Quantitative analysis of the activation mechanism of the multicomponent growth-factor receptor Ret


Cytokines and growth factors signal by modulating the interactions between multiple receptor components to form an activated receptor complex. The quantitative details of the activation mechanisms of this important class of receptors are not well understood. Using receptor phosphorylation measurements in live cells, as well as mathematical modeling and data fitting, we have characterized the multistep mechanism by which the GDNF-family neurotrophin artemin (ART), together with its co-receptor GDNF-family receptor α3 (GFRα3), brings about activation of the Ret receptor tyrosine kinase through formation of a pentameric signaling complex: ART–(GFRα3)2–(Ret)2. By systematically varying the concentrations of ART and cell-surface GFRα3, we establish both the sequence of steps by which the signaling complex forms and the affinities of all the steps, including the two-dimensional affinities of the steps involving protein-protein interactions between membrane-bound species. Our results reveal the ways in which the individual binary interactions involved in the activation of a multicomponent receptor govern the receptor's functional properties.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: ART interacts with the receptor tyrosine kinase Ret and the GPI-linked co-receptor GFRα3 to form a pentameric activated receptor complex.
Figure 2: ART binding to GFRα3 and Ret on cells.
Figure 3: Bell-shaped dose-response curve for ART.
Figure 4: Dependence of Ret phosphorylation on the concentrations of ART and GFRα3.
Figure 5: Possible pathways for activation of Ret by ART and GFRα3.
Figure 6: Effect of ART on the distribution of receptor species on the cell surface.


  1. 1

    Hendriks, B.S., Orr, G., Wells, A., Wiley, H.S. & Lauffenburger, D.A. Parsing ERK activation reveals quantitatively equivalent contributions from epidermal growth factor receptor and HER2 in human mammary epithelial cells. J. Biol. Chem. 280, 6157–6169 (2005).

    CAS  Article  Google Scholar 

  2. 2

    Pearce, K.H., Jr, Cunningham, B.C., Fuh, G., Teeri, T. & Wells, J.A. Growth hormone binding affinity for its receptor surpasses the requirements for cellular activity. Biochemistry 38, 81–89 (1999).

    CAS  Article  Google Scholar 

  3. 3

    Sarkar, C.A., Lowenhaupt, K., Wang, P.J., Horan, T. & Lauffenburger, D.A. Parsing the effects of binding, signaling, and trafficking on the mitogenic potencies of granulocyte colony-stimulating factor analogues. Biotechnol. Prog. 19, 955–964(2003).

    CAS  Article  Google Scholar 

  4. 4

    Wilkinson, J.C., Stein, R.A., Guyer, C.A., Beechem, J.M. & Staros, J.V. Real-time kinetics of ligand/cell surface receptor interactions in living cells: binding of epidermal growth factor to the epidermal growth factor receptor. Biochemistry 40, 10230–10242 (2001).

    CAS  Article  Google Scholar 

  5. 5

    Stahl, N. & Yancopoulos, G.D. The alphas, betas, and kinases of cytokine receptor complexes. Cell 74, 587–590 (1993).

    CAS  Article  Google Scholar 

  6. 6

    Heldin, C.H. Dimerization of cell surface receptors in signal transduction. Cell 80, 213–223 (1995).

    CAS  Article  Google Scholar 

  7. 7

    Lindsay, R.M. & Yancopoulos, G.D. GDNF in a bind with known orphan: accessory implicated in new twist. Neuron 17, 571–574 (1996).

    CAS  Article  Google Scholar 

  8. 8

    Baloh, R.H. et al. Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFRα3-RET receptor complex. Neuron 21, 1291–1302 (1998).

    CAS  Article  Google Scholar 

  9. 9

    Rosenblad, C. et al. In vivo protection of nigral dopamine neurons by lentiviral gene transfer of the novel GDNF-family member neublastin/artemin. Mol. Cell. Neurosci. 15, 199–214 (2000).

    CAS  Article  Google Scholar 

  10. 10

    Masure, S. et al. Enovin, a member of the glial cell-line-derived neurotrophic factor (GDNF) family with growth promoting activity on neuronal cells. Existence and tissue-specific expression of different splice variants. Eur. J. Biochem. 266, 892–902 (1999).

    CAS  Article  Google Scholar 

  11. 11

    Kotzbauer, P.T. et al. Neurturin, a relative of glial-cell-line-derived neurotrophic factor. Nature 384, 467–470 (1996).

    CAS  Article  Google Scholar 

  12. 12

    Milbrandt, J. et al. Persephin, a novel neurotrophic factor related to GDNF and neurturin. Neuron 20, 245–253 (1998).

