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Biochem.
J.
(1990)
270,
383-390
(Printed
in
Great
Britain)
Receptors
for
insulin
and
insulin-like
growth
factor-I
can
form
hybrid
dimers
Characterisation
of
hybrid
receptors
in
transfected
cells
Maria
A.
SOOS,*
Jonathan
WHITTAKER,t
Reiner
LAMMERS,t
Axel
ULLRICHt
and
Kenneth
SIDDLE*§
*
Department
of
Clinical
Biochemistry,
University
of
Cambridge,
Addenbrookes
Hospital,
Hills
Road,
Cambridge
CB2
2QR,
U.K.,
tDivision
of
Endocrinology,
Department
of
Medicine,
SUNY
at
Stony
Brook,
Stony
Brook,
NY
11794-8154,
U.S.A.,
and
I
Department
of
Molecular
Biology,
Max-Planck
Institut
fur
Biochemie,
8033
Martinsried,
Federal
Republic
of
Germany
We
have
demonstrated
the
formation
of
hybrid
insulin/insulin-like
growth
factor-I(IGF-I)
receptors
in
transfected
rodent
fibroblasts,
which
overexpress
human
receptors,
by
examining
reactivity
with
species-
and
receptor-specific
monoclonal
antibodies.
In
NIH
3T3
and
Rat
1
fibroblasts,
endogenous
IGF-I
receptors
were
unreactive
with
anti-(human
insulin
receptor)monoclonal
antibodies
(47-9,
25-49,
83-14,
83-7,
18-44).
However,
in
transfected
cells
expressing
high
levels
of
insulin
receptors,
60-80
%
of
high-affinity
IGF-I
receptors
reacted
with
these
antibodies,
as
assessed
either
by
inhibition
of
ligand
binding
in
intact
cells
or
by
precipitation
of
solubilized
receptors.
Conversely,
endogenous
insulin
receptors
in
NIH
3T3
cells
were
unreactive
with
anti-(IGF-I
receptor)
antibodies
aIR-3
and
16-13.
However,
approx.
50%
of
high-affinity
insulin
receptors
reacted
with
these
antibodies
in
cells
expressing
high
levels
of
human
IGF-I
receptors.
The
hybrid
receptors
in
transfected
cells
bound
insulin
or
IGF-I
with
high
affinity.
However,
responses
to
these
ligands
were
asymmetrical,
in
that
binding
of
IGF-I
inhibited
subsequent
binding
of
insulin,
but
prior
binding
of
insulin
did
not
affect
the
affinity
for
IGF-I.
The
existence
of
hybrid
receptors
in
normal
tissues
could
have
important
implications
for
metabolic
regulation
by
insulin
and
IGF-I.
INTRODUCTION
Insulin
and
insulin-like
growth
factor-I
(IGF-I)
exert
their
biological
effects
by
binding
to
specific
plasma
membrane
receptors
which
show
many
similarities
of
structure
and
function.
Each
receptor
is
synthesized
initially
as
a
proreceptor
polypeptide
which
is
processed
by
glycosylation
and
proteolytic
cleavage
to
give
a-
and
/3-subunits,
the
mature
and
functional
receptors
being
symmetrical
structures
of
two
disulphide-linked
(a,f)
units
(reviewed
in
Czech,
1985;
Rechler
&
Nissley,
1985;
Duronio
&
Jacobs,
1988;
Yarden
&
Ullrich,
1988).
The
a-subunit
is
extracellular
and
contains
the
ligand
binding
site.
The
transmembrane
fl-subunit
possesses
ligand-stimulated
tyrosine
kinase
activity
which
appears
to
be
an
essential
component
of
the
signalling
pathways
mediating
hormone
action
(reviewed
in
Rosen,
1987;
Zick,
1989).
The
cloning
and
sequencing
of
cDNAs
coding
for
the
respective
proreceptors
has
confirmed
that
receptors
for
insulin
and
IGF-I
are
the
products
of
separate
genes,
with
substantial
similarity
of
amino
acid
sequence
(Ullrich
et
al.,
1985,1986).
There
has
been
debate
as
to
the
basis
of
the
overlapping
but
apparently
distinct
biological
effects
of
insulin
and
IGF-I
(Froesch
et
al.,
1985).
