sequence alignment of gdf15 from human, … figure 2 gdf15 decreases meal size but not meal ......

Supplementary Figure 1 Sequence alignment of GDF15 from human, cynomolgus monkey, and mouse. Sequence alignment of mature GDF15 from human (UniProt ID Q99988), cyno (RefSeq ID XP_005588497.1), and mouse (UniProt ID Q9Z0J7) and human GDNF (UniProt ID P39905) over a comparable region. Human sequence start site defined as in a previous report.36. Start for cyno and mouse sequences selected by similarity to human sequence. Alignment was colored with Jalview. Residues matching the consensus sequence are assigned a dark blue background; whereas residues differing from the consensus but with a positive Blosum62 score have a light blue background. Overall background intensity for a given column is adjusted based on the calculated conservation index at that position. Yellow boxes surround conserved cysteine residues. Similarity and identity matrices generated with the program MOE are shown to the right for the aligned GDF15 sequences. Black dots below the sequence of GDNF represent GDNF residues having any atom within 5 Å of GFRA1 in PDB 3FUB (chains B and A, respectively)28.

Nature Medicine: doi:10.1038/nm.4392

Supplementary Figure 2 GDF15 decreases meal size but not meal frequency in mice. (a) Meal number, (b) average amount consumed during each meal and the (c) average duration of meals in the six-hour period after a single subcutaneous administration of recombinant human or mouse GDF15 to lean mice (n = 7 per group). Data is reported as average ± s.e.m.. *P < 0.05 compared to PBS, one-way ANOVA with post hoc Tukey’s test.


Supplementary Figure 3 Serum concentration of HSA-GDF15 in male cynomolgus monkeys after a single subcutaneous administration. n = 8 per group. Data shown is average ± s.e.m..


Supplementary Figure 4 TGFβ receptor profiling upon GDF15 treatment using the PathHunterTM TGFβ family receptor dimerization assay. (a) Summary of receptor pairs tested for dimerization upon GDF15 treatment by PathHunterTM TGFβ family receptor dimerization assay (DiscoveRx). (b) Representative PathHunterTM TGFβ family receptor dimerization assay curve showing the dose dependent signal achieved when cells co-expressing ALK5 and TGFβII were treated with increasing concentrations of positive control TGFβ1 or GDF15. Data is shown as average ± s.d. of technical replicates.


Supplementary Figure 5 FACS analyses of hits identified in Retrogenix Membrane Protein Screen and receptors related to GFRAL. (a) Summary of the five membrane proteins identified as potential Fc-GDF15 binding partners in a Retrogenix Cell Microarray Screen of ~4493 membrane proteins. (b) Fc-GDF15 binding to HEK293 cells transiently expressing the potential GDF15 binding partners identified in a Retrogenix screen (listed in (a)) detected by FACS. Unstained cells are shown in solid gray, cells treated with Fc alone are shown in the dotted line and 40nM Fc-GDF15 exposed cells are represented by the black line. Histograms are representative of at least two independent experiments. Percentage of cells with Fc-GDF15 binding is listed above the black bar.


Supplementary Figure 6 Assessment of GDF15 binding to GFRAL related GDNF family receptors. (a) Sequence alignment about GFRAL D2 domain from human (UniProt ID Q6UXV0), cyno (UniProt ID G7P2W4), and mouse (UniProt ID Q6SJE0) and rat GFRA1 (UniProt ID Q62997) over a comparable region. Alignment was colored with Jalview. Residues matching the consensus sequence are assigned a dark blue background; whereas residues differing from the consensus but with a positive Blosum62 score have a light blue background. Overall background intensity for a given column is adjusted based on the calculated conservation index at that position. Yellow boxes surround conserved cysteine residues. Similarity and identity matrices are shown to the right for the aligned GFRAL sequences. Black dots below the sequence of GFRA1 represent GFRA1 residues having any atom within 5 Å of GDNF in PDB 3FUB (chains A and B, respectively)28. (b) Fc-GDF15 binding to HEK293 cells transiently expressing GDNF family receptors, GFRA1-4 detected by FACS. Unstained cells are shown in solid gray, cells treated with Fc alone are shown in the dotted line and 40nM Fc-GDF15 exposed cells are represented by the black line. Histograms are representative of at least two independent experiments. Percentage of cells with Fc-GDF15 binding is listed above the black bar.


