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Rho Residue 43 is a Structural Determinant of GEF Activity and Selectivity
Author(s) -
Goetzinger Chris J.,
Sloan Christina M.,
Quinn Clancy V.,
Ellerbroek Shawn M.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb295
Subject(s) - rhoa , guanine nucleotide exchange factor , rhob , rhoc , isoleucine , valine , chemistry , biochemistry , stereochemistry , amino acid , gtpase , leucine , signal transduction
RhoA, RhoB, and RhoC GTPases share over 80% sequence identity but diverge in cellular function. In previous work, we hypothesized that variation at amino acid residue 43 between RhoA/B (valine) and RhoC (isoleucine) was a structural impediment to XPLN activation of RhoC. In this work, we have systematically studied a putative role of Rho residue 43 as a structural determinant of guanine nucleotide exchange factor (GEF) activity. Specifically, we performed in vitro analysis of GEF activity (VAV2, XPLN, Dbs, GEFT, and Dbl) against both normal RhoA/B/C proteins and those mutated at residue 43. Substitution of val43 with an isoleucine in RhoA reduced 40‐50% of GEF catalyzed exchange, except for GEFT (reduced only 10‐20%). Substitution of val43 with an isoleucine in RhoB had variable effect on exchange, with Vav2 and GEFT modestly sensitive (10% decrease) and XPLN‐stimulated exchange reduced 80%. On the other hand, substitution of ile43 with a valine in RhoC promoted all GEF catalyzed exchange (1.3‐4.0 fold increases). Substitution of a polar threonine at residue 43 significantly reduced all RhoA GEF exchange 60‐80%, nearly eliminated RhoB GEF exchange (80‐100% decrease), but had little to no affect on RhoC exchange, with the exception of GEFT (reduced 75%). In support, RhoA V43T displayed a reduced phenotype when expressed in NIH3T3 fibroblasts, whereas constitutively active RhoA V43T, 63L was phenotypical equivalent to RhoA 63L. Together, these findings support the hypothesis that a divergence at residue 43 between RhoA/B and RhoC is a structural determinant of GEF activity and selectivity. This work was supported with funds from the RJ McElroy Trust.