Structure-function studies of GDNF and other members of the TGF-beta superfamily
Author: Eketjäll, Susanna
Date: 2002-11-12
Location: Rockefeller, Nobels väg 11
Time: 9.00
Department: Institutionen för neurovetenskap / Department of Neuroscience
Abstract
The glial cell line-derived neurotrophic factor (GDNF) family is a
distant subclass of the TGF-beta superfamily. The GDNF family of ligands,
consisting of GDNF, neurturin (NTN), artemin (ART) and persephin (PSP),
are potent survival and differentiation factors for neurons in the
central and peripheral nervous systems. These ligands signal through a
tyrosine kinase receptor, Ret and an accessory receptor subunit, the GDNF
family receptor alphas (GFRas). There are four GFRas known to date
(GFRalpha1 to 4). GDNF preferentially binds to GFRalpha1, while NTN, ART,
and PSP bind to GFRalpha2, 3 and 4, respectively, but with some degree of
cross talk. It has been postulated that the ligand first associates with
the GFRalpha and that only then is Ret recruited to the complex, becoming
autophosphorylated on several cytoplasmic tyrosine residues.
Phosphotyrosine residues on active Ret form a platform for the
recruitment of multiple adaptor and effector proteins. There are two main
isoforms of Ret, Ret9 and Ret51, differing at their C-terminal sequence.
We have assessed the ability of different GDNF mutants to bind to
GFRalpha1 and induce tyrosine phosphorylation in Ret. Hydrophobic and
negatively charged residues in the tips of GDNF fingers 1 and 2, were
found to be important for receptor interaction. Unexpectedly some of the
mutants that lost their affinity for GFRalpha1 were still able to induce
Ret tyrosine phosphorylation. These mutants however, were not able to
activate Ret in cells not expressing GFRalpha1, indicating that GFRalpha1
was still required even if GDNF was unable to bind. These results led us
to propose a model including two distinct binding sites for GDNF: one
formed by GFRalpha1 alone, requires hydrophobic and acidic residues in
finger 1 and 2, and another by a pre-associated GFRalpha1/Ret complex,
which requires acidic residues in finger 1.
Several mutations have been found in the GDNF gene of patients with
Hirschsprung disease (HSCR). We have characterized the effects of these
mutants on the ability of GDNF to bind to and activate its receptors.
Although none of the four mutations analyzed appeared to affect the
ability of GDNF to activate Ret, two of them resulted in a significant
reduction in the binding affinity of GDNF for GFRalpha1. Indicating that,
although none of the GDNF mutations identified so far in HSCR patients
are per se likely to result in HSCR, two of these mutations may, in
conjunction with other genetic lesions, contribute to the pathogenesis of
this disease.
We have demonstrated that Ret51 associates more strongly, than Ret9, with
the ubiquitin ligase Cbl, leading to increased ubiquitylation and faster
turnover of active Ret51. The association of Cbl with Ret is indirect and
mediated through Grb2. A constitutive complex of Grb2 and Cbl can be
recruited to both receptor isoforms via docking and tyrosine
phosphorylation of Shc. However, Ret 51, but not Ret9, can in addition
recruit Cbl via direct interaction of the Grb2/Cbl complex with
phosphorylated Tyr-1096, unique to the Ret51. Interestingly, this same
phosphotyrosine also allows Ret51 to recruit the adaptor protein CrkL,
leading to prolonged activation of MAP kinases ERK1 and ERK2 upon
activation of Ret51 in neuronal cells. Our results have therfore
established distinct signaling mechanisms by Ret51 and Ret9.
Taking advantage of the conserved pattern of cysteine residues in the
TGF-beta superfamily, we sought to identify new members of this family,
using a novel search engine called Motifer. We identified two genes,
provisionally named Motifer Derived Factors (MDF451 and MDF628), in the
public human genome database. The MDFs can only be found in genomes of
primates and not in other species such as rodents. Both genes are
expressed in human fetal brain and cerebellum, but so far we have been
unable to isolate full-length cDNA for either of the two MDFs. Further
analysis of upstream sequence in MDF628 revealed STOP codons in frame
with the TGF-beta like reading frame, thereby ruling out the capacity of
this gene to encode a TGF-beta family member. We are suggesting that the
genes identified may have recently appeared in evolution, in a common
ancestor of the primate lineage, perhaps by duplication of a
GDNF/TGF-beta-like gene.
List of papers:
I. Eketjall S, Fainzilber M, Murray-Rust J, Ibanez CF (1999). "Distinct structural elements in GDNF mediate binding to GFRalpha1 and activation of the GFRalpha1-c-Ret receptor complex. " EMBO J 18(21): 5901-10
Pubmed
II. Eketjall S, Ibanez CF (2002). "Functional characterization of mutations in the GDNF gene of patients with Hirschsprung disease. " Hum Mol Genet 11(3): 325-9
Pubmed
III. Scott RP, Eketjall S, Aineskog H, Ibanez CF (2005). "Distinct turnover of alternatively-spliced isoforms of the RET kinase receptor mediated by differential recruitment of the Cbl ubiquitin ligase." J Biol Chem Jan 27: Epub ahead of print
Pubmed
IV. Eketjall S, Jornvall H, Lonnerberg P, Kobayashi S, Ibanez CF (2004). "Recent evolutionary origin within the primate lineage of two pseudogenes with similarity to members of the transforming growth factor-beta superfamily. " Cell Mol Life Sci 61(4): 488-96
Pubmed
I. Eketjall S, Fainzilber M, Murray-Rust J, Ibanez CF (1999). "Distinct structural elements in GDNF mediate binding to GFRalpha1 and activation of the GFRalpha1-c-Ret receptor complex. " EMBO J 18(21): 5901-10
Pubmed
II. Eketjall S, Ibanez CF (2002). "Functional characterization of mutations in the GDNF gene of patients with Hirschsprung disease. " Hum Mol Genet 11(3): 325-9
Pubmed
III. Scott RP, Eketjall S, Aineskog H, Ibanez CF (2005). "Distinct turnover of alternatively-spliced isoforms of the RET kinase receptor mediated by differential recruitment of the Cbl ubiquitin ligase." J Biol Chem Jan 27: Epub ahead of print
Pubmed
IV. Eketjall S, Jornvall H, Lonnerberg P, Kobayashi S, Ibanez CF (2004). "Recent evolutionary origin within the primate lineage of two pseudogenes with similarity to members of the transforming growth factor-beta superfamily. " Cell Mol Life Sci 61(4): 488-96
Pubmed
Issue date: 2002-10-22
Publication year: 2002
ISBN: 91-7349-331-7
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