Fibronectin leucine rich transmembrane proteins

The FLRT family of proteins is a rather young family. Not only because it was discovered in 1999 in an in silico screen for human muscle ECM interacting proteins (Lacy, et al. 1999), but also because orthologous genes have been found only in vertebrates,

such as in Xenopus, zebrafish and mouse. The FLRT family is comprised of three

members, FLRT1, FLRT2 and FLRT3 (Lacy, et al. 1999). The three members show a rather high degree of structural homology. These putative type 1 transmembrane proteins with a size of about 650 to 675 amino acids, have an extracellular domain, a transmembrane region and a rather short (about 100 aa long) intracellular C-terminal

tail. The extracellular domain consists of 10 leucine rich repeats (LRR), flanked by a characteristic N- and C-terminal LRR, respectively, and a fibronectin type 3 (FN3)

domain (Figure 8). This characteristic domain structure gave the name to the entire protein family, FLRT (Fibronectin Leucine Rich Transmembrane protein). However, the intracellular stretch shows no known protein interaction motive. Nevertheless, several putative phosphorylation sites have been predicted and two highly conserved lysine residues were implicated in Rnd1 (a Rho GTPase) interaction (Ogata, et al. 2007).

Figure 8 The domain architecture of FLRT3

The name FLRT is derived from the protein’s domain structure. FLRT proteins are putative type 1

transmembrane proteins carrying 10 leucine rich repeats (LRR) flanked by a N- and a C-terminal LRR and a fibronectin type 3 domain on its extracellular region. After the transmembrane domain, a short

C-terminal domain extends into the cytoplasm without any known protein homology domain but with two highly conserved lysine residues.

N/C-term LRR _{leucine rich repeat} fibronectin type 3 transmembrane conserved KK residues

Although a great effort has been put into the understanding of the biological roles of FLRT proteins, only a limited but steadily increasing number of studies have been published so far. Yet, research has mainly focused thus far on the biological functions of FLRT3, and almost no published data is available on the functions of FLRT1 or FLRT2. FLRT3, in turn, has been found to be upregulated in PNS neurons after injury (Tanabe, et al. 2003); Robinson et al. 2004), and to promote neurite outgrowth of cerebellar granule cells when grown on a FLRT3 expressing surface layer (Tsuji, et al. 2004). In Xenopus embryos, xFLRT3 (the Xenopus homologue) was shown to function in the FGFR pathway (Figure 9 A). Injection of xFLRT3 RNA into Xenopus embryos led to ectopic expression of Xbra and Fgf8. This is mediated through the interaction of the FN3 domain of FLRT3, with the extracellular domain of the FGFR1 in presence of the intracellular C-terminal region of FLRT3, followed by downstream activation of the MAP- kinase pathway (Böttcher, et al. 2004). Furthermore, a second study performed in the laboratory of Christof Niehrs showed that FLRT proteins can interact homotypically and are able to promote cell sorting in transfected cells via their LRRs (Karaulanov, et al. 2006) (Figure 9 C). An additional function of FLRT3 was found in conjunction with the Nodal- like TGF-beta signal Activin (Figure 9 B). In Xenopus, FLRT3 is involved in a pathway controlling the subcellular localization of C-cadherin in animal cap cells. Upon activation through Activin, FLRT3 and the Rho-type GTPase Rnd1 directly bind and mediate the internalization of C-cadherin via endocytosis. In overexpression experiments, massive detachment of cells was evident after injection of with xFLRT3 or Rnd1 RNA (Ogata 2007). The possible biological roles of FLRT3 have so far mainly been addressed in cell culture or amphibian embryo experiments. Initially, very little data on the function of FLRT3 in mouse was available. Haines and co-workers provided some insights into the expression pattern of

FLRT3 during postimplantation development. In addition, they have confirmed the possible

cell-adhesion function of FLRT3, as well as an interaction with FGFRs (Haines, et al. 2006). The available data on the functions of FLRT3 shows a rather diverse picture. FLRT3, a potential ECM interacting protein, is expressed on regenerating PNS neurons and can facilitate the outgrowth of neurites. Further in vitro studies have implicated a capability of FLRT3 for homotypic binding and mediation of cell sorting. In combination with

FGF or activin signalling, it can also influence the formation of the embryonic body

axis either by posteriorizing effects or by disturbing axis elongation, at least in the amphibian embryo. Aside from the expression pattern of FLRT3 in mouse embryos, the function in mammalian development remained largely unknown. Only very recently, a publication has claimed a possible role for FLRT3 in the developing mouse embryo. The loss of FLRT3 during development was suggested to result in DE migration defects that are independent of FGF signalling. But no possible mechanism of how FLRT3 is

B

C

Figure 9 Proposed functions of FLRT3

Although the function of FLRT3 is not yet clear, several roles for FLRT3 have been demonstrated thus far. (A) In Xenopus embryos, FLRT3 interacts with FGFR1 and enhances the signalling function of FGFR1 via the MAPKinase pathway. (B) In addition, during Xenopus gastrulation, FLRT3, in cooperation with Rnd1, is required to regulate C-cadherin levels present in the cell membrane. This pathway is activated through TGF-beta signalling. (C) It has been shown in heterologous cell systems that FLRT3 is able to bind in a homotypic manner and can mediate cell sorting. How this effect is accomplished is so far unclear

2. Results