Investigating the function of single-pass leucine-rich repeat transmembrane proteins in cell signalling and early neural development

Abstract

Single-pass leucine-rich (LRR) repeat transmembrane proteins contain a diverse number of repeating motifs of approximately 24 amino acid residues with a large number being conserved leucines. Flrt expression is observed in the developing embryo in important developmental regions such as the central nervous system and developing skeletal muscle. Knockout of Flrt3 during embryogenesis results in early embryonic lethality making in vivo analysis of endogenous Flrt3 function difficult. No cell based model exists for studying Flrt function. Flrt family members have previously been shown to interact with FgfR1 and 2, with the Flrt1 FgfR1 interaction resulting in an increase in FgfR1 signalling activity. Immunofluorescent microscopy reveals that Flrt3 from mouse (Mus musculus) co-localises with FgfR1 both intracellularly and at the plasma membrane, with the interaction resulting in a trend of increased FgfR1 signalling being observed, and phosphorylation of tyrosine residues within Flrt3. An attempt was made to identify domains of the protein important in the trend of increased FgfR1 signalling, but no domains could be identified as contributing to this outcome. To study endogenous Flrt3 function, the P19 embryonic carcinoma retinoic acid-induced neural differentiation model was used, and the results showed a rapid and robust induction of Flrt3 mRNA and protein expression. A region of the promoter between 4 kb and 6 kb upstream of the Flrt3 start site was found to be partially responsible for the induction of Flrt3. Interestingly, this response element was not within a region of promoter that showed conservation among higher-order mammals. An effect of increased Flrt3 expression during neural differentiation was observed, resulting in decreased MAPK pathway activation. Induction of Flrt3 is found to occur prior to that of Sox1, accepted to be one of the first genes up-regulated in early neurectoderm differentiation, yet was found to be not solely responsible for the induction of Sox1. The individual cell expression of Flrt3 and Sox1 was analysed by immunofluorescence, although it did not reveal details regarding induction of Sox1 in cells with increased Flrt3 expression. The potential for a common feature of single-pass LRR transmembrane proteins to function as modulators of receptor signalling during embryonic development was investigated, using Lrrtm3 and FgfR1 as an example. Lrrtm3 was investigated as a modulator of FgfR1 signalling due to overlapping region of expression with FgfR1 in the developing embryo. Lrrtm3 was found to co-localise and form an interaction with FgfR1, with this interaction resulting in an increase in FgfR1 signalling. The data obtained in this thesis provides further insight into not only the role of the Flrt protein family as FgfR1 modulators, but potentially identifies a role for similar, if not all single-pass LRR transmembrane proteins as regulators of receptor signalling during embryonic development. While the results of Chapter 3 and 5 were obtained using a protein over-expression system, the first model for studying endogenous Flrt3 was identified and characterised in Chapter 4, providing the opportunity to study Flrt3 function during development with protein expression levels closely resembling those that are found in the embryo.