SYX/PLEKHG5, A Rhoa Guanine Exchange Factor Involved in Cell Migration and Angiogenesis
Plekhg5 first emerged as a partial cDNA clone KIAA0720 in the HUGE (Human Unidentified Gene-Encoded) database of large proteins analyzed by the Kazusa Human cDNA Project in Japan (http://www.kazusa.or.jp/huge). It was initially characterized as a RhoA-specific GEF and a potential oncogen named GEF720 (de Toledo et al. 2003). Independently, the rat cDNA of the same protein (named Tech) was characterized also as a RhoA-specific GEF that was highly expressed in cortical and hippocampal neurons (Marx et al. 2005). Two full-length cDNA clones of the mouse ortholog were identified as splice variants (named Syx1 and Syx2) and characterized as participants in cell motility (Liu and Horowitz 2006).
Splice Variants and Domain Structure
The majority of the known splice-variants of Plekhg5 are produced by the initiation of transcription at alternate 5′ start codons (http://www.uniprot.org/uniprot/O94827). These five transcripts code for variants ranging in length from 930 to 1083 residues. Interestingly, the sixth variant is generated by 3′ splicing, very close to the stop codon. The shorter splice variant lacks the two 3′ codons. The absence of the two carboxy-terminus residues in the shorter splice variant removes the PDZ-binding motif and abolishes binding to the PDZ adaptor proteins synectin (Liu and Horowitz 2006) and Mupp1/Patj (Ernkvist et al. 2009). The expression level of the shorter splice variant is negligible in most cell types, except in lines of glioblastoma multiforme, where it reached close to 10% of the expression level of the longer splice variant (Liu and Horowitz 2006). The absence of the PDZ-binding domain resulted in a diffuse rather than targeted distribution of the overexpressed shorter splice variant. RhoA activity in these cells was similarly diffuse and higher than the basal RhoA activity in cells expressing the longer splice variant.
Major Sites of Expression and Subcellular Location
Northern blots of mouse tissue showed that plekhg5 is expressed primarily in the brain and to a lesser extent in the heart (de Toledo et al. 2003). More recent Northern blotting pinpointed the expression of plekhg5 in the brain to the hippocampus and cortex (Marx et al. 2005). Quantitative PCR showed that the highest expression level in the human nervous system was in the peripheral nerves (Maystadt et al. 2007). Gene array studies reported a more widely distributed expression of human plekhg5, with similar expression levels in the brain, heart, skeletal muscle, lung, kidney, liver, pancreas, and the prostate (Yanai et al. 2005).
Plekhg5 was observed in three main cellular compartments. Plekhg5 is present in the cytoplasm, associated with endocytic vesicles and with angiomotin, primarily but not exclusively in the perinuclear region. Plekgh5 collocated with Rab13, suggesting it traffics together with tight junction proteins (Wu et al. 2011). It was also observed at tight junctions where it collocated with ZO1, and in the leading edge of endothelial cells.
Plekhg5 (pleckstrin homology domain containing family with Rho GEF domain 5) is a member of the Dbl Rho GEFs. Plekhg5 conforms to the canonic Dbl structure, that is, a Dbl homology (DH) domain that catalyzes GDP removal from Rho GTPases, followed by a pleckstrin homology (PH) domain. The PH domain is commonly involved in GEF auto-regulation and targeting. The carboxy-terminus of Plekhg5 (SEV*) conforms to the consensus sequence of neuronal nitric oxide synthase (nNOS) class 1 PDZ-binding motifs. Plekhg5 is widely expressed and highly conserved in vertebrates, but is not expressed in Drosophila melanogaster and in the nematode Caenorhabditis elegans. Among vertebrates, it is expressed in the zebrafish (XM_001923778.1, 75% homology), chicken (XP_423692.2, partial sequence, 92% homology), mouse (AY605057.1, 86% homology), rat (AY499658.1, 87% homology), cow (XM_867846.3, 90% homology), horse (XP_001915300.1, 90% homology), dog (XM_536728.2, 91% homology), and chimpanzee (XP_514339, 85% homology). Plekhg5 was characterized as RhoA-specific versus RhoB, RhoC, RhoG, Rac1-3, and Cdc42 (de Toledo et al. 2003). The RhoA-specificity was confirmed in an independent study (Marx et al. 2005).
