Science - USA (2022-02-18)

The near-uniform competence of Fz iso-
forms for Gpr124/Reck-dependent Wnt7a/b
signaling reveals that these ligands lose their
capacity to distinguish Fz receptors when
bound by Reck within the Gpr124/Reck/Fz/
Lrp5/6 complex. Because Wnt ligands discrim-
inate Fz receptors primarily via site 2 ( 15 ), we
suspected that Wnt7a/b activity at the BBB
might be dominated by site 1 of the N-terminal

domain (NTD). Supporting this hypothesis,

chimeric Wnt ligands composed of Wnt7a

NTD fused to a C-terminal domain (CTD)

derived from another Wnt were competent

for Reck binding and Gpr124/Reck-dependent

signaling (fig. S4) ( 19 , 21 ).

This suggested that in contrast to the sys-

temic“off-target”situation, where both con-

tact sites 1 and 2 are strictly required for Wnt

signaling, Wnt7a/b-Fz interaction at site 2

might become dispensable in the context of

Gpr124/Reck-stimulated signaling. Accord-

ingly, Wnt7aNTD, a hemisected Wnt7a variant,

retained partial activity on Gpr124/Reck/Fz1-

mediated signaling while showing no stimu-

lation of Fz5 signaling, used as a paradigm

“off-target”readout (Fig. 1B). Furthermore, mu-

tating three of the five Wnt7a residues involved

Martinet al.,Science 375 , eabm4459 (2022) 18 February 2022 2 of 11

Fig. 1. Engineering Wnt7a
ligands into highly specific
Gpr124/Reck activators.
(A) Backbone model of murine
Wnt7a based on the crystal
structure of XWnt8a ( 15 ) and
schematics of the receptor
complexes mediating
Wnt7a/b/b-catenin signaling.
(B) Relative STF (super
top flash) luciferase activity of
Wnt7a and its depicted var-
iants on Fz5 or Gpr124/Reck/
Fz1. (C) Position and color-coded
Gpr124/Reck/Fz1 or Fz5 STF
activities of 147 V5-tagged
single-residue Wnt7a variants.
Gpr124/Reck agonists (blue
dots) were defined as variants
with >70% Gpr124/Reck/Fz1
and <10% Fz5 activity. (D) Front,
lateral, and back views of the
Wnt7a surface model with
integrated color-coded activities
of all 147 single-residue
variants. Agonists are in blue.
(E)b-Catenin signaling assays
using STF cells transfected with
any of the 10 Fz receptors or
Gpr124/Reck, together with
Wnt7a, Wnt7aNTD, Wnt7aK190A,
or the empty pCS2 vector.
(F) Phenotypic scoring of stage
36 Xenopus laevisembryos
injected into the ventral vegetal
region of the four-cell embryo
with 15 pg of the indicated
mRNA. Arrowhead indicates
duplicated axis. (G) Phenotypic
scoring of 3-dpf zebrafish larvae
injected at the one-cell stage
with Wnt7a, Wnt7aNTD, or
Wnt7aK190AmRNA at the indi-
cated doses. Colored arrow-
heads refer to the phenotypic
classes on the right. Wnt7a and
variants are V5-tagged. Data
are means ± SD. ***P< 0.001.
Amino acid abbreviations: A, Ala;
E, Glu; F, Phe; I, Ile; K, Lys;
L, Leu; M, Met; P, Pro; R, Arg;
T, Thr; V, Val; W, Trp; Y, Tyr.

E

Relative luciferase activity

1

1.5

0.5

2

2.5

3

0

Wnt7a Wnt7aNTD Wnt7aK190A pCS2

Fz1

Fz2

Fz3

Fz4

Fz5

Fz6

Fz7

Fz8

Fz9

Fz10

Gpr124/Reck

pCS2

***

***

***

***

***

0 50 1000 50 1000 50 100

Fz5 Gpr124/Reck/Fz1

Relative^050100

luciferase

activity

(%)

***

Wnt7a Wnt7aS206A Wnt7aNTD Wnt7aCTD

NTD

ThumbSite1

Linker

domain CTD

PalmitoleicS206 S206A

acid

Site2Index

Wnt7a

NTD

Site1

Thumb

Linker

domain CTD

PalmitoleicS206

acid

Site2

Index

A

W322A

T266A

K229A

K231AV236A

K190A

K212A

I160A

K164A

V99AE239AY260A

P263A

F75A

Fz, Lrp5/6, Gpr124, Reck

Fz (site 1/2)+Lrp5/6

High (Wnt7a, Wnt7b)

Limited

+Reck

p p

Gpr124

Reck domainLinker

Fz, Lrp5/6

Fz (site 1/2)+Lrp5/6

Limited

Fz5, Fz8

Frizzled

(Fz)

Lrp5/6

p

systemic

"off-target"

BBB

"on-target"

Receptor complex

Wnt7 contacts

Wnt specificity

Frizzled specificity

Wnt7a/b

B

Wnt7a

F

G

51

11011021031101102103 RNA (pg)

56 23 67 40 9373 80 52 39 40

343843

29 98 99

Phenotype class (%)

Wnt7a Wnt7aNTDWnt7aK190A

Wnt7aWnt7a

NTD

1101102103

n= 43 36 98 50514145

20

0

40

60

80

100

Phenotype class (%)

n=

20

0

40

60

80

100

unaffected

eyeless

dysmorphic

unaffected

duplicated

axis

Wnt7a

Wnt7aNTD

Wnt7aK190A

Wnt7a Wnt7aNTD Wnt7aK190A

1 pg

10 pg

100 pg

1 pg

10 pg

100 pg

10 pg

100 pg

1 pg

Wnt7a

K190A

K190A

K273A

K95AK296A

K255A

CD

Gpr124/Reck/Fz1

relative luciferase activity

Gpr124/Reck/Fz1

Fz5

Wnt7a

Gpr124/Reck agonists (25)

Gpr124/Reck/Fz1 signaling

Agonists

Fz5 signaling

NTD

90°

NTD

Linker Linker

CTD CTD

147 Wnt7a single-residue variant positions 90°

Luciferase activity

Fz5 relative luciferase activity

0 31

SP V5

206238266278 349

pCS2

< 10% 10<35% 35<70% > 70%

W322A

E64A

M68A

R222A

T338A

I172A I59A L70A

E72A

F75A

T266AK190A

A58R

K164A

I160A

F162A

P263A

Y260A

R189AI172A

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