Supplementary Figure 7 Gfral expression in mice. (a) Gfral expression in the central nervous system of wild-type, Gfral+/- and Gfral-/- mice determined by qPCR. (n = 3 WT, 6 Gfral+/- and 5 Gfral-/-, data is shown as average ± s.e.m.). (b) In situ hybridization of Gfral in a sagittal section of mouse brain at postnatal day 56 reported in the Allen Mouse Brain Atlas39. Black arrow indicates area postrema. Image can be found at http://mouse.brain-map.org/gene/show/126377.


http://mouse.brain-map.org/gene/show/126377

http://mouse.brain-map.org/gene/show/126377

Supplementary Figure 8 Expression of Gfral in humans. (a) Gfral expression in a panel of human tissues determined by qPCR. Data is reflective of one pool of each tissue and is shown relative to PPIA. (b) Gfral expression determined by a query of publicly available RNAseq data at the GTEx Portal. (c) Anti-GFRAL antibody immuno-reactive neurons detected in the area postrema (AP) and nucleus of the solitary tract (NTS) of human medulla. Inset on bottom left shows a neuron in that panel at higher magnification. IgG and no primary incubation samples serve as negative controls.


Supplementary Figure 9 Targeted deletion of Gfral in mice. Lexicon targeting strategy reported for generation of Taconic Biosciences Model TF3754 resulting in Gfral-/- mice used in these studies. Red text refers to the identification and location of the primers used to verify genetic targeting.


Supplementary Figure 10 Homology modeling of GDF15 and GFRAL interaction.

Homology models of GDF15 (grey and tan surface), GFRAL D1 domain (pink cartoon,

labeled) and GFRAL D2/D3 domains (pink cartoon, labeled) generated using as template

structures BMP-6 from PDB 2QCW25, GFRA1 D2 domain from PDB 3FUB28, and the

GFRA1 D2/D3 domains from PDB 3FUB, respectively, are shown following structural

alignment to and replacement of GDNF and GFRA1 in an EM and SAXS-based model of

the GDNF : GFRA1 : RET ternary complex (PDB 4UX829). GDF15 was subsequently

translated 3 Å along the GDF15 two-fold axis in the direction of the schematically-drawn

membrane to partially relieve clash with GFRAL as modeled. RET cadherin-like domains

(CLDs) 1-4 are as modeled in PDB 4UX8 (green cartoon labeled). Green ellipsoids

represent the RET C-terminal cysteine-rich domain (CRD) (scale not implied) and

approximate regions of EM density that were proposed previously29 to correspond to the

same. Stalk, transmembrane, and intracellular regions were added in Coot40 or as CGO

elements in Pymol (The PyMOL Molecular Graphics System, Version 1.7 Schrödinger,

LLC) and are not intended to indicate conformation. GDF15 residues I89 (blue surface,

labeled) and W32 (orange surface, labeled) are centrally located on surfaces anticipated

to be involved in GFRAL and RET engagement, respectively.


a.

b.

Supplementary Figure 11 Full scans of western blots in (a) Fig. 5d and (b) Fig. 5e.

Empty Vector GFRAL

198--

98--

62--

49--

38--

28--

14--

GFRAL Empty Vector GFRAL

198--

98--

62--

49--

38--

28--

14--

RET

Empty Vector GFRAL

198--

98--

62--

49--

38--

28--

14--

6--

pRET

Empty Vector GFRAL

198--

98--

62--

49--

38--

28--

14--

pERK1/2

Empty Vector GFRAL

198--

98--

62--

49--

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pAKTEmpty Vector GFRAL

198--

98--

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pPLCg

62--

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b-actin

49--

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GFRAL

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pPLCg

pERK1/2

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pAKT

62--

49--

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b-actin


a.

b.

c.

Supplementary Figure 12 Full scans of western blots in (a) Fig. 6a, (b) Fig. 6b and (c) Fig. 6c.

98--

62--

49--

38--

28--

14--

Empty Vector GFRAL

siNTC

siRET

+++ +- -- - - - ++

GFRAL

PBS NRTN PBS GDF15

62--

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+++ +- -- - - - ++

RET

b-actin

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+++ +- -- - - - ++

pAKT

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SPP86

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GFRAL

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SPP86

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b-actin

Empty Vector GFRAL

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Total AKT

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RET

111--

210--

71--

55--

41--

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GFRAL


Supplementary Figure 13 Summary of Gating Strategy used in flow cytometry experiments. (a) Cells were first selected based on size and cellular composition using forward (FSC-A) vs. side scatter pulse area (SSC-A) (gate P1), (b, c) aggregates and doublets were excluded based on forward (b) and side (c) scatter pulse height vs. their respective width parameters (FSC-H vs. FSC-W and SSC-H vs. SSC-W, gates P2 and P3), (d) dead cells were further excluded by 4’,6’-diamidino-2-phenylindole (DAPI) staining, and (e) Fc-GDF15 fusion protein binding positive population was gated based on the Alexa Fluor 647 signal of the isotype-matched Fc-alone protein as a negative control. Gating (a) to (e) was applied to all datasets, expect for datasets for Supplementary Fig. 5b where gating (d) was omitted because live/dead DAPI stain was not performed.


