The Faciogenital Dysplasia Gene Product FGD1 Functions as a Cdc42Hs-specific Guanine-Nucleotide Exchange Factor

Abstract

The Rho family of small GTP-binding proteins plays important roles in the regulation of actin cytoskeleton organization and cell growth. Activation of these GTPases involves the replacement of bound GDP with GTP, a process catalyzed by the Dbl-like guanine-nucleotide exchange factors, all of which seem to share a putative catalytic motif termed the Dbl homology (DH) domain, followed by a pleckstrin homology (PH) domain. Here we have examined the role of a Dbl-like molecule, the faciogenital dysplasia gene product (FGD1), which when mutated in its Dbl homology domain, cosegregates with the developmental disease Aarskog-Scott syndrome. We report that a polypeptide of FGD1 encompassing the DH and PH domains can bind specifically to the Rho family GTPase Cdc42Hs and stimulates the GDP-GTP exchange of the isoprenylated form of Cdc42Hs. Microinjection of this FGD1 polypeptide into Swiss 3T3 fibroblast cells induces the formation of peripheral actin microspikes, similar to that previously observed when cells were injected with a constitutively active form of Cdc42Hs. This effect of FGD1 on actin organization is readily inhibited by coinjection of a dominant-negative mutant of Cdc42Hs. Examination of NIH 3T3 cells expressing the FGD1 fragment revealed that similar to cells expressing Dbl, two independent elements downstream of Cdc42Hs, the Jun NH 2 -terminal kinase and the p70 S6 kinase, became activated. Hence, our results indicate that FGD1, through its DH and PH domains, acts as a Cdc42Hs-specific guanine-nucleotide exchange factor and suggest that the Cdc42Hs GTPase may have a role in mammalian development.The Rho family of small GTP-binding proteins have been implicated in multiple signaling pathways leading to cytoskeleton reorganization (1) and cell growth (2). RhoA has been shown to be involved in actin stress fiber and focal adhesion formation (3); Rac1 is required for membrane ruffling and lamellipodia formation induced by growth factors (4); and Cdc42Hs has been demonstrated to mediate the induction of peripheral actin microspikes (PAM) 1 and filopodia by bradykinin (5). In fibroblast cells, Cdc42Hs/Rac1/RhoA have been suggested to work in a hierarchical cascade mediating these changes in the actin cytoskeleton (6). In addition, Rho, Rac, and Cdc42Hs all seem to be involved in the transcriptional regulation by serum response factor (7) and in the regulation of cell cycle progression (8). Particularly, Cdc42Hs and Rac participate in the p21-activated kinase-mediated Jun N-terminal kinase (JNK) activation (9, 10) and, through an independent pathway, in the regulation of p70 S6 kinase (S6K) (11).The activation of Rho proteins requires the exchange of bound GDP for GTP (12), a process catalyzed by a growing family of guanine-nucleotide exchange factors (GEFs) for which the Dbl oncoprotein is a prototype (13). These putative GEFs share the structural arrangement of an ϳ200-amino acid motif, the Dbl homology (DH) domain, followed by a second putative signaling motif, the pleckstrin homology (PH) domain (13). The ...

Identification of Rho GTPase-dependent Sites in the Dbl Homology Domain of Oncogenic Dbl That Are Required for Transformation

“…Production and purification of the Sf9 insect cell expressed GST-Dbl and DH mutants were performed similarly as described (17). The concentration and integrity of purified proteins were estimated by Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis using bovine serum albumin as a standard.…”

“…The sequences of mutagenized cDNA inserts were confirmed by automated DNA sequencing. The BamHI fragments encoding the DH-PH module of onco-Dbl mutants were subsequently subcloned into the BglII and BamHI sites of pVL1392 vector together with the cDNAs encoding the glutathione S-transferase (GST) for insect cell expression (17) or into the BamHI site of the mammalian pZipneo vector for transfection into NIH 3T3 cells (18). The same BamHI fragments of the wild type and mutant Dbl were also cloned into the pKH3 vector, which contains a triple hemagglutinin (HA) tag at the N terminus for transient expression in Cos-7 cells (19).…”

FGD2, a CDC42-specific Exchange Factor Expressed by Antigen-presenting Cells, Localizes to Early Endosomes and Active Membrane Ruffles

“…FGD2 Has RhoGEF Activity for Cdc42-Other Fgd family members have been shown to have exchange activity toward Cdc42 directly (FGD1 (11) and FGD4 (17,18)) and Rac1 indirectly (FGD4 (18)), suggesting that FGD2 might have similar specificity. Alignment of the "specificity patch" region of the DH domain sequences of FGD2 with other Cdc42-specific RhoGEFs (ASEF, h-PEM2, intersectin) further supported this prediction (Fig.…”

“…Fgd1 was identified as the gene defective in the Aarskog-Scott syndrome, an X-chromosome-linked disorder involving multiple developmental defects, including bone and body malformations (15). Studies have demonstrated that FGD1 is a guanine nucleotide exchange factor for the GTPase Cdc42, which plays important roles in cytoskeletal regulation and the organization of actin filaments (16,17). FGD3 and FGD4 have also been shown to modulate the actin cytoskeleton and to be Cdc42-specific exchange factors (18,19).…”

Phenotypic and molecular characterisation of the Aarskog–Scott syndrome: a survey of the clinical variability in light of FGD1 mutation analysis in 46 patients

“…3 The FGD1 gene encodes a guanine nucleotide exchange factor (GEF) for members of the Rho/Rac family of small GTP-binding proteins, binding specifically to the Rho GTPase Cdc42 and stimulating the GDP -GTP exchange of the isoprenylated form of Cdc42. 4 Studies suggest that an FGD1/Cdc42 signalling pathway is involved in the cytoskeletal organisation and, ultimately, in the skeletal formation and morphogenesis. 5 In the past, the clinical diagnosis of AAS has been made in a relatively large number of males based on the clinical dysmorphological findings.…”