    CAS  Article  Google Scholar 

  13. 13

    Lapchak, P.A. Therapeutic potentials for glial cell line-derived neurotrophic factor (GDNF) based upon pharmacological activities in the CNS. Rev. Neurosci. 7, 165–176 (1996).

    CAS  Article  Google Scholar 

  14. 14

    Unsicker, K. GDNF: a cytokine at the interface of TGF-βs and neurotrophins. Cell Tissue Res. 286, 175–178 (1996).

    CAS  Article  Google Scholar 

  15. 15

    Gardell, L.R. et al. Multiple actions of systemic artemin in experimental neuropathy. Nat. Med. 9, 1383–1389 (2003).

    CAS  Article  Google Scholar 

  16. 16

    Orozco, O.E., Walus, L., Sah, D.W., Pepinsky, R.B. & Sanicola, M. GFRα3 is expressed predominantly in nociceptive sensory neurons. Eur. J. Neurosci. 13, 2177–2182 (2001).

    CAS  Article  Google Scholar 

  17. 17

    Andres, R. et al. Multiple effects of artemin on sympathetic neurone generation, survival and growth. Development 128, 3685–3695 (2001).

    CAS  PubMed  Google Scholar 

  18. 18

    Jing, S. et al. GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-α, a novel receptor for GDNF. Cell 85, 1113–1124 (1996).

    CAS  Article  Google Scholar 

  19. 19

    Eigenbrot, C. & Gerber, N. X-ray structure of glial cell-derived neurotrophic factor at 1.9 A resolution and implications for receptor binding. Nat. Struct. Biol. 4, 435–438 (1997).

    CAS  Article  Google Scholar 

  20. 20

    Silvian, L. et al. Artemin crystal structure reveals insights into heparan sulfate binding. Biochemistry 45, 6801–6812 (2006).

    CAS  Article  Google Scholar 

  21. 21

    Trupp, M. et al. Functional receptor for GDNF encoded by the c-ret proto-oncogene. Nature 381, 785–789 (1996).

    CAS  Article  Google Scholar 

  22. 22

    Vega, Q.C., Worby, C.A., Lechner, M.S., Dixon, J.E. & Dressler, G.R. Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis. Proc. Natl. Acad. Sci. USA 93, 10657–10661 (1996).

    CAS  Article  Google Scholar 

  23. 23

    Worby, C.A. et al. Glial cell line-derived neurotrophic factor signals through the RET receptor and activates mitogen-activated protein kinase. J. Biol. Chem. 271, 23619–23622 (1996).

    CAS  Article  Google Scholar 

  24. 24

    Treanor, J.J. et al. Characterization of a multicomponent receptor for GDNF. Nature 382, 80–83 (1996).

    CAS  Article  Google Scholar 

  25. 25

    Wrana, J.L. et al. TGF β signals through a heteromeric protein kinase receptor complex. Cell 71, 1003–1014 (1992).

    CAS  Article  Google Scholar 

  26. 26

    Boulanger, M.J., Chow, D.C., Brevnova, E.E. & Garcia, K.C. Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp130 complex. Science 300, 2101–2104 (2003).

    CAS  Article  Google Scholar 

  27. 27

    Dustin, M.L. et al. Low affinity interaction of human or rat T cell adhesion molecule CD2 with its ligand aligns adhering membranes to achieve high physiological affinity. J. Biol. Chem. 272, 30889–30898 (1997).

    CAS  Article  Google Scholar 

  28. 28

    Whitty, A. et al. Interaction affinity between cytokine receptor components on the cell surface. Proc. Natl. Acad. Sci. USA 95, 13165–13170 (1998).

    CAS  Article  Google Scholar 

  29. 29

    Bromley, S.K. et al. The immunological synapse and CD28–CD80 interactions. Nat. Immunol. 2, 1159–1166 (2001).

    CAS  Article  Google Scholar 

  30. 30

    Gavutis, M., Jaks, E., Lamken, P. & Piehler, J. Determination of the two-dimensional interaction rate constants of a cytokine receptor complex. Biophys. J. 90, 3345–3355 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Carmillo, P. et al. Glial cell line-derived neurotrophic factor (GDNF) receptor α-1 (GFRα1) is highly selective for GDNF versus artemin. Biochemistry 44, 2545–2554 (2005).

    CAS  Article  Google Scholar 

  32. 32

    Sadick, M.D. et al. Kinase receptor activation (KIRA): a rapid and accurate alternative to end-point bioassays. J. Pharm. Biomed. Anal. 19, 883–891 (1999).

