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Molecular Modeling of the Extracellular Domain of the RET Receptor Tyrosine Kinase Reveals Multiple Cadherin-like Domains and a Calcium-binding Site*

  • Jonas Anders
    Footnotes
    Affiliations
    Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden
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  • Svend Kjær
    Footnotes
    Affiliations
    Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden
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  • Carlos F. Ibáñez
    Correspondence
    To whom correspondence should be addressed: Div. of Molecular Neurobiology, Dept. of Neuroscience, Karolinska Inst., Retzius väg 8, 17177 Stockholm, Sweden. Tel.: 46-8-728-7660; Fax: 46-8-33-9548
    Affiliations
    Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden
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  • Author Footnotes
    * This work was supported by Swedish Medical Research Council Grant K99-33X-10908-06C, Vth Framework Program of the European Union Grant QLRT-1999-00099, and grants from the Göran Gustafssons Stiftelse and the Karolinska Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Supported by Grant An 338/1-1s from the Deutsche Forschungsgemeinschaft.
    § Supported by funds from Centrala Försökdjursnämnden.
Open AccessPublished:September 21, 2001DOI:https://doi.org/10.1074/jbc.M104968200
      Using bioinformatic tools, mutagenesis, and binding studies, we have investigated the structural organization of the extracellular region of the RET receptor tyrosine kinase, a functional receptor for glial cell line-derived neurotrophic factor (GDNF). Multiple sequence alignments of seven vertebrate sequences and one invertebrate RET sequence delineated four distinct N-terminal domains, each of about 110 residues, containing many of the consensus motifs of the cadherin fold. Based on these alignments and the crystal structures of epithelial and neural cadherins, we have generated molecular models of each of the four cadherin-like domains in the extracellular region of human RET. The modeled structures represent realistic models from both energetic and geometrical points of view and are consistent with previous observations gathered from biochemical analyses of the effects of Hirschsprung's disease mutations affecting the folding and stability of the RET molecule, as well as our own site-directed mutagenesis studies of RET cadherin-like domain 1. We have also investigated the role of Ca2+ in ligand binding by RET and found that Ca2+ ions are required for RET binding to GDNF but not for GDNF binding to the GFRα1 co-receptor. In agreement with these results, RET, but not GFRα1, was found to bind Ca2+ directly. Our results indicate that the overall architecture of the extracellular region of RET is more closely related to cadherins than previously thought. The models of the cadherin-like domains of human RET represent valuable tools with which to guide future site-directed mutagenesis studies aimed at identifying residues involved in ligand binding and receptor activation.
      GDNF
      glial cell line-derived neurotrophic factor
      HSCR
      Hirschsprung's disease
      TBS
      Tris-buffered saline
      CLD
      cadherin-like domain
      E-cadherin
      epithelial cadherin
      N-cadherin
      neural cadherin
      Endo H
      endoglycosidase H
      The RET receptor tyrosine kinase is an unusual receptor from many points of view. It cannot by itself bind its ligand, GDNF,1 unless in a complex with another protein, the glycosyl phosphatidylinositol-anchored receptor GFRα1 (
      • Airaksinen M.S.
      • Titievsky A.
      • Saarma M.
      ,
      • Saarma M.
      ). In contrast with other receptor tyrosine kinases, there appears to be only one RET homologue in all species investigated so far. All members of the GDNF ligand family utilize RET as a signal transducing receptor subunit, with specificity being determined by cooperation between RET and different members of the GFRα family of glycosyl phosphatidylinositol-anchored receptors (
      • Airaksinen M.S.
      • Titievsky A.
      • Saarma M.
      ,
      • Saarma M.