These
might
be
a
consequence
of
inherently
different
signalling
capacities
of
the
respective
receptors,
or
of
differences
in
receptor
distribution
among
tissues
with
different
capacities
for
metabolic
response
to
a
given
signal.
Insulin
and
IGF-I
induce
the
phosphorylation
of
common
endogenous
substrates
for
the
receptor
tyrosine
protein
kinase
(Izumi
et
al.,
1987;
Kadowaki
et
al.,
1987).
No
differences
in
inherent
signalling
capacity
were
apparent
when
human
insulin
and
IGF-I
receptors
were
expressed
in
Chinese
hamster
ovary
cells
following
cDNA
transfection
(Steele-Perkins
et
al.,
1988).
However,
expression
of
normal
and
chimaeric
receptors
in
NIH
3T3
fibroblasts
revealed
differences
in
signalling
potential
of
the
insulin-
and
IGF-I-
receptor
cytoplasmic
domains,
at
least
for
stimulation
of
DNA
synthesis
(Lammers
et
al.,
1989).
It
has
recently
been
reported
that
in
human
tissues
and
cell
lines
expressing
both
insulin
and
IGF-I
receptors,
a
proportion
of
receptors
exists
as
hybrid
(a/Ja'/3')
structures
(Soos
&
Siddle,
1989;
Moxham
et
al.,
1989).
The
existence
of
hybrid
receptors
could
have
important
functional
consequences
if
such
structures
are
capable
of
binding
and
responding
to
both
insulin
and
IGF-I.
However,
other
work
has
suggested
the
existence
of
two
distinct
IGF-I
receptor
polypeptides
as
a
possible
basis
for
signalling
by
insulin
via
the
IGF-I
receptor
(Garofalo
&
Rosen,
1989;
Alexandrides
&
Smith,
1989).
The
availability
of
rodent
cell
lines
transfected
with
human
insulin-
or
IGF-I-receptor
cDNA
(Whittaker
et
al.,
1987;
McClain
et
al.,
1987;
Lammers
et
al.,
1989)
has
allowed
us
to
examine
the
consequences
for
receptor
assembly
when
one
type
of
receptor
is
overexpressed
relative
to
the
other.
We
have
demonstrated
conclusively
the
ability
of
insulin-
and
IGF-I-receptor
subunits
to
assemble
as
interspecies
hybrids
in
these
cells,
by
using
species-
and
receptor-
specific
monoclonal
antibodies.
We
have
also
been
able
to
Vol.
270
Abbreviations
used:
IGF-I,
insulin-like
growth
factor-I;
WGA,
wheat-germ
agglutin;
PEG,
poly(ethylene
glycol);
DMEM,
Dubecco's
modified
Eagle's
medium;
PBS,
phosphate-buffered
saline;
k.i.u.,
kallikrein-inactivating
unit;
rIR/hIGFR
and
hIR/rIGFR,
complexes
between
rodent
insulin
receptor
and
human
IGF-I
receptor,
and
human
insulin
receptor
and
rodent
IGF-I
receptor,
respectively.
§
To
whom
correspondence
should
be
addressed.
383
M.
A.
Soos
and
others
examine
some
of
the
ligand-binding
properties
of
these
hybrid
receptors,
and
to
show
that
both
insulin
and
IGF-I
are
bound
with
high
affinity.
EXPERIMENTAL
Materials
Bovine
insulin
(for
displacement
studies)
was
from
Sigma
Chemical
Co.,
Poole,
Dorset,
U.K.,
and
highly
purified
des-
amido-free
bovine
insulin
(for
iodination)
was
a
gift
from
Dr.
D.
Brandenburg,
Deutsches
Wollforschungsinstitut,
Aachen,
Germany. Recombinant
human
IGF-I
was
generously
provided
by
Ciba-Geigy,
Basle,
Switzerland.
Proteinase
inhibitors,
BSA,
methotrexate,
Sephadex
G-50
and
wheat-germ-agglutinin-
Sepharose
(WGA-Sepharose)
were
from
Sigma,
poly(ethylene
glycol)
(PEG)
6000
was
from
BDH
Chemicals,
Dagenham,
Essex,
U.K.
and
Na1251I
(IMS
30)
was
from
Amersham
Inter-
national,
Aylesbury,
Bucks.,
U.K.