    CAS  Article  Google Scholar 

  33. 33

    Coulpier, M., Anders, J. & Ibanez, C.F. Coordinated activation of autophosphorylation sites in the RET receptor tyrosine kinase: importance of tyrosine 1062 for GDNF mediated neuronal differentiation and survival. J. Biol. Chem. 277, 1991–1999 (2002).

    CAS  Article  Google Scholar 

  34. 34

    Fuh, G. et al. Rational design of potent antagonists to the human growth hormone receptor. Science 256, 1677–1680 (1992).

    CAS  Article  Google Scholar 

  35. 35

    Whitty, A. & Borysenko, C.W. Small molecule cytokine mimetics. Chem. Biol. 6, 107–118 (1999).

    Article  Google Scholar 

  36. 36

    Ilondo, M.M. et al. Receptor dimerization determines the effects of growth hormone in primary rat adipocytes and cultured human IM-9 lymphocytes. Endocrinology 134, 2397–2403 (1994).

    CAS  Article  Google Scholar 

  37. 37

    Matthews, D.J., Topping, R.S., Cass, R.T. & Giebel, L.B. A sequential dimerization mechanism for erythropoietin receptor activation. Proc. Natl. Acad. Sci. USA 93, 9471–9476 (1996).

    CAS  Article  Google Scholar 

  38. 38

    Kuzmic, P. Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal. Biochem. 237, 260–273 (1996).

    CAS  Article  Google Scholar 

  39. 39

    Sanicola, M. et al. Glial cell line-derived neurotrophic factor-dependent RET activation can be mediated by two different cell-surface accessory proteins. Proc. Natl. Acad. Sci. USA 94, 6238–6243 (1997).

    CAS  Article  Google Scholar 

  40. 40

    Simons, K. & Toomre, D. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31–39 (2000).

    CAS  Article  Google Scholar 

  41. 41

    Paratcha, G. & Ibanez, C.F. Lipid rafts and the control of neurotrophic factor signaling in the nervous system: variations on a theme. Curr. Opin. Neurobiol. 12, 542–549 (2002).

    CAS  Article  Google Scholar 

  42. 42

    Tansey, M.G., Baloh, R.H., Milbrandt, J. & Johnson, E.M., Jr. GFRα-mediated localization of RET to lipid rafts is required for effective downstream signaling, differentiation, and neuronal survival. Neuron 25, 611–623 (2000).

    CAS  Article  Google Scholar 

  43. 43

    Jencks, W.P. Binding energy, specificity, and enzymic catalysis: the circe effect. Adv. Enzymol. 43, 219–410 (1975).

    CAS  PubMed  Google Scholar 

Download references


We thank A. Rossomando and B.J. Gong for providing rat ART, C. Graf for providing the anti-GFRα3 antibodies GFRA3B and GFRA3N, J. Campos-Rivera for assistance with FACS measurements and E. Day for helpful discussions.

Author information




S.S. performed all of the experiments, data analysis, mathematical modeling and simulations, except for the experiment shown in Supplementary Figure 5, which was done by P.C. Experiments were designed and interpreted, and the manuscript was written, by S.S. and A.W., with substantial help and advice from P.C. throughout.

Note: Supplementary information is available on the Nature Chemical Biology website.

Corresponding author

Correspondence to Adrian Whitty.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Quantification of Ret and GFRα3 expression on NB41A3-mGFRα3 cells by FACS. (PDF 50 kb)

Supplementary Fig. 2

Contribution of ligand-dependent trafficking to the shape of the ART-dependent dose-response curves measured by KIRA ELISA. (PDF 48 kb)

Supplementary Fig. 3

Binding of blocking antibody GFRA3B to GFRα3 as measured by flow cytometry. (PDF 28 kb)

Supplementary Fig. 4

Plots of experimental receptor activation data with best fits to equilibrium models 1–4. (PDF 78 kb)

Supplementary Fig. 5

Comparison of Ret phosphorylation quantified by western blot and KIRA ELISA. (PDF 64 kb)

Supplementary Methods

Description of methods and representative input file for the DynaFit modeling. (PDF 39 kb)

Supplementary Data

Estimated parameters with standard errors for models 1–4 in the global fitting analysis. (PDF 45 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Schlee, S., Carmillo, P. & Whitty, A. Quantitative analysis of the activation mechanism of the multicomponent growth-factor receptor Ret. Nat Chem Biol 2, 636–644 (2006). https://doi.org/10.1038/nchembio823

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing