Deciphering ret signalling in cell biology and development

The rearranged during transformation (RET) tyrosine kinase regulates a plethora of biological processes such as cell survival, proliferation and migration and is essential for the normal development of several organs such as the sensory, enteric and sympathetic nervous systems and the kidneys. After RET activation by its ligands several intracellular tyrosine residues are phosphorylated and serve as binding sites for adaptor proteins that activate different downstream signalling pathways. One prominent binding site is tyrosine 1062. This residue is part of a binding motif for the phosphotyrosine binding (PTB) proteins DOK1-6, FRS2 and SHCA,B,C. The binding of PTB adaptors depends on the amino acids N-terminal of the tyrosine, and this feature can be utilised to engineer adaptor-specific receptors.

RET is known to be recruited into cholesterol-rich membrane domains upon activation, but the mechanism and biological importance of this translocation were previously unknown. In Paper I, we analyse the influence of the membrane domain localization of RET and its adaptors on their signalling characteristics. We show that the lipid raft-associated FRS2 recruits RET to lipid raft domains, while SHC localizes it to other membrane regions. A lipid raft-bound SHC (SHCMLS) resembles FRS2 both in signalling, translocation of RET and biological functionality, with diminished support of cell survival and increased migration of SHCMLS compared to normal SHC. In contrast to SHC, both FRS2 and SHCMLS functions depend on lipid raft integrity.

RET signalling is important for the development of several organ systems. In particular Y1062 plays a role in both the enteric and sympathetic nervous system and in nephrogenesis, however the specific roles of the different Y1062 binding proteins in vivo were unknown. In Paper II I investigate the role of RET signalling via DOK, FRS2 or SHC from Y1062 in vivo. Ret9Frs/9Frs mice show severe enteric aganglionosis, reduced soma size of dorsal root ganglion (DRG) neurons and mechanical hypersensitivity at early postnatal stages. Ret9Shc/9Shc mice on the other hand show a misregulation of sensory markers together with a hypersensitivity for cold and itch stimuli. In the sympathetic nervous system, Ret9Frs/9Frs animals display a reduced repression of cholinergic markers, with unchanged noradrenergic specification. We conclude that the studied adaptors have tissue- and cell type-specific roles and that they are main regulators of cell type specification both in the sensory and sympathetic nervous system.

One central process during sympathetic nervous system development is the segregation of the noradrenergic and cholinergic lineages. While several regulating factors are known, the knowledge about how they are organized into a regulatory network is incomplete and is still missing several regulatory elements. In Paper III we investigate the gene regulatory network that controls this segregation. We show that sympathetic progenitors are a hybrid population expressing markers of both the cholinergic and noradrenergic lineage and that the homeobox transcription factor HMX1 is required both for the repression of the expression of Ret and other cholinergic markers and for the maintenance of noradrenergic marker expression. RET on the other hand maintains cholinergic marker expression and supresses HMX1.

List of scientific papers

I. Lundgren TK, Lübke M, Stenqvist A, Ernfors P. Differential membrane compartmentalization of RET by PTB-adaptor Engagement. FEBS Journal 2008 May 275 (9), 2055-2066.
https://doi.org/10.1111/j.1742-4658.2008.06360.x

II. Lübke M, Furlan A, Eleuteri B, Abdo H, Lundgren TK, Li L, Lal M, Oddsson A, Tryggvason K, Ernfors P. Rewiring RET to specific PTB adaptor pathways leads to distinct developmental deficits. [Manuscript]

III. Furlan A, Lübke M, Adameyko I, Lallemend F, Ernfors P. The transcription factor HMX1 and growth factor receptor activities control sympathetic neurons diversification. EMBO Journal 2013 April 32 (11), 1613-1625.
https://doi.org/10.1038/emboj.2013.85