Hydroxyapatite
was
pur-
chased
from
Bio-Rad
Laboratories,
Watford,
Herts.,
U.K.
All
tissue
culture
reagents
and
Geneticin
(G418
sulphate)
were
from
Gibco
Ltd.,
Paisley,
Scotland,
U.K.
Sheep
anti-(mouse
IgG)
antibodies
were
coupled
to
aminocellulose
to
obtain
immuno-
adsorbents
as
described
previously
(Soos
et
al.,
1986).
Radioiodinations
Mono-1251-insulin
with
a
specific
radioactivity
of
100-200
uCi/,#g
was
prepared
from
highly
purified
bovine
insulin
as
described
by
Linde
et
al.
(1981).
IGF-I
was
iodinated
to
a
specific
activity
of
50-150,1Ci/1sg
using
a
stoichiometric
chloramine-T
method
(Roth,
1975)
and
was
purified
by
gel
filtration
on
Sephadex
G-50
to
separate
125I-IGF-I
from
free
[125
]iodide.
Antibodies
Monoclonal
antibodies
specific
for
human
insulin
receptors
(Soos
et
al.,
1986)
were
purified
from
ascites
fluids
by
precipitation
with
(NH4)2SO4
followed
by
chromatography
on
hydroxyapatite
(Stanker
et
al.,
1985).
Monoclonal
antibody
16-13,
specific
for
human
IGF-I
receptors,
was
obtained
following
fusion
of
NSO
myeloma
cells
with
spleen
cells
from
a
mouse
immunized
with
mouse
fibroblasts
overexpressing
human
IGF-I
receptors
(IGF-I-R/3T3
cells)
using
standard
techniques
(Galfre
&
Milstein,
1981).
Monoclonal
antibody
aIR-3
(also
specific
for
human
IGF-I
receptors)
was
kindly
donated
by
Dr.
Steven
Jacobs
(Wellcome
Research
Laboratories,
Research
Triangle
Park,
NC,
U.S.A.).
Cell
culture
All
the
cell
lines
were
grown
in
medium
with
10
%
(v/v)
foetal
calf
serum,
penicillin
(100
units/ml)
and
streptomycin
(100
jug/ml).
Rat
1
cells
(untransfected)
(McClain
et
al.,
1987)
were
grown
in
Dulbecco's
modified
Eagle's
medium
(DMEM).
The
cell
lines
NIH
3T3
NEO
(mock-transfected),
NIH
3T3
HIR3.5
(Whittaker
et
al.,
1987)
and
IGF-I-R/3T3
(Lammers
et
al.,
1989)
were
routinely
grown
in
DMEM
plus
0.4
mg
of
Geneticin/ml.
HIRc-B
cells
(McClain
et
al.,
1987)
were
grown
in
F12/DMEM
(1:1,
v/v)
plus
500
nM-methotrexate
and
0.4
mg
of
Geneticin/ml.
Cultures
were
maintained
in
a
humidified
atmosphere
of
air/CO2
(19:
1)
at
37
'C.
For
the
1251-hormone
binding
studies,
cells
were
seeded
on
to
24-well
tissue
culture
plates
at
2
x
104
cells
per
well.
They
were
cultured
for
2
days
to
approximately
half
confluence
for
1251-insulin
binding
to
NIH
3T3
HIR3.5
and
HIRc-B
cells
and
for
125I-IGF-I
binding
to
IGF-I-R/3T3
cells,
and
for
3
days
to
confluence
for
all
other
binding
studies.
Receptor
preparations
Receptors
from
NIH
3T3
NEO,
NIH
3T3
HIR3.5
and
IGF-
I-R/3T3
cell
lines
were
partially
purified
from
approx.
3
x
108
cells.
After
washing
with
phosphate-buffered
saline
(PBS;
150
mM-NaCl/10
mM-sodium
phosphate,
pH
7.4),
cells
were
removed
from
flasks
by
scraping
into
PBS
containing
proteinase
inhibitors
[0.2
mM-phenylmethanesulphonyl
fluoride,
0.4
mg
of
benzamidine/ml,
1
4g
of
leupeptin/ml,
1
/g
of
pepstatin/ml,
1
4g
of
antipain/ml,
200
kallikrein-inactivating
units
(k.i.u.)
of
aprotinin/ml
and
0.2
mg
of
bacitracin/mll.
The
cell
pellet
after
centrifugation
(150
g,
7
min)
was
solubilized
for
1
h
at
4
°C
in
20
ml
of
0.05
M-Hepes,
pH
7.4,
containing
the
proteinase
in-
hibitors
and
1
%
(v/v)
Triton
X-100.
The
supernatant
after
centrifugation
(50000
g
for
30
min,
4
°C)
was
diluted
1:
1.5
with
0.05
M-Hepes,
pH
7.4,
containing
0.15
M-NaCl,
0.025
M-MgCl2
and
proteinase
inhibitors,
and
mixed
for
16-20
h
at
4
°C
with
1
ml
of
WGA-Sepharose.
The
WGA-Sepharose
was
then
washed
with
10-20
ml
of
0.05
M-Hepes,
pH
7.4,
containing
0.15
M-NaCl
and
0.1
%
Triton
X-100
before
elution
with
0.5
M-N-acetyl-
glucosamine
in
the
same
buffer.
The
single
protein
peak
was
pooled
for
use
in
assays.
Solubilized
Rat
1
and
HIRc-B
receptors
were
prepared
by
solubilizing
approx.
107
cells
in
1.5
ml
of
0.05
M-Hepes,-pH
7.4,
containing
the
proteinase
inhibitors
and
1
%
Triton
X-100
as
described
above.
This
Triton
X-100
extract
was
used
without
further
purification.
1251-Hormone
binding
assays
Cells
were
washed
twice
with
PBS
before
addition
of
insulin,
IGF-I
or
antibody
in
0.25
ml
of
modified
DMEM
(containing
25
mM-Hepes,
pH
7.8,
and
7
mM-sodium
bicarbonate)
supplemented
with
1
mg
of
BSA/ml
and
250
k.i.u.
of
aprotinin/ml.
After
30
min
at
4
°C,
0.05
ml
of
1251-insulin
or
125I.
IGF-I
(approx.
30000
d.p.m.)
was
added.
After
a
further
4
h,
cells
were
washed
twice
with
ice-cold
PBS
and
solubilized
with
0.03
%
SDS
for
determination
of
radioactivity.
Non-specific
binding
was
determined
in
the
presence
of
I
#sM-insulin
or
0.1
zM-
IGF-I
as
appropriate.
Binding
to
solubilized
receptors
was
performed
as
described
previously
(Soos
&
Siddle,
1989),
except
that
receptors
were
incubated
with
1251I-insulin
or
1251I-IGF-I
(approx.
30000
d.p.m.
in
a
total
volume
of
0.25
ml),
together
with
unlabelled
hormone,
for
18
h
at
4
'C.
Receptor-bound
radioactivity
was
determined
by
precipitation
with
PEG
6000
(Baron
&
Sonksen,
1982).
Non-
specific
binding
was
determined
in
the
presence
of
1
aM-insulin
or
0.1
,M-IGF-I
as
appropriate.
The
concentration
of
Triton
X-100
in
all
soluble
binding
assays
was
maintained
at
0.05
%.
Co-precipitation
of
receptor-1251-hormone
complexes
Assays
were
performed
as
described
in
Soos
et
al.
(1986)
by
preincubating
solubilized
receptors
with
1251-insulin
or
125I-IGF-I
(approx.
30000
d.p.m.
in
a
total
volume
of
0.1
ml)
for 18
h
at
4
'C
before
addition
of
0.1
ml
of
antibody
for
a
further
6
h.
To
establish
the
specificity
of
binding
of
labelled
hormones,
unlabelled
hormones
were
included
in
the
first
incubation.
Antibody-bound
radioactivity
was
determined
using
a
sheep
anti-(mouse
IgG)
adsorbent
as
previously
described
(Soos
et
al.,
1986).
Total
receptor-bound
radioactivity
was
measured
by
precipitation
with
PEG
6000
(Baron
&
Sonksen,
1982).
