Abstract
Rearranged during transfection (RET), a receptor tyrosine kinase that is activated by the glial cell line-derived neurotrophic factor family ligands (GFLs), plays a crucial role in the development and function of the nervous system and additionally is required for kidney development and spermatogenesis. RET encodes a transmembrane receptor that is 20 exons long and produces two known protein isoforms differing in C-terminal amino acid composition, referred to as RET9 and RET51. Studies of human pheochromocytomas identified two additional novel transcripts involving the skipping of exon 3 or exons 3, 4, and 5 and are referred to as RET(Δ) (E3) and RET(Δ) (E345), respectively. Here we report the presence of Ret(Δ) (E3) and Ret(Δ) (E345) in zebrafish, mice, and rats and show that these transcripts are dynamically expressed throughout development of the CNS, peripheral nervous system, and kidneys. We further explore the biochemical properties of these isoforms, demonstrating that, like full-length RET, RET(ΔE3) and RET(ΔE345) are trafficked to the cell surface, interact with all four GFRα co-receptors, and have the ability to heterodimerize with full-length RET. Signaling experiments indicate that RET(ΔE3) is phosphorylated in a similar manner to full-length RET. RET(ΔE345), in contrast, displays higher baseline autophosphorylation, specifically on the catalytic tyrosine, Tyr(905), and also on one of the most important signaling residues, Tyr(1062) These data provide the first evidence for a physiologic role of these isoforms in RET pathway function.
Highlights
The presence of two major Ret isoforms, RET9 and RET51, has been extensively described in the literature, and a third isoform, RET43, has been observed in humans [7,8,9]
We show that Ret⌬E3 and Ret⌬E345 transcripts are conserved in vertebrates and that the mRNA and proteins of these splice variants are expressed throughout the nervous system in mice
Exons 3, 4, and 5 of Ret Are Not Highly Conserved, but Ret⌬E3 and Ret⌬E345 Transcripts Are Observed in Several Organisms— It has been described previously that the intracellular domain of Rearranged during transfection (RET), which contains a tyrosine kinase domain, is more highly conserved than the extracellular domain [13]
Summary
We show that Ret⌬E3 and Ret⌬E345 transcripts are conserved in vertebrates and that the mRNA and proteins of these splice variants are expressed throughout the nervous system in mice. We show that these isoforms are trafficked to the cell surface and that both isoforms interact with all four GFR␣s. We find that RET⌬E3 is phosphorylated to a similar level as full-length RET and is activated in a GDNF-dependent manner. RET⌬E345 displays higher baseline autophosphorylation, on the catalytic tyrosine Tyr905, and on an additional signaling tyrosine, Tyr1062. RET⌬E345 is not activated in a GDNF-dependent manner. Taken together, these isoforms may have unique and important unidentified roles in the development and maintenance of the nervous system and kidneys as well as in the pathophysiology of neuroendocrine gland diseases
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