Supplementary Table 1 qPCR of Gfral in human tissues. Data is reflective of one pool of each tissue and reported as cycle threshold (Ct).

Sample Name Tissue Source PPIA CT Gfral CT

Testis Pooled from 7 Asians/Caucasians, ages: 24-87 22.233 24.432

Adipose Pooled from 18 male/female Caucasians, ages: 22-61;

cause of death: sudden death 22.828 25.674

Fetal Brain Pooled from 21 spontaneously aborted male/female

Caucasians, ages: 26-40 weeks 20.651 32.845

Smooth Muscle Pooled from 4 Asian males, ages: 21-36 22.724 33.458

Colon Pooled from 3 male Asians, ages: 24-29 23.021 33.784

Thymus Pooled from 2 male Caucasians, ages: 18-57 21.993 34.602

Skeletal Muscle 20-year-old male Caucasian 24.815 34.643

Small Intestine Pooled from 5 male/female Caucasians, ages: 20-61 22.547 34.957

Stomach 50-year-old male Caucasian 23.976 35.269

Fetal Liver Pooled from 3 male/female Asians, ages: 20-38 weeks 23.621 35.504

Bone Marrow Pooled from 4 male/female Caucasians, ages: 58-76 25.255 35.616

Spleen Pooled from 15 male/female Caucasians, ages: 22-69 23.898 35.713

Adrenal Gland Pooled from 62 male/female Caucasians, ages: 15-61 24.356 35.866

Prostate Pooled from 12 Caucasians, ages: 20-58 23.194 36.875

Brain (whole) Pooled from 4 male Asians, ages: 21-29 22.468 37.765

Uterus Pooled from 8 Caucasians, ages: 23-63 24.752 37.788

Lung Pooled from 3 male/female Caucasians, ages: 32-61 23.898 38.003

Salivary Gland Pooled from 24 male/female Caucasians, ages: 16-60 25.050 38.291

Heart Pooled from 3 male Caucasians, ages: 30, 30, 39 24.465 38.876

Kidney 40-year-old female Caucasian 21.793 Undetermined

Liver Pooled from 3 male Asians, ages: 24-64 24.048 Undetermined

Placenta Pooled from 3 Caucasians, ages: 23-30 23.899 Undetermined

Pancreas Pooled from 1 male Caucasian, age 35, cause of death:

sudden death 23.736 Undetermined


Supplementary Table 2 Metabolic characteristics of lean Gfral-/- mice. Data is reported as average ± s.e.m., n = 18 WT and 14 Gfral-/- for both males and females at 3 mos., n = 26 WT and 27 Gfral-/- for 6 mo. female body weight, n = 25 WT and 28 Gfral-/- for 6 mo. male body weight and n = 24 WT and 21 Gfral-/- for 6 mo. male food intake. P values were obtained using an unpaired t test.

Male Female

Age Parameter WT Gfral-/- P value WT Gfral-/- P value

3

mos.

Body weight (g) 27.4 ± 0.6 24.9 ± 0.5 0.0075 19.9 ± 0.3 19.8 ±

0.3 0.9024

Body length (mm) 97.33 ± 0.6 94.9 ± 0.6 0.0071 92.4 ± 0.6 92.3 ±

0.6 0.8559

Fed Blood Glucose

(mg/dL) 156.3 ± 5.3 160.1 ± 6.2 .06351

134.7 ±

4.3

127.8 ±

2.8 0.1729

OGTT – Glucose

∆AUC(0-120min)

7818 ±

962.1

8238 ±

1480.0 0.8061

3368 ±

684.1

4582 ±

790.6 0.2535

OGTT – Glucose

Total AUC(0-120min)

26208 ±

919.6

26035 ±

1506 0.9210

21198 ±

632.6

21206 ±

891.5 0.9939

Fat mass (g) 3.4 ± 0.4 2.7 ± 0.2 0.1016 2.4 ± 0.1 2.5 ± 0.1 0.6321

Fat mass (% of

body) 12.2 ± 1.0 10.6 ± 0.7 0.2394 12.1 ± 0.6

12.5 ±

0.6 0.6258

Lean mass (g) 22.5 ± 0.3 20.7 ± 0.4 0.0029 16.1 ± 0.3 15.9 ±

0.2 0.5870

Lean mass (% of

body) 82.3 ± 1.0 83.2 ± 0.9 0.5158 81.0 ± 0.5

80.2 ±

0.6 0.3065

24 hr food intake (g) 3.9 ± 0.2 3.6 ± 0.2 0.4318 4.7 ± 0.3 4.3 ± 0.2 0.2605

6

mos.