RESULTS
Relative
levels
of
insulin
and
IGF-I
receptors
in
cell
lines
Estimates
of
numbers
of
receptors
in
the
cell
lines
studied
are
summarized
in
Table
1.
These
values
take
no
account
of
whether
ligand
binding
is
to
homomeric
or
hybrid
receptors.
The
NIH
1990
384
Insulin/insulin-like
growth
factor-I
receptor
hybrids
Table
1.
Numbers
of
insulin
and
IGF-I-
receptors
in
transfected
cell
lines
Values
indicate
receptor
numbers
per
cell.
Data
are
taken
from
*Hoffman
et
al.
(1989),
t
McClain
et
al.
(1987),
Maegawa
et
al.
(1988)
and
t
Lammers
et
al.
(1989).
Rodent
receptors
were
deter-
mined
in
corresponding
untransfected
cells,
but
levels
were
very
similar
in
transfected
cells
where
measured.
l0-'
x
Receptor
no.
Rodent
receptors
Human
receptors
(untransfected
cells)
(transfected
cells)
Cell
line
Insulin
IGF-I
Insulin
IGF-I
NIH
3T3
HIR3.5*
<
3
180
3000
-
HIRc-Bt
1.7
120
1250
-
IGF-I-R/3T3T
5.4
3.4
-
1311
3T3
and
Rat
1
fibroblasts
transfected
with
insulin
receptor
cDNA
possess
a
moderately
high
number
of
endogenous
rodent
IGF-I
receptors
but
relatively
few
insulin
receptors.
The
NIH
3T3
subline
used
for
transfection
with
IGF-I
receptor
cDNA
clearly
differs
from
that
used
for
insulin
receptor
transfection
in
having
low
levels
of
endogenous
receptors
for
both
insulin
and
IGF-I.
The
levels
of
human
receptors
in
transfected
cells
greatly
exceeded
those
of
endogenous
receptors
in
all
cases.
The
NIH
3T3
HIR3.5
cell
line
(Ebina
et
al.,
1985;
Whittaker
et
al.,
1987)
was
transfected
with
a
cDNA
sequence
encoding
a
receptor
with
an
additional
12-amino-acid
segment
compared
with
that
ex-
pressed
in
the
Rat
1
HIRc-B
cells
(Ullrich
et
al.,
1985;
McClain
et
al.,
1987).
There
is
no
evidence
in
any
of
these
cells
that
type
II
IGF
receptors
(Froesch
et
al.,
1985)
contribute
significantly
to
125I-IGF-I
binding.
Thus
125I-IGF-I
binding
was
effectively
displaced
by
insulin,
albeit
at
high
concentrations,
as
expected
for
reactivity
with
'classical'
IGF-I
receptors.
Further,
type
II
receptors,
because
of
their
totally
different
structure
compared
with
insulin-
and
IGF-I-receptors,
would
not
be
expected
to
react
with
any
of
the
antibodies
used
in
this
study.
Binding
studies
with
intact
cells
overexpressing
human
insulin
receptors
Monoclonal
anti-(insulin
receptor)
antibodies
47-9
and
25-49
were
tested
for
effects
on
ligand
binding
to
normal
fibroblasts
(NIH
3T3,
Rat
1)
and
to
cells
transfected
with
human
insulin
receptor
cDNA.
These
antibodies
recognize
distinct
epitopes
on
the
human
insulin
receptor
and
inhibit
insulin
binding
(Soos
et
al.,
1986).
They
do
not
react
with rodent
insulin
receptors
(Soos
et
al.,
1986)
nor
with
human
IGF-I
receptors
in
IGF-I-R/3T3
cells
(results
not
shown).
As
expected,
therefore,
these
antibodies
had
no
effect
on
the
binding
of
IGF-I
(Fig.
1)
or
insulin
(results
not
shown)
to
mock-transfected
NIH
3T3
NEO
or
untransfected
Rat
1
cells.
The
IGF-I
binding
sites
behaved
as
a
homogeneous
population,
in
showing
inhibition
of
tracer
binding
by
low
concentrations
of
unlabelled
IGF-I
[concn.
giving
50
%
inhibition
of
binding
(IC50)
0.5-0.6
nM],
but
only
by
high
concentrations
of
insulin
(IC50
400-1000
nm,
Fig.
