Rac1 Selectively Binds a Specific Lamellipodin Isoform via a Noncanonical Helical Interface
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Abstract
Lamellipodin (Lpd) is a multifunctional adapter protein that regulates cell migration and adhesion by recruiting Ena/VASP proteins to the leading edge and modulating actin polymerization. The interaction of Lpd and Rho family or Ras family GTPases is crucial for regulating actin dynamics. Contrary to previous assumptions that the main Lpd isoform interacts with Rac1, here we show that strong and specific binding to Rac1 is instead mediated by the short isoform Lpds. This interaction is dependent on Rac1’s GTPase activity and a short insertion (cs2) within the coiled-coil (CC) region unique to the Lpds isoform. Structural modeling and mutagenesis analyses further reveal that Lpds engages Rac1 through a noncanonical, single-helix binding mode distinct from the classical helical pair configuration. Our results reveal a novel isoform-dependent GTPase:effector binding mode and suggest a critical regulatory pathway that may represent a promising therapeutic target in Rac1-driven cancer progression.
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targeted therapies
It would be very illuminating to study how these different isoforms may differentially impact Rac1 activity in vitro. If their expression levels are similar in certain cell types, does one directly activate rac1 while the other indirectly activates Rac1? If the Lpd isoform doesn't bind directly to Rac1, how does it enhance Rac1 activity?
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predicted interaction of the Lpds:Rac1 complex is structurally robust and biologically reliable
It might be useful to comment on why your structural predictions above (of just the csRAPH domain) gave you a different hypothesis than predicting the structure of the full Lpds in complex with Rac1. What do you get for structural predictions when you just look at Lpds alone?
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or neither (isoforms #2, #5, #10
It would also be interesting to examine expression patterns of all of these isoforms.
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exhibit markedly stronger interaction with csRAPH of Lpds than wild-type Rac1
This is a really nice pulldown! It would be cool to see binding curves for these interactions, too.
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A Lpds construct (1-PH) containing the intact RA-PH module
What was your rationale for this construct in particular? Why not just express the RA-PH, or why not the full-length construct?
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The result suggests that the Lpds interact with Rac1 as a monomer, and the dimerization through the CC region does not contribute significantly to in the interaction of Lpds and Rac1.
I think this only suggests that Lpds doesn't heterodimerize, but Lpds could still associate with itself as a dimer to facilitate interaction with Rac1. It would be interesting if you could purify it and determine its tendency to dimerize that way.
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Rac1 preferentially binds the Lpds isoform
I would be careful with conclusions here - just because something coIPs with a protein doesn't mean it binds directly. There could be other proteins in the mix that facilitate this interaction/complex.
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