AbstractThe glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) is one of three neurotrophic factor families found in the nervous system. The GFLs, along with their receptors, play important roles in axon guidance, synapse formation, neuronal survival, neural regeneration, and are implicated in a number of neurological diseases.1 There are four GFLs: GDNF, Neurturin, Artemin, and Persephin. These ligands are known to bind to the co-receptors GFRα-1, GFRα-2, GFRα-3, and GFRα-4, respectively, through which they activate the common signal transducer, the receptor tyrosine kinase RET (Rearranged during Transformation).2 A fi fth co-receptor, GDNF Receptor-α-Like (GFRAL) was identifi ed in 2005, but the function of this orphan receptor is currently unknown.3 We have synthesized recombinant human GFRAL and tested the hypothesis that GFRAL interacts with known GFLs to change the dynamics of GFLs and their receptor networks. Both ELISA binding studies and cell-based assays were carried out using standard methods.4,5 Our results show that Neurturin binds to GFRAL with a high affi nity (EC50 = 8 ng/mL, N=4). Artemin binds to GFRAL with a low affi nity (EC50 = 2 mg/mL, N=2). GDNF and Persephin have only minimal or no binding to GFRAL (N=3). Addition of GFRAL proteins at a concentration of 0.5 µg/mL in cell culture media stimulated Neurturin-induced cell proliferation in the SH-SY5Y human neuroblastoma cell line (EC50 of Neurturin = 0.5 µg/mL, N=4). This effect of GFRAL is similar to that of GFRα-2 (EC50 of Neurturin = 1 µg/mL, N=4), although the maximal response by GFRAL is lower than that of GFRα-2. GFRα-2 dose-dependently increased the effect of GFRAL in stimulating the Neurturin-induced cell proliferation in SH-SY5Y cells (N=2). On the contrary, addition of GFRAL did not affect the stimulatory effect of GFRα-2 on Neurturin-induced stimulation of SH-SY5Y cell proliferation (N=2). This is the fi rst study to demonstrate that GFRAL interacts with GFLs, primarily Neurturin and Artemin, and can play a major role in GFLs and receptor networks involved in neuronal processes. These data also show that the biologically active recombinant GFRAL protein can be used to identify new GFRAL ligands and as a tool to investigate GDNF-related signaling mechanisms. The identifi cation of this novel interaction between GFRAL and GDNF family ligands will enhance our understanding of the mechanisms and applications of GLFs and their receptors in neurodegenerative and neuropsychiatric diseases.

IntroductionThe GFLs, which include GDNF, Neurturin, Artemin, and Persephin, have been shown to be critical for the proliferation, migration, and differentiation of several neuronal populations, as well as axon guidance and neurite branching, synaptogenesis, and adult synaptic plasticity. The GFLs signal through a multimolecular protein complex that includes receptors of the GFRα family and the RET receptor tyrosine kinase. Although some cross-talk exists, GFLs preferentially signal as homodimers through their cognate GFRα receptor. GFRα-1, GFRα-2, GFRα-3, and GFRα-4 are the co-receptors for GDNF, Neurturin, Artemin, and Persephin, respectively. Following ligand binding, the GFL-GFRα complexes associate with the extracellular domain of RET, which cause RET to dimerize, autophosphorylate, and stimulate the Ras/MAPK, PI 3-Kinase/Akt, and PLC/Ca2+ signaling pathways. Recently, a fi fth co-receptor for GFLs, termed GFRAL, has been identifi ed. However, the ligand and function of this receptor has yet to be determined. This study investigated a possible role of GFRAL in GFL-GFRα signaling.

Hypothesis: GFRAL interacts with known GFLs and may change the dynamics of GFLs and their receptor networks.

Signifi cance: Determination of such an interaction is important for better understanding the dynamics of GFLs and their receptors and for identifying the unknown ligand of GFRAL.

Results Summary• GFRAL binds to Neurturin with an EC50 in the order of ng/mL.

• GFRAL binds to Artemin with an EC50 in the order of µg/mL.

• GFRAL only weakly binds to GDNF.