1).
The
level
of
IGF-I
binding
to
NIH
3T3
HIR3.5
and
HIRc-B
cells
was
similar
to
that
in
the
corresponding
untransfected
cell
lines.
However,
in
the
cells
expressing
human
insulin
receptors,
antibodies
47-9
and
25-49
inhibited
not
only
the
binding
of
insulin
(Figs.
2c
and
3c)
but
also
the
binding
of
IGF-I
(Figs.
2d
and
3d).
Maximum
inhibition
of
insulin
binding
was
95-98
%
and
of
IGF-I
binding
it
was
70-800%.
The
antibody
concentration-dependence
of
binding
inhibition
was
similar
for
both
ligands
with
antibody
25-49,
but
antibody
47-9
was
consistently
approximately
2-fold
less
potent
with
IGF-I
than
-
0
0
0
C
._
c
\
~~b
10-11
10-10
10-9
10-8
10-7
10-6
100-
80-
60-
40-
20-
10-11
10-10
10-9
10-8
10-i
10-6
Concentration
(M)
Fig.
1.
Inhibition
of
125I-IGF-I
binding
to
NIH
3T3
NEO
cells
Binding
of
labelled
IGF-I
to
cells
was
measured
in
the
presence
of
the
indicated
concentrations
of
unlabelled
insulin
(O),
IGF-I
(0),
47-9
(A\)
or
25-49
(A)
as
described
in
the
Experimental
section.
Data
points
are
the
means
of
duplicate
incubations
within
a
representative
experiment.
Specific
binding
is
expressed
as
a
per-
centage
of
that
in
the
absence
of
unlabelled
ligand.
Total
cell-bound
'25I-IGF-I
was
2.7
%
and
non-specific
binding
was
0.2
%
of
the
total
radioactivity.
Very
similar
results
were
obtained
with
Rat
1
fibroblasts.
'25I-IGF-I
binding
was
inhibited
by
low
concentrations
of
unlabelled
IGF-I
(IC50
0.5
nM)
and
high
concentrations
of
insulin
(IC50
400
nM),
whereas
antibodies
47-9
and
25-49
were
without
effect.
with
insulin.
Monovalent
Fab
fragments
of
antibodies
47-9
and
25-49
(10-100
nM)
inhibited
binding
of
both
IGF-I
and
insulin
to
the
same
maximum
extent
as
bivalent
antibodies.
Anti-(insulin
receptor)
antibodies
which
did
not
inhibit
insulin
binding
(e.g.
83-7)
were
without
effect
on
IGF-I
binding
(results
not
shown).
In
both
transfected
cell
types
the
binding
of
'25I-IGF-I
appeared
to
be
heterogeneous
in
terms
of
inhibition
by
unlabelled
insulin
(Figs.
2b
and
3b).
Approx.
75
%
of
IGF-I
binding
was
inhibited
by
low
concentrations
of
IGF-I
(IC50
0.5-1
nM)
but
only
by
high
concentrations
of
insulin
(IC50
approx.
800
nM).
Thus
most
of
the
IGF-I
binding
sites
still
showed
the
properties
expected
of
high-affinity
IGF-I
receptors,
as
in
the
untransfected
cells.
However,
a
fraction
(approx.
25
%)
of
IGF-I
binding
was
inhibited
by
low
insulin
concentrations
(IC50
approx.
0.2
nM)
and
was
assumed
to
reflect
low-affinity
binding
of
IGF-I
to
the
large
excess
of
insulin
receptors.
There
was
a
significant
difference
in
the
reactivity
of
IGF-I
with
insulin
receptors
in
the
two
different
transfected
cell
lines.
Under
the
conditions
of
these
experiments,
unlabelled
IGF-I
was
considerably
more
potent
at
inhibiting
tracer
binding
in
HIRc-B
cells
(IC50
approx.
20
nm,
Fig.
3a)
than
in
NIH
3T3
HIR3.5
cells
(IC50
>
100
nm,
Fig.
2a),
although
displacement
by
unlabelled
Vol.
270
385
M.
A.
