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. 1997 Jun 10;94(12):6238-43.
doi: 10.1073/pnas.94.12.6238.

Glial cell line-derived neurotrophic factor-dependent RET activation can be mediated by two different cell-surface accessory proteins

M Sanicola  1 , C Hession, D Worley, P Carmillo, C Ehrenfels, L Walus, S Robinson, G Jaworski, H Wei, R Tizard, A Whitty, R B Pepinsky, R L Cate
Affiliations

Glial cell line-derived neurotrophic factor-dependent RET activation can be mediated by two different cell-surface accessory proteins

M Sanicola et al. Proc Natl Acad Sci U S A. .

Abstract

Glial cell line-derived neurotrophic factor (GDNF)-dependent activation of the tyrosine kinase receptor RET is necessary for kidney and enteric neuron development, and mutations in RET are associated with human diseases. Activation of RET by GDNF has been shown to require an accessory component, GDNFR-alpha (RETL1). We report the isolation and characterization of rat and human cDNAs for a novel cell-surface associated accessory protein, RETL2, that shares 49% identity with RETL1. Both RETL1 and RETL2 can mediate GDNF dependent phosphorylation of RET, but they exhibit different patterns of expression in fetal and adult tissues. The most striking differences in expression observed were in the adult central and peripheral nervous systems. In addition, the mechanisms by which the two accessory proteins facilitate the activation of RET by GDNF are quite distinct. In vitro binding experiments with soluble forms of RET, RETL1 and RETL2 demonstrate that while RETL1 binds GDNF tightly to form a membrane-associated complex which can then interact with RET, RETL2 only forms a high affinity complex with GDNF in the presence of RET. This strong RET dependence of the binding of RETL2 to GDNF was confirmed by FACS analysis on RETL1 and RETL2 expressing cells. Together with the recent discovery of a GDNF related protein, neurturin, these data raise the possibility that RETL1 and RETL2 have distinctive roles during development and in the nervous system of the adult. RETL1 and RETL2 represent new candidate susceptibility genes and/or modifier loci for RET-associated diseases.

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Figures

Figure 1
Figure 1
Comparative analysis of RETL1 and RETL2 protein sequences. Alignment of rat and human RETL1 and RETL2 predicted protein sequences. Residues conserved in all four sequences are boxed. Sequences were aligned using the clustal method (16).
Figure 2
Figure 2
RETL1 and RETL2 can mediate GDNF dependent phosphorylation of RET. NB41A3 cells were transfected with the indicated plasmid and 48 hrs later treated with or without GDNF. Cells were lysed, precipitated with a mAb to RET, and subjected to Western blot analysis with either a polyclonal antibody to RET (Lower) or a mAb against phosphotyrosine (Upper).
Figure 3
Figure 3
Detection of RETL1 and RETL2 interactions with GDNF and RET by direct binding. GDNF-coated microtiter plates were treated with serial dilutions of RETL1–Ig (▪), RETL2–Ig (•), or a control Fc fusion protein with the extracellular domain of LFA3 (▴) in the presence or absence of RET–AP. Samples were tested for complex formation either directly by measuring AP activity using the chemiluminescence substrate disodium 3-(4-methoxyspiro{1, 2-dioxetane-3, 2′-(5′-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate, or indirectly by probing for Fc in the complex using an anti-human Fc specific antibody conjugated with HRP and the chromogenic substrate 2–2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (measured at 405 nm). (a) Samples were incubated in the absence of RET–AP and detected through the anti-Fc-HRP conjugate. (b) samples were incubated in the presence of RET–AP and detected through the anti-Fc-HRP conjugate. (c) samples were incubated in the presence of RET–AP and detected using the AP readout.
Figure 4
Figure 4
Binding of GDNF and RET–Ig to cells expressing RETL1 and RETL2 evaluated by FACS. 293-EBNA cell lines expressing RETL1 (b and e) and RETL2 (c and f) or transformed with the CH269 vector (a and d) were established. (a–c) The ability of these cell lines to bind GDNF was evaluated using a mAb to GDNF as described in the experimental methods. In each of these panels, curves on the left show cells incubated in the absence of GDNF. (d–f) The ability of these cell lines to bind RET-Ig was evaluated using an antibody against the human Fc region as described in the experimental methods. Curves labeled RET–Ig show cells incubated with RET–Ig in the absence of GDNF; curves labeled RET–Ig + GDNF show cells incubated with RET–Ig in the presence of GDNF. In each of these panels, control curves (which fall on top of the curves labeled RET–Ig) show cells incubated in the absence of both RET–Ig and GDNF.
Figure 5
Figure 5
Expression of RETL1 and RETL2 mRNA in rat tissues. Northern blots (17) contained 4 μg per lane of poly(A)+ mRNA from the indicated tissue. RETL1 and RETL2 cDNA probes of 1.3 kb and 1.4 kb, respectively, were generated by random priming (18). Human β-Actin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) control oligonucleotide probes (CLONTECH) were 5′ end-labeled with 32P using T4 polynucleotide kinase.
Figure 6
Figure 6
Expression of RETL1 AND RETL2 mRNA in human brain. Northern blots containing mRNA from the indicated neuronal tissues (Clontech) were hybridized at 68°C in ExpressHyb (CLONTECH) and washed in 0.1× standard saline citrate (0.15 M sodium chloride/0.015 M sodium citrate, pH 7.0), 0.1% SDS at 50°C. RETL1 and RETL2 cDNA probes of 1.1 kb and 1.2 kb, respectively, were generated by random priming (18).
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