      ). Both gain- and loss-of-function mutations in the RETgene have been identified in human diseases. Mutations inRET of patients with multiple endocrine neuroplasias type 2A and 2B and familial medullary thyroid carcinoma induce constitutive activation of the RET tyrosine kinase and lead to congenital and sporadic cancers in neuroendocrine organs (
      • Edery P.
      • Eng C.
      • Munnich A.
      • Lyonnet S.
      ,
      • Santoro W.
      • Carlomagno F.
      • Melillo R.M.
      • Billaud W.
      • Vecchio G.
      • Fusco A.
      ). On the other hand, loss-of-function mutations in RET cause a dominant genetic disorder of neural crest development known as Hirschsprung's disease (HSCR), which results in the death of neurons in distal segments of the enteric nervous systems and colon aganglionosis (
      • Carlomagno F.
      • Devita G.
      • Berlingieri M.T.
      • Defranciscis V.
      • Melillo R.M.
      • Colantuoni V.
      • Kraus M.H.
      • Difiore P.P.
      • Fusco A.
      • Santoro M.
      ).
      The extracellular region of the RET molecule is peculiar compared with that found in other receptor tyrosine kinases in that it lacks leucine repeats, immunoglobulin, and fibronectin-like domains that are common in many other such receptors. The only recognizable motif within its over 600-residue-long extracellular region so far appears to be a stretch of 110 residues with similarity to members of the cadherin family of Ca2+-dependent cell adhesion molecules (
      • Iwamoto T.
      • Taniguchi M.
      • Asai N.
      • Ohkusu K.
      • Nakashima I.
      • Takahashi M.
      ,
      • Schneider R.
      ). Cadherins comprise a large and divergent superfamily with at least six subfamilies and several more divergent members, characterized on the basis of their domain architecture, genomic structure, and phylogenetic relationships (
      • Nollet F.
      • Kools P.
      • van Roy F.
      ). The extracellular region of cadherins is formed by a variable number of repeated modules (cadherin domains) of about 110 residues, all sharing a common consensus sequence and often with a Ca2+-binding site present in between each of the domains. Several crystal structures of cadherin domains from epithelial and neural cadherins have been solved (
      • Tamura K.
      • Shan W.S.
      • Hendrickson W.A.
      • Colman D.R.
      • Shapiro L.
      ,
      • Overduin M.
      • Harvey T.S.
      • Bagby S.
      • Tong K.I.
      • Yau P.
      • Takeichi M.
      • Ikura M.
      ,
      • Shapiro L.
      • Fannon A.M.
      • Kwong P.D.
      • Thompson A.
      • Lehmann M.S.
      • Grubel G.
      • Legrand J.F.
      • Als-Nielsen J.
      • Colman D.R.
      • Hendrickson W.A.
      ,
      • Nagar B.
      • Overduin M.
      • Ikura M.
      • Rini J.M.
      ). Ca2+ binding appears to help to linearize and rigidify the structure and, at least according to one report, to promote dimerization of cadherin domains (
      • Nagar B.
      • Overduin M.
      • Ikura M.
      • Rini J.M.
      ). Results from several laboratories have indicated that lateral dimerization or clustering of cadherins may increase their adhesivity (see, for example, Ref.
      • Tamura K.
      • Shan W.S.
      • Hendrickson W.A.
      • Colman D.R.
      • Shapiro L.
      ). As originally proposed, the segment of similarity between RET and cadherins would extend from the middle of one cadherin domain to the next over a Ca2+-binding motif (
      • Iwamoto T.
      • Taniguchi M.
      • Asai N.
      • Ohkusu K.
      • Nakashima I.
      • Takahashi M.
      ). However, because the remaining portion of the RET extracellular region did not appear to display any obvious similarity to cadherins or to any other protein, the actual relationship between RET and cadherins has been unclear.
      In the present study, we have used a battery of bioinformatic tools together with mutagenesis and binding studies to explore the structural organization of the extracellular region of RET. These studies allow us to propose a model for the overall architecture of the extracellular region of the RET molecule, as well as homology-based model structures of four of the five identified domains in this region.

      Acknowledgments

      We thank Judith Murray-Rust and Neil McDonald for valuable comments on the manuscript.

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