Soos
and
others
(a)
0
b
0
0
%.I
--%
10-9
O
-___
100'
80'-
60-
40-
20
-
10-8
10-7
l'o-6
(c)
100
80
-
60-
40-
20-
0O
(b)
b
10-7
10-6
(d)
10-11
10-11
Concentration
(M)
Fig.
2.
Inhibition
of
'25I-hormone
binding
to
NIH
3T3
HIR
3.5
cells
Binding
of
labelled
insulin
(a,c)
and
IGF-I
(b,d)
to
cells
was
measured
in
the
presence
of
the
indicated
concentrations
of
unlabelled
insulin
(0),
IGF-I
(0),
47-9
(A)
or
25-49
(A)
as
described
in
the
Experimental
section.
The
means
+
S.E.M.
of
three
independent
experiments,
performed
in
duplicate,
are
shown.
Specific
binding
is
expressed
as
a
percentage
of
that
in
the
absence
of
unlabelled
ligand.
Total
cell-bound
radioactivity
was
36
+
2
%
('25I-insulin)
and
4.1
+
0.4
%
(125I-IGF-I),
and
non-specific
binding
was
0.23
+
0.13
%
(.251-insulin)
and
0.33
+
0.04
%
('25I-IGF-I)
of
the
total
radioactivity.
101
8
6
4
2
10-7
10-6
(c)
100
80
I
z
%%60
\t\
I-
8
\
40
I
-11
10-1°
10-9 10-8 10-7
16
20
10-11
-10
0
9
10-8
0-7
100
0
~~~~~~~~~~~(b)
0
-~~~~~~
50,
.
.
X
0
~~~~~~~~~%0
0o-
10-11
10-10
10-9
10-8
10-7
(d)
10-11
10-1Q
10-9
10-8
10-7
10-6
Concentration
(M)
Fig.
3.
Inhibition
of
l25l-hormone
binding
to
HIRc-B
cells
Binding
of
labelled
insulin
(a,c)
and
IGF-I
(b,d)
to
cells
was
measured
in
the
presence
of
the
indicated
concentrations
of
unlabelled
insulin
(0),
IGF-I
(-),
47-9
(A)
or
25-49
(A)
as
described
in
the
Experimental
section.
The
means
+
S.E.M.
of
three
independent
experiments
performed
in
duplicate
are
shown.
Specific
binding
is
expressed
as
a
percentage
of
that
in
the
absence
of
unlabelled
ligand.
Total
cell-bound
radioactivity
was
34+7.9%
(.251-insulin)
and
10.9
+
2.1
%
(125I-IGF-I),
and
non-specific
binding
was
0.16
+
0.07
%
(.251-insulin)
and
0.45
+
0.0
%
(125I-IGF-I)
of
the
total
radioactivity.
1990
100-
80
!
60
-
40
-
20-
0-1l
10-10
-
0
0
0)
~Ro
C
._
c
100-
80-
60-
40-
20-
10-11
100
0
a
U
0
4-
.n
100
c
80
60
40
20
60
10-6
386
"h
I
t
'A
-
----v
0i
6-9
i
.
8
i
.-7
10-6
i
;-lo
i
;-g
i
.-8
-1.-7
1
-6
10-11
10-10
10-9
10-8
Insulin/insulin-like
growth
factor-I
receptor
hybrids
(a)
0
-
-0
O
5
100
-
80
-
0
0
"S
'o
i
.
.
I
r
I
10-11
1
0-l°
1
0-9
1
0-8
10-7
10-6
60
-
40
-
20
-
(b)
0""
\
-
0O
_
I
Concentration
(M)
Fig.
4.
Inhibition
of
'l25l-ormone
binding
to
partially
purified
receptors
Binding
of
.25I-IGF-I
to
WGA-Sepharose-purified
NIH
3T3
HIR
3.5
receptors
(a)
and
of
.25I-insulin
to
WGA-Sepharose-purified
IGF-I-R/3T3
receptors
(b)
was
measured
in
the
presence
of
the
indicated
concentrations
of
unlabelled
insulin
(0)
or
IGF-I
(@)
as
described
in
the
Experimental
section.
Data
points
are
the
means
of
duplicate
incubations
within
a
representative
experiment.
