses hydrophobic matching to stabilize two conformations of a peptide comprising the EphA2 TM domain and a portion of the intracellular juxtamembrane (JM) segment. The two conformations exhibit different TM crossing angles, resembling the ligand-dependent and ligand-independent states. We developed a single-molecule technique using SMALPs to measure dimerization in membranes. We observed that the signaling lipid PIP2 promotes TM dimerization, but only in the small crossing angle state, which we propose corresponds to the ligand-independent conformation. In this state the two TM are almost parallel, and the positively charged JM segments are expected to be close to each other, causing electrostatic repulsion. The mechanism PIP2 uses to promote dimerization might involve alleviating this repulsion due to its high density of negative charges. Our data reveal a conformational coupling between the TM and JM regions, and suggest that PIP2 might directly exert a regulatory effect on EphA2 activation in cells that is specific to the ligand-independent conformation of the receptor.Competing Interest StatementThe authors have declared no competing interest.TMtransmembraneJMjuxtamembraneRTKreceptor tyrosine kinaseALSamyotrophic lateral sclerosisMDmolecular dynamicsHRheptad repeatGZglycine zipperSMALPsstyrene maleic acid lipid particlesTEMtransmission electron microscopyTIRFtotal internal reflection fluorescenceOCDoriented circular dichroismFRETFörster Resonance Energy TransferANOVAanalysis of variancePSphosphatidylserineDTTdithiothreitolMALDI-TOFmatrix assisted laser desorption ionization time of flightHPLChigh performance liquid chromatography