Recruitment of Eph receptors into signaling clusters does not require ephrin contact.
Eph receptors and their cell membrane-bound ephrin ligands regulate cell positioning and thereby establish or stabilize patterns of cellular organization. Although it is recognized that ephrin clustering is essential for Eph function, mechanisms that relay information of ephrin density into cell biological responses are poorly understood. We demonstrate by confocal time-lapse and fluorescence resonance energy transfer microscopy that within minutes of binding ephrin-A5-coated beads, EphA3 receptors assemble into large clusters. While remaining positioned around the site of ephrin contact, Eph clusters exceed the size of the interacting ephrin surface severalfold. EphA3 mutants with compromised ephrin-binding capacity, which alone are incapable of cluster formation or phosphorylation, are recruited effectively and become phosphorylated when coexpressed with a functional receptor. Our findings reveal consecutive initiation of ephrin-facilitated Eph clustering and cluster propagation, the latter of which is independent of ephrin contacts and cytosolic Eph signaling functions but involves direct Eph-Eph interactions.
Wimmer-Kleikamp
Sabine H
SH
Dept. of Biochemistry and Molecular Biology, P.O. Box 13D, Monash University, Clayton, Victoria 3800, Australia.
Janes
Peter W
PW
Squire
Anthony
A
Bastiaens
Philippe I H
PI
Lackmann
Martin
M
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D036386:Ephrin-A5 / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D046911:Macromolecular Substances; D018715:Microscopy, Video; D011806:Quinolinium Compounds / Q000737:chemistry / Q000378:metabolism; D036121:Receptor, EphA3 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors
C481052:Alexa fluor 546; D036386:Ephrin-A5; D046911:Macromolecular Substances; D011806:Quinolinium Compounds; D011993:Recombinant Fusion Proteins; D036121:Receptor, EphA3