• GFRAL does not bind to Persephin.

• GFRAL acts as a co-receptor enabling Neurturin-induced proliferation of SH-SY5Y human neuroblastoma cells.

• GFRAL-enabled-Neurturin-induced proliferation of SH-SY5Y human neuroblastoma cells is increased in the presence of GFRα-2.

Conclusion• GFRAL interacts with known GFLs, mainly, Neurturin and Artemin.

• Interaction between GFRAL and Neurturin enables Neurturin-induced proliferation of SH-SY5Y cells through the RET signaling pathway.

• Tissue expression of GFRAL may potentially affect local dynamics of GFLs and receptor networks.

• Identifi cation of interactions between GFRAL and GFLs should be helpful in guiding biochemical and bioinformatics effort to uncover the missing ligand of GFRAL.

GRLs Binding to GFRAL

Neurturin Binding to GFRα-2

Putative Interactions Between GFLs and GFRs

Update to GFLs and GFRs Interactions

GFRα-2 Stimulates Neurturin-Induced Cell Proliferation

GFRAL Affects Neurturin-Induced Cell Proliferation

Differential Interaction Between GDNF Receptor-α-like (GFRAL) Protein and GDNF Family Ligands on Binding and Neuroblastoma Cell Proliferation Assays

Guangjian Wang, Liwen Xiong, Tatsiana Gerassenkov, Vassili Kalabokis, Anthony Person | R&D Systems, Inc., 614 McKinley Place NE, Minneapolis, MN, 55413

RnDSy-lu-2945

For research use or manufacturing purposes only. PS_GWang SfN 2016_9624

Neurturin Binds GFRAL. Recombinant Human GFRAL was coated onto a microplate well at 1 µg/mL. Recombinant Human Neurturin (Catalog # 1297-NE) binds GFRAL in a dose-dependent manner (N=4).

Neurturin Binds GFRα-2. Recombinant Human GFRα-2 (Catalog # 613-FR) was coated onto a microplate well at 1 µg/mL. Recombinant Human Neurturin (Catalog # 1297-NE) binds GFRα-2 in a dose-dependent manner (N=4).

GFRAL Stimulates Neurturin-Induced Cell Proliferation. Proliferation of SH-SY5Y human neuroblastoma cells was assessed following treatment with increasing concentrations of Recombinant Human Neurturin (Catalog # 1297-NE) while in the presence of 0.5 µg/mL of Recombinant Human GFRAL. Cultures also contained 10 µM of retinoic acid (N=4).

GFRα-2 Enhances the Effects of GFRAL on Neurturin-Induced Cell Proliferation. Proliferation of SH-SY5Y human neuroblastoma cells was assessed following treatment with increasing concentrations of Recombinant Human Neurturin (Catalog # 1297-NE) while in the presence of 0.5 µg/mL of Recombinant Human GFRAL (blue line). Addition of 0.2 µg/mL (red line) and 0.5 µg/mL (green line) of Recombinant Human GFRα-2 (Catalog # 613-FR) enhanced the effects of GFRAL on Neurturin-induced cell proliferation. Cultures also contained 10 µM of retinoic acid (N=2).

GFRAL Does Not Affect the Stimulatory Effects of GFRα-2 on Neurturin-Induced Cell Proliferation. Proliferation of SH-SY5Y human neuroblastoma cells was assessed following treatment with increasing concentrations of Recombinant Human Neurturin (Catalog # 1297-NE) while in the presence of 0.5 µg/mL of Recombinant Human GFRα-2 (Catalog # 613-FR; blue line). Addition of 0.2 µg/mL (red line) and 0.5 µg/mL (green line) of Recombinant Human GFRAL had no effect on the stimulatory effects of GFRα-2 on Neurturin-induced cell proliferation. Cultures also contained 10 µM of retinoic acid (N=2).

