The C-terminal basic tail of RhoG assists the guanine nucleotide exchange factor trio in binding to phospholipids.
The multidomain protein Trio regulates among others neuronal outgrowth and axonal guidance in vertebrates and invertebrates. Trio contains two Dbl-homology/pleckstrin homology (DH/PH) tandem domains that activate several RhoGTPases. Here, we present the x-ray structure of the N-terminal DH/PH, hereafter TrioN, refined to 1.7-A resolution. We show that the relative orientations of the DH and PH domains of TrioN and free Dbs are similar. However, this relative orientation is dissimilar to Dbs in the Dbs/Cdc42 structure. In vitro nucleotide exchange experiments catalyzed by TrioN show that RhoG is approximately 3x more efficiently exchanged than Rac and support the conclusion that RhoG is likely the downstream target of TrioN. Residues 54 and 69, which are not conserved between the two GTPases, are responsible for this specificity. Dot-blot assay reveals that the TrioN-PH domain does not detectably bind phosphatidylinositol 3,4-bisphosphate, PtdIns(3,4)P(2), or other phospholipids. This finding is supported by our three-dimensional structure and affinity binding experiments. Interestingly, the presence of RhoG but not Rac or a C-terminal-truncated RhoG mutant allows TrioN to bind PtdIns(3,4)P(2) with a micromolar affinity constant. We conclude the variable C-terminal basic tail of RhoG specifically assists the recruitment of the TrioN-PH domain to specific membrane-bound phospholipids. Our data suggest a role for the phosphoinositide 3-kinase, PI 3-kinase, in modulating the Trio/RhoG signaling pathway.
Skowronek
Karlheinz R
KR
Department of Physiology and Biophysics, Stony Brook University, Health Sciences Center, Stony Brook, New York 11794-8661, USA.
Guo
Fukun
F
Zheng
Yi
Y
Nassar
Nicolas
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray; D017931:DNA Primers; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020741:rho GTP-Binding Proteins
D017931:DNA Primers; D020662:Guanine Nucleotide Exchange Factors; D010743:Phospholipids; D010750:Phosphoproteins; C075397:RHOG protein, human; D017346:Protein Serine-Threonine Kinases; C099628:TRIO protein, human; D020558:GTP Phosphohydrolases; D020741:rho GTP-Binding Proteins