Fat cadherins constitute a subclass of the large cadherin family characterized by the presence of 34 cadherin motifs. To date, three mammalian Fat cadherins have been described; however, only limited information is known about the function of these molecules. This paper describes the second fat cadherin in Drosophila, fat-like (ftl). ftl is the true orthologue of vertebrate fat-like genes, whereas the previously characterized tumor suppressor cadherin, fat, is more distantly related as compared with ftl. Ftl is a large molecule of 4705 amino acids. It is expressed apically in luminal tissues such as trachea, salivary glands, proventriculus, and hindgut. Silencing of ftl results in the collapse of tracheal epithelia giving rise to breaks, deletions, and sac-like structures. Other tubular organs such as proventriculus, salivary glands, and hindgut are also malformed or missing. These data suggest that Ftl is required for morphogenesis and maintenance of tubular structures of ectodermal origin and underline its similarity in function to a reported lethal mouse knock-out of fat1 where glomerular epithelial processes collapse. Based on these results, a model is proposed where Ftl acts as a spacer to keep tubular epithelia apart rather than the previously described adhesive properties of the cadherin superfamily (Castillejo-Lopez, 2004).
FAT, a new member of the human cadherin super-family, has been isolated from the T-leukemia cell line J6. The predicted protein closely resembles the Drosophila tumor suppressor Fat, which is essential for controlling cell proliferation during Drosophila development. The gene has the potential to encode a large transmembrane protein of nearly 4600 residues with 34 tandem cadherin repeats, five EGF-like repeats, and a laminin A-G domain. The cytoplasmic sequence contains two domains with distant homology to the cadherin catenin-binding region. Northern blotting analysis of J6 mRNA has demonstrated a full-length, approximately 15-kb, FAT message in addition to several 5'-truncated transcripts. In addition to its presence in J6 cells, in situ hybridization has revealed FAT mRNA expression in epithelia and in some mesenchymal compartments. Furthermore, higher levels of expression were observed in fetal, as opposed to adult, tissue, suggesting that its expression may be developmentally regulated in these tissues. FAT shows homologies with a number of proteins important in developmental decisions and cell:cell communication and is the first Fat-like protein reported in vertebrates. The gene encoding FAT was located by in situ hybridization on chromosome 4q34-q35. It is proposed that this family of molecules is likely to be important in mammalian developmental processes and cell communication (Dunne, 1995).
The expression during rat embryogenesis of the protocadherin fat, the murine homolog of a Drosophila tumor suppressor gene, has been examined. The sequence encodes a large protocadherin with 34 cadherin repeats, five epidermal growth factor (EGF)-like repeats containing a single laminin A-G domain and a putative transmembrane portion followed by a cytoplasmic sequence. This cytoplasmic sequence shows homology to the ß-catenin binding regions of classical cadherin cytoplasmic tails and also ends with a PDZ domain-binding motif. In situ hybridization studies at E15 show that fat is predominately expressed in fetal epithelial cell layers and in the CNS, although expression is also seen in tongue musculature and condensing cartilage. Within the CNS, expression is seen in the germinal regions and in areas of developing cortex, and this neural expression pattern is also seen at later embryonic (E18) and postnatal stages. No labelling was seen in adult tissues except in the CNS, where the remnant of the germinal zones, as well as the dentate gyrus, continue to express fat (Ponassi, 1999).
The entire sequence of the mouse Fat ortholog (mFat1) is presented. mFat1 protein consists of 4,588 amino acids with 34 cadherin repeats, 27 potential N-glycosylation sites, five EGF repeats and a laminin A G-motif in its extracellular domain. A single transmembrane region is followed by a cytoplasmic domain containing putative catenin-binding sequences. mFat1 shows high homology to human FAT and lesser homology to Drosophila Fat. The sequence of this giant cadherin suggests that it is unlikely to have a homophilic adhesive function, but may mediate heterophilic adhesion or play a signaling role. Expression analysis shows that the mfat1 gene is expressed early in pre-implantation mouse development, at the compact eight cell stage. Whole-mount and section in situ analyses show that transcripts are widely expressed throughout post-implantation development, most notably in the limb buds, branchial arches, forming somites, and in particular in the proliferating ventricular zones in the brain, being down-regulated as cells cease dividing. RT-PCR detects widespread expression in the adult, suggesting a role in proliferation and differentiation of many tissues and cell types (Cox, 2000).
Slit diaphragms are intercellular junctions of podocytes of the renal glomerulus. The molecular composition of slit diaphragms is still elusive. Slit diaphragms are characterized by the presence of a wide intercellular space. The morphological feature is shared by desmosomes and adherens junctions, which contain members of the cadherin superfamily. Thus, it has been hypothesized that some components of slit diaphragms belong to the cadherin superfamily. Consequently, cDNA encoding FAT has been isolated from reverse-transcribed (RT) glomerular cDNA by homology polymerase chain reaction (PCR) using primers based on conserved sequences in cadherin molecules. FAT is a novel member of the cadherin superfamily with 34 tandem cadherin-like extracellular repeats, and it closely resembles the Drosophila tumor suppressor fat. Expression of FAT was examined in glomeruli of the adult rat kidney by the ribonuclease protection assay and in situ hybridization. To localize the FAT protein in podocytes minutely, affinity-purified antibody was prepared against FAT by immunizing rabbits against an oligopeptide corresponding to the C-terminal 20 amino acids. Expression of FAT mRNA was detected in total RNA from glomeruli. In situ hybridization revealed significant signals in podocytes. Western blot analysis using solubilized glomeruli showed a single band, in which the molecular weight was more than 500 kD. Immunostaining of cultured epithelial cells from rat kidney (NRK52E) revealed FAT accumulation in cell-cell contact sites. In the glomerulus, FAT staining is observed distinctly along glomerular capillary walls. Double-label immunostaining using monoclonal antibody against slit diaphragms (mAb 5-1-6) showed identical localization of anti-FAT antibody and mAb 5-1-6. Furthermore, the double-label immunogold technique with ultrathin cryosections demonstrates that gold particles for FAT cytoplasmic domain are located at the base of slit diaphragms labeled by mAb 5-1-6 and that the cytoplasmic domain of FAT colocalizes with ZO-1, a cytoplasmic component associated with slit diaphragms. Thus the molecular structure of FAT and its colocalization with 5-1-6 antigen and ZO-1 indicate that FAT is a component of slit diaphragms (Inoue, 2001).
Using computer-based, motif-trap screening, a third member of the mammalian fat family, fat3, has been identified. Human and rat fat3 are also similar to the Drosophila tumor suppressor gene fat. The rat fat3 gene encodes a large protein of 4555 amino acids with 34 cadherin domains, 4 epidermal growth factor (EGF)-like motifs, a laminin A-G motif, and a cytoplasmic domain. Each member of the fat family is differentially expressed in the central nervous system during development. While both fat3 mRNA and fat1 mRNA are abundantly expressed in the fetal brain, the expression of MEGF1/fat2 mRNA is restricted to the postnatal cerebellum. fat3 mRNA and protein expression in the brain peaks at E15 during embryonic development. During this time, robust fat3 immunoreactivity is also observed in the spinal cord. These data suggest that the fat3 protein plays an important role in axon fasciculation and modulation of the extracellular space surrounding axons during embryonic development (Mitsui, 2002).
Cell migration requires integration of cellular processes resulting in cell polarization and actin dynamics. Previous work using tools of Drosophila genetics suggested that protocadherin Fat serves in a pathway necessary for determining cell polarity in the plane of a tissue. This study identifies mammalian FAT1 as a proximal element of a signaling pathway that determines both cellular polarity in the plane of the monolayer and directs actin-dependent cell motility. FAT1 is localized to the leading edge of lamellipodia, filopodia, and microspike tips where FAT1 directly interacts with Ena/VASP proteins that regulate the actin polymerization complex. When targeted to mitochondrial outer leaflets, FAT1 cytoplasmic domain recruits components of the actin polymerization machinery sufficient to induce ectopic actin polymerization. In an epithelial cell wound model, FAT1 knockdown decreases recruitment of endogenous VASP to the leading edge and results in impairment of lamellipodial dynamics, failure of polarization, and an attenuation of cell migration. FAT1 may play an integrative role regulating cell migration by participating in Ena/VASP-dependent regulation of cytoskeletal dynamics at the leading edge and by transducing an Ena/VASP-independent polarity cue (Moeller, 2004).
Fat cadherins form a distinct subfamily of the cadherin gene superfamily and are featured by their unusually large extracellular domain. This work investigated the function of a mammalian Fat cadherin. Fat1 is localized at filopodial tips, lamellipodial edges, and cell-cell boundaries, overlapping with dynamic actin structures. RNA interference-mediated knockdown of Fat1 results in disorganization of cell junction-associated F-actin and other actin fibers/cables, disturbance of cell-cell contacts, and also inhibition of cell polarity formation at wound margins. Furthermore, Ena/vasodilator-stimulated phosphoproteins were identified as a potential downstream effector of Fat1. These results suggest that Fat1 regulates actin cytoskeletal organization at cell peripheries, thereby modulating cell contacts and polarity (Tanoe, 2004).
The significance of cadherin superfamily proteins in vascular smooth muscle cell (VSMC) biology is undefined. Recent studies of the Fat1 protocadherin show that expression in VSMCs increases significantly after arterial injury or growth factor stimulation. Fat1 knockdown decreases VSMC migration in vitro, but surprisingly, enhances cyclin D1 expression and proliferation. Despite limited similarity to classical cadherins, the Fat1 intracellular domain (Fat1IC) interacts with beta-catenin, inhibiting both its nuclear localization and transcriptional activity. Fat1 undergoes cleavage and Fat1IC species localize to the nucleus; however, inhibition of the cyclin D1 promoter by truncated Fat1IC proteins corresponds to their presence outside the nucleus, which argues against repression of beta-catenin-dependent transcription by nuclear Fat1IC. These findings extend recent observations about Fat1 and migration in other cell types, and demonstrate for the first time its anti-proliferative activity and interaction with beta-catenin. Because it is induced after arterial injury, Fat1 may control VSMC functions central to vascular remodeling by facilitating migration and limiting proliferation (Hou, 2006).
The cadherin superfamily protein Fat1 is known to interact with the EVH1 domain of mammalian Ena/VASP. This study demonstrates that the scaffolding proteins Homer-3 and Homer-1 also interact with the EVH1 binding site of hFat1 in vitro; binding of Homer-3 and Mena to hFat1 is mutually competitive. Endogenous Fat1 binds to immobilised Homer-3 and endogenous Homer-3 binds to immobilised Fat1. Both, endogenous and over-expressed Fat1 exhibit co-localisation with Homer-3 in cellular protrusions and at the plasma membrane of HeLa cells. Since Homer proteins and Fat1 have been both linked to psychic disorders, their interaction may be of patho-physiological importance (Schreiner, 2006).
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