GFRα-2 Stimulates Neurturin-Induced Cell Proliferation. Proliferation of SH-SY5Y human neuroblastoma cells was assessed following treatment with increasing concentrations of Recombinant Human Neurturin (Catalog # 1297-NE) while in the presence of 0.5 µg/mL of Recombinant Human GFRα-2 (Catalog # 613-FR). Cultures also contained 10 µM of retinoic acid (N=4).

Artemin Binds GFRAL. Recombinant Human Artemin (Catalog # 2589-AR) was coated onto a microplate well at 1 µg/mL. Recombinant Human GFRAL binds Artemin in a dose-dependent manner (N=2).

GDNF Exhibits Minimal Binding to GFRAL. Recombinant Human GFRAL was coated onto a microplate well at 1 µg/mL and incubated with increasing concentrations of Recombinant Human GDNF (Catalog # 212-GD). There was minimally binding of GDNF to GFRAL (N=3).

Persephin Does Not Bind GFRAL. Recombinant Human Persephin (Catalog # 2388-PS) was coated onto a microplate well at 1 µg/mL and incubated with increasing concentrations of Recombinant Human GFRAL. Persephin did not bind to GFRAL (N=3).

117.02

KeyPSPN PersephinGDNF Glial Cell Line-Derived Neurotrophic FactorRET Rearranged during TransformationNRTN Neurturin

ARTN ArteminGFR GDNF Family ReceptorGFRAL GDNF Receptor-α-Like

KeyPSPN PersephinGDNF Glial Cell Line-Derived Neurotrophic FactorRET Rearranged during TransformationNRTN Neurturin

ARTN ArteminGFR GDNF Family ReceptorGFRAL GDNF Receptor-α-Like

Bind

ing

(Mea

n O.

D.)

3

2.5

2

1.5

1

0.5

0

Neurturin (µg/mL)0.10.010.0011e-4

NRTN

GFRα-4GFRα-1 GFRα-2 GFRα-3 GFRAL

RET

Signaling

?

GDNFPSPN ARTN ?

NRTN

GFRα-4GFRα-1 GFRα-2 GFRα-3 GFRAL

RET

Signaling

GDNFPSPN ARTN ?

Bind

ing

(Mea

n O.

D.)

1.31

2.5

2

1.5

1

0.5

0

Neurturin (µg/mL)0.10.010.0011e-4

Cell

Prol

ifera

tion

(RFU

)

200

0

400

600

800

1200

1000

1400

Neurturin (µg/mL)1e-4 0.001 0.01 0.1 1 10 100

Cell

Prol

ifera

tion

(RFU

)

3000

2500

2000

1500

1000

500

0

Neurturin (µg/mL)1e-4 0.001 0.01 0.1 1 10 100

Neurturin (µg/mL)0.1

Cell

Prol

ifera

tion

(RFU

)

500

0

1000

1500

2000

2500

1e-4 0.001 0.01 1 10 100

Bind

ing

(Mea

n O.

D.)

1

0

2

3

GFRAL (µg/mL)1001010.1

Bind

ing

(Mea

n O.

D.)

3

2.5

2

1.5

1

0.5

0

GDNF (µg/mL)1010.10.01

Cell

Prol

ifera

tion

(RFU

)

200

0

400

600

800

1000

1200

1400

Neurturin (µg/mL)0.001 0.01 0.1 1 10 1001e-4

Bind

ing

(Mea

n O.

D.)

1

0

2

3

GFRAL (µg/mL)1001010.1

References1. Ibáñez, C.F. and J.O. Andressoo (2016) Neurobiol. Dis. [Epub ahead of print] doi:

10.1016/j.nbd.2016.01.021.

2. Wang, X. (2013) Biochim. Biophys. Acta 1834:2205.

3. Li, Z. et al. (2005) J. Neurochem. 95:361.

4. Hishiki, T. et al. (1998) Cancer Res. 58:2158.

5. Nishiuchi, R. et al. (2006) Matrix Biol. 25:189.

EC50=0.001–0.004 µg/mL

EC50=0.006–0.147 µg/mL

Neurturin : GFRAL

Artemin : GFRAL

GDNF : GFRAL

Persephin : GFRAL

EC50=2.22–3.36 µg/mL