In vitro studies showed that Plekhg5 is essential in endothelial cell migration and tube formation (Liu and Horowitz 2006). Knockdown of the zebrafish plekhg5 ortholog resulted in growth arrest of the intersegmental vessels and alteration of the morphology of the tip cells of these vessels (Garnaas et al. 2008). Instead of a tapered end that projected long filopodia, the tip cells in the knockdown zebrafish had a round end from which a dense array of thin filopodia protruded in all directions. The loss of filopodia directionality suggests that the growth arrest of the intersegmental vessels was caused, at least in part, by a guidance defect. The plekhg5 −/− mouse was viable and fertile, but had a subtle angiogenic defect of a similar nature to the phenotype of the zebrafish plekhg5 knockdown – sparseness of arterioles and capillaries. Probably because of reduced perfusion, the pumping function of the plekhg5 −/− heart was significantly lower than the wild-type heart. Further in vitro studies showed that plekhg5 silencing in endothelial cells was accompanied by loss of the ZO1 scaffold protein from tight junctions, and an increase in monolayer permeability (unpublished data). Accordingly, electron microscopy showed that tight junctions in the plekhg5 −/− coronary capillaries were missing or not fully formed. In vitro studies showed that plekhg5 silencing in endothelial cells was accompanied by loss of the ZO1 scaffold protein from tight junctions, and an increase in monolayer permeability (unpublished data).
High throughput screening of genes that activate nuclear factor κ B (NFκB) signaling identified plekhg5 among a group of 299 genes (Matsuda et al. 2003). In agreement, in vitro studies with epithelial cells found that overexpression of plekhg5-produced a tenfold increase in NFκB activity (Maystadt et al. 2007). A point mutation (F703S) in the PH domain of human plekhg5 was found to be a marker of a previously uncharacterized autosomal recessive lower motor neuron disease prevalent in a large inbred family from sub-Saharan Africa (Maystadt et al. 2007; Maystadt et al. 2006). Further studies revealed that overexpressed mutant plekhg5 in murine motor neuronal cells (but not in human epithelial cells) formed large cytoplasmic aggregates (Maystadt et al. 2007). Conceivably, and similar to other degenerative diseases of the nervous system, the aggregation of the mutant Plekhg5 could have damaged the motor neurons in the affected individuals (Ross and Poirier 2005). At this point, no nervous system defects were reported in the plekhg5 −/− mouse.
Relatively little is known on Plekhg5 regulation. Truncation of the first 248 amino-terminal residues together with the carboxy-terminal region immediately downstream of the PH domain conferred constitutive activity on Plekgh5 (Marx et al. 2005). Since both the amino and carboxy-terminus regions were truncated, it is not yet possible to determine if auto-regulation requires one or both regions. No Plekgh5 phosphorylation sites have been reported yet.
The phenotype of the zebrafish plekhg5 knockdown has been described above. The loss of filopodia directionality suggests that the growth arrest of intersegmental vessels was caused, at least in part, by a guidance defect. The Plekhg5 −/− mouse was viable and fertile, but had an angiogenic defect consisting of sparseness of arterioles and capillaries. Probably because of reduced perfusion, the pumping function of the plekhg5 −/− heart was significantly lower than that of the wild-type heart. Further in vitro studies showed that plekhg5 silencing in endothelial cells was accompanied by loss of the ZO1 scaffold protein from tight junctions and an increase in monolayer permeability (unpublished observations).
Interactions with Ligands and Other Proteins
Plekhg5 binds RhoA (Marx et al. 2005), in agreement with its specificity for RhoA as an effector. A recent study reported that Plekhg5 binds Rnd3 (alternatively named RhoE), a constitutively active GTPase of intrinsically low GTP hydrolysis rate (Goh and Manser 2010). Rnd3 is thought to act as a RhoA antagonist by activating the GTPase activating protein p190 RhoGAP (Wennerberg et al. 2003). Rnd3 expressed endogenously in mouse embryonic stem cells binds to a 70 amino acid-long domain in the N-terminus region of Plekhg5 that bears similarity to the Raf1 Ras-binding domain (Goh and Manser 2010).
Two PDZ adaptor proteins are known to bind the carboxy-terminus of Plekhg5: synectin (Liu and Horowitz 2006) and Mupp1 (Ernkvist et al. 2009; Estevez et al. 2008) as well as the Mupp1 paralog Patj (Ernkvist et al. 2009). The binding site of Plekhg5 to Mupp1 is either the 10th or 13th PDZ domain, or both (Estevez et al. 2008), whereas Plekhg5 binds to the 10th PDZ domain of Patj (Ernkvist et al. 2009). Both Mupp1 and Patj bind angiomotin, which couples Plekhg5 to members of the Par-6/Par-3 apico-basal polarity complex (Ernkvist et al. 2009; Wells et al. 2006). This angiomotin-associated complex includes the Cdc42-specific GTPase-activating protein Rich1, and the scaffold protein Pals1. In turn, Pals1 and Patj link angiomotin and Plekhg5 to the Crumbs complex. Hence, Plekhg5 is member of a large signaling complex involved in maintaining cell polarity and tight junctions.
Syx/Plekhg5 is a ubiquitously expressed RhoA guanine exchange factor whose functions are known only partially. Despite its high expression level in the brain, its specific neural function is not fully understood. While the highest site of expression appears to be in cortical and hippocampal neurons of the central nervous system, a point mutation in human PLEKHG5 results in an autosomal recessive lower motor neuron disease. In contrast, animal models suggest that one of the primary functions of Syx/Plekhg5 is in angiogenesis.
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