The Long and Short Isoforms of Ret Function as Independent Signaling Complexes

Brian A. Tsui-Pierchala,Jeffrey Milbrandt,Rebecca C. Ahrens,Robert J. Crowder,Eugene M. Johnson

doi:10.1074/jbc.m203580200

Abstract

Ret, the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family ligands (GFLs), is alternatively spliced to yield at least two isoforms, Ret9 and Ret51, which differ only in their C termini. To identify tyrosines in Ret that are autophosphorylation sites in neurons, we generated antibodies specific to phosphorylated Y905Ret, Y1015Ret, Y1062Ret, and Y1096Ret, all of which are autophosphorylated in cell lines. All four of these tyrosines in Ret became phosphorylated rapidly upon activation by GFLs in sympathetic neurons. These tyrosines remained phosphorylated in sympathetic neurons in the continued presence of GFLs, albeit at a lower level than immediately after GFL treatment. Comparison of GFL activation of Ret9 and Ret51 revealed that phosphorylation of Tyr(905) and Tyr(1062) was greater and more sustained in Ret9 as compared with Ret51. In contrast, Tyr(1015) was more highly phosphorylated over time in Ret51 than in Ret9. Surprisingly, Ret9 and Ret51 did not associate with each other in sympathetic neurons after glial cell line-derived neurotrophic factor stimulation, even though they share identical extracellular domains. Furthermore, the signaling complex associated with Ret9 was markedly different from the Ret51-associated signaling complex. Taken together, these data provide a biochemical basis for the dramatic functional differences between Ret9 and Ret 51 in vivo.

Highlights

Trophic factors sculpt the nervous system during development by regulating neuronal number, size, and phenotype
The receptor tyrosine kinase for the glial cell linederived neurotrophic factor family ligands (GFLs), is alternatively spliced to yield at least two isoforms, Ret9 and Ret51, which differ only in their C termini
When Ret9 and Ret51 complexes were compared with each other from sympathetic neurons treated with glial cell linederived neurotrophic factor (GDNF), we found that these complexes displayed more striking differences than immune complexes purified from CHP126 cells that express both Ret9 and Ret51 (Fig. 6B)

Summary

EXPERIMENTAL PROCEDURES

Sympathetic Neuron Cultures and Treatments—Sympathetic neurons from the rat superior cervical ganglia were dissociated and maintained in vitro as described previously [26]. Phosphotyrosine antibodies not specific to the particular tyrosine in Ret were removed from the eluate by counter-purifying the eluate from the prior two columns over a third column produced by using the three other unrelated PYRet peptides. These eluates were dialyzed and concentrated with a Centriprep centrifugal device (Millipore). The peptides were: Ret, CGRISHAFTRF; Ret, CMVSPSAAKLMDTFDS, both of which are only contained in that particular Ret isoform For immunoprecipitation using these Ret and Ret antibodies, antiRet and anti-Ret were covalently bound to an agarose support to avoid contamination of the immunoprecipitate with IgG released from the protein A beads after detergent solubilization. The Y1096F Ret mutant was generously provided by Jack Dixon and Carolyn Worby [7]

RESULTS

Ret Long and Short Isoforms Assemble Unique Complexes

DISCUSSION