Abstract
Rat (r) PDZRhoGEF, initially identified as a glutamate transporter EAAT4-associated protein, is a member of a novel RhoGEF subfamily. The N terminus of the protein contains a PDZ and a proline-rich domain, two motifs known to be involved in protein-protein interactions. By using the yeast two-hybrid approach, we screened for proteins that interact with the N terminus of rPDZRhoGEF. The light chain 2 of microtubule-associated protein 1 (LC2) was the only protein identified from the screen that does not contain a type I PDZ-binding motif at its extreme C terminus (-(S/T)Xphi-COOH, where phi is a hydrophobic amino acid). However, the C terminus does conform to a type II-binding motif (-phiXphi). We report here that rPDZRhoGEF interacts with LC2 via the PDZ domain, and the interaction is abolished by mutations in the carboxylate-binding loop. The specificity of the interaction was confirmed using GST fusion protein pull-down assays and coimmunoprecipitations. Expression of rPDZRhoGEF mutants that are unable to interact with proteins via the carboxylate-binding loop induced changes in cell morphology and actin organization. These mutants alter the activation of RhoGTPases, and coexpression of dominant-negative RhoGTPases prevent the morphological changes. Furthermore, in cells expressing wild type rPDZRhoGEF, drug-induced microtubule depolymerization produces changes in cell morphology that are similar to those induced by rPDZRhoGEF mutants. These results indicate that modulation of the guanine nucleotide exchange activity of rPDZRhoGEF through interaction with microtubule-associated protein light chains may coordinate microtubule integrity and the reorganization of actin cytoskeleton. This coordinated action of the actin and microtubular cytoskeletons is essential for the development and maintenance of neuronal polarity.
Highlights
Rat (r) PDZRhoGEF, initially identified as a glutamate transporter EAAT4-associated protein, is a member of a novel RhoGEF subfamily
We found that full-length light chain clone E12 (LC1) cDNA fused to the GAL4(AD) grew on dropout media, and quantification of -galactosidase activity using the o-nitrophenyl -D-galactoside assay revealed that both LC1 and LC2 interact strongly with the N terminus of rPDZRhoGEF (Fig. 1B)
The specificity of the yeast interaction was confirmed as expression of T7 fused to GAL4(AD) yielded no growth on dropout media or -galactosidase activity when coexpressed with GAL4(DB)-rPDZRhoGEF-(1–186)
Summary
Rat (r) PDZRhoGEF, initially identified as a glutamate transporter EAAT4-associated protein, is a member of a novel RhoGEF subfamily. In cells expressing wild type rPDZRhoGEF, drug-induced microtubule depolymerization produces changes in cell morphology that are similar to those induced by rPDZRhoGEF mutants These results indicate that modulation of the guanine nucleotide exchange activity of rPDZRhoGEF through interaction with microtubule-associated protein light chains may coordinate microtubule integrity and the reorganization of actin cytoskeleton. Rat PDZRhoGEF (rPDZRhoGEF), initially termed GTRAP48, was identified from a yeast two-hybrid screen of an adult rat cDNA library as a protein that interacts with the C terminus of EAAT4, a neuronal glutamate transporter [18].
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