Body weight (g) 32.4 ± 0.7 32.1 ± 0.7 0.7545 27.2 ± 0.8 26.7 ±

0.5 0.6221

24 hr food intake (g) 4.4 ± 0.1 4.4 ± 0.2 0.9894 ND ND n/a


Supplementary Table 3 Expression of Gfral and related genes in multiple cell lines determined by qPCR. PPIB (Cyclophilin B) serves as a housekeeping gene indicative of relative cDNA concentration. Samples with Cts > 40 were considered negative for Gfral message. Cells with qPCR results of Cts < 37 were further tested for GFRAL protein by western blot analysis or by functional GFRAL activity by Fc-GDF15 binding in a FACS based assay. HEK-GFRAL Clone 4 is a stably transfected cell line overexpressing human Gfral that served as a positive control in these experiments. nt = not tested. Neg = no signal, Pos = positive signal.

Cycle Threshold (Ct) for designated genes

Cell Line PPIB GFRAL RET GDF15 GFRA1 GFRA2 GFRA3 GFRA4 Western Binding

HEK-293 21.0 >40 31.5 28.6 27.3 28.1 30.5 38.1 Neg Neg

HEK-GFRAL Clone 4 21.0 20.3 32.0 30.1 25.4 27.9 29.3 >40 Pos Pos

1321-N1 19.8 34.8 >40 29.5 33.0 35.3 37.7 >40 Neg Neg

LNCaP 20.1 >40 34.2 20.1 37.1 35.7 34.7 >40 nt nt

Daudi 21.9 >40 35.7 34.5 >40 33.7 36.0 >40 nt nt

MIA PaCa-2 19.9 >40 30.7 28.0 >40 34.7 32.1 >40 nt nt

SK-N-AS 18.5 >40 20.8 26.6 31.8 21.9 31.6 >40 Neg Neg

SK-BR-3 20.7 >40 37.1 27.4 34.0 32.7 38.8 >40 nt nt

DU-145 22.1 >40 33.1 28.9 35.3 32.3 32.8 >40 Neg nt

HT-29 20.1 >40 >40 23.2 >40 >40 37.1 >40 nt nt

MDA-MB-435 19.9 >40 >40 21.6 34.8 38.8 33.7 >40 Neg nt

SW-480 20.9 35.1 35.2 24.0 36.3 34.5 30.6 >40 nt Neg

SW-620 20.2 >40 39.9 25.3 >40 38.2 26.0 >40 nt nt

RPTEC 21.1 >40 34.8 23.6 21.6 34.8 35.0 >40 nt nt

H2052 21.0 >40 28.8 23.9 23.4 35.2 32.5 >40 nt nt

HepG2 21.1 >40 >40 22.2 >40 >40 32.2 >40 nt nt

KLE 21.6 36.4 35.2 28.8 21.5 34.2 29.4 >40 Neg nt

H2066 20.6 37.7 37.4 22.1 25.0 35.8 33.3 >40 nt nt

HeLa 18.3 >40 >40 25.7 39.9 39.1 33.9 >40 Neg nt

THP-1 18.4 >40 25.9 28.2 38.4 30.6 32.0 >40 Neg nt

SU-DHL-6 20.2 >40 >40 34.3 >40 34.7 31.3 >40 Neg nt

HUH-7 18.2 >40 >40 22.1 24.7 >40 33.4 >40 Neg nt

PANC-1 18.5 >40 26.2 20.8 33.7 30.9 30.2 >40 Neg nt

MCF-7 17.0 >40 23.1 18.9 17.3 25.7 27.0 >40 Neg nt

Colo205 15.7 >40 31.4 20.2 >40 >40 28.8 >40 Neg nt

Caco-2 20.1 34.2 34.7 24.0 37.4 34.6 28.0 >40 Neg nt

DLD1/FFAR1 21.0 >40 36.2 19.1 >40 38.7 35.4 >40 Neg nt

A431 20.8 >40 35.7 25.7 39.7 >40 35.3 >40 Neg nt

SK-N-SH 20.4 >40 25.0 24.6 28.3 27.0 29.7 >40 Neg nt

SK-N-DZ 21.0 >40 25.4 32.1 25.8 20.7 25.9 >40 Neg nt

SK-N-FI 20.5 >40 21.8 27.3 25.3 22.0 31.4 >40 Neg nt

T98 19.3 37.5 33.1 26.1 23.6 30.2 33.5 37.5 nt nt

U118MG 19.5 37.4 39.1 25.6 35.4 31.1 35.2 36.6 nt nt


Supplemental References:

39. Lein, E.S., et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature

445, 168-176 (2007). 40. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta

crystallographica. Section D, Biological crystallography 60, 2126-2132 (2004).


sequence alignment of gdf15 from human, … figure 2 gdf15 decreases meal size but not meal ......

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