A conserved hydrophobic surface of the LARG pleckstrin homology domain is critical for RhoA activation in cells.
Leukemia associated Rho guanine nucleotide exchange factor (LARG) activates RhoA in response to signals received by specific classes of cell surface receptors. The catalytic core of LARG is a Dbl homology (DH) domain whose activity is modulated by an adjacent pleckstrin homology (PH) domain. In this study, we used a transcriptional assay and confocal microscopy to examine the roles of several novel structural features of the LARG DH/PH domains, including a conserved and exposed hydrophobic patch on the PH domain that mediates protein-protein interactions in crystal structures of LARG and its close homolog PDZ-RhoGEF. Mutation of the hydrophobic patch has no effect on nucleotide exchange activity in vitro, but abolished the ability of LARG to activate RhoA and to induce stress fiber formation in cultured cells. The activity of these mutants could be rescued by fusion with exogenous membrane-targeting domains. However, because membrane recruitment by activated G alpha(13) subunits was not sufficient to rescue activity of a hydrophobic patch mutant, the LARG PH domain cannot solely contribute to membrane targeting. Instead, it seems likely that the domain is involved in regulatory interactions with other proteins near the membrane surface. We also show that the hydrophobic patch of the PH domain is likely important for the activity of all Lbc subfamily RhoGEFs.
Aittaleb
Mohamed
M
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109-2216, USA.
Gao
Guang
G
Evelyn
Chris R
CR
Neubig
Richard R
RR
Tesmer
John J G
JJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002522:Chlorocebus aethiops; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D064067:Rho Guanine Nucleotide Exchange Factors; D016415:Sequence Alignment; D015398:Signal Transduction; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
C577222:ARHGEF12 protein, human; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020742:rhoA GTP-Binding Protein