Specific
binding
is
expressed
as
a
percentage
of
that
in
the
absence
of
unlabelled
ligand.
Total
receptor-bound
radioactivity
was
9.4
%
(a)
and
7.1
%
(b),
and
non-specific
binding
was
4
%
(a)
and
0.9%
(b).
insulin
was
very
similar
in
both
cell
types.
The
basis
of
the
apparently
higher
cross-reaction
of
IGF-I
with
human
insulin
receptors
expressed
in
Rat
1
compared
with
NIH
3T3
HIR3.
cells
is
unknown.
This
might
reflect
the
differences
in
recepto
sequence
or
differences
in
glycosylation
which
influence
binding
affinity
for
IGF-I
but
not
insulin.
The
conclusion
from
these
experiments
is
that
approx.
60-70
%
of
endogenous
rodent
IGF-I
receptors
became
reactive
with
anti-
(insulin
receptor)
specific
antibodies
when
a
10-20-fold
excess
of
human
insulin
receptors
was
co-expressed.
This
is
consistent
with
the
idea
that
a
large
fraction
of
IGF-I
binding
sites
were
in
hybrid
receptors
(a6z'fl')
in
which
binding
of
ligand
to
the
a'-
subunit
was
inhibited
by
binding
of
antibody
or
Fab
to
particular
epitopes
on
a.
In
other
respects,
including
affinity
for
IGF-I
and
insulin,
these
IGF-I
binding
sites
were
very
similar
to
IGF-I
receptors
in
untransfected
cells.
Binding
studies
with
solubilized
receptors
from
cells
overexpressing
human
insulin
receptors
Receptors
solubilized
and
partially
purified
from
transfected
cells
were
studied
in
order
to
permit
the
investigation
of
reactivity
with
antibodies
which
did
not
necessarily
inhibit
ligand
binding.
In
these
experiments,
125I-IGF-I
or
l25l-insulin
was
preincubated
with
receptor
before
determination
of
the
fraction
of
receptor-ligand
complexes
which
was
immunoprecipitable.
Solubilized
receptors
from
NIH
3T3
HIR3.5
and
HIRc-B
cells
behaved
similarly
to
those
from
the
intact
cells
in
terms
of
inhibition
of
1251I-ligand
binding
by
unlabelled
insulin
and
IGF-I.
The
different
potency
of
cross-reaction
of
IGF-I
with
insulin
receptors
from
the
two
cell
lines
was
still
apparent
after
solubilization.
However,
the
minor
component
of
1251I-IGF-I
binding
which
was
inhibitable
by
low
concentrations
of
insulin
in
intact
cells
was
not
apparent
with
solubilized
receptors
(Fig.
4a).
This
probably
reflects
the
fact
that
conditions
for
separation
of
bound
and
free
ligand
in
the
soluble
receptor
assays
(PEG
precipitation)
do
not
detect
the
low-affinity
component
of
IGF-I
binding
because
of
the
dissociation
during
precipitation
and
washing.
Binding
of
125I-IGF-I
in
this
assay
therefore
reflects
only
high-affinity
IGF-I
receptors
(IC50
0.4
nm
for
IGF-I
and
40-150
nm
for
insulin;
Fig.
4a).
Antibodies
for
three
different
insulin
receptor
epitopes
(83-7
and
83-14,
a-subunit;
18-44,
fl-subunit)
precipitated
not
only
receptor-'251-insulin
complexes
but
also
the
bulk
of
receptor-'25I-
IGF-I
complexes
from
solubilized
NIH
3T3
HIR3.5
and
HIRC-
B
cells
(Table
2).
None
of
the
antibodies
reacted
at
all
with
Table
2.
Reaction
of
antibodies
with
receptor-'251-hormone
complexes
WGA-Sepharose-purified
receptors
(NIH
3T3
HIR3.5
and
IGF-I-R/3T3
cells)
or
Triton
X-
100-solubilized
receptors
(HIRc-B)
were
preincubated
with
'25I-insulin
or
125I-IGF-I
before
addition
of
10
nM-antibody.
Total
and
immunoreactive
receptor-bound
radio-
activity
was
determined
as
described
in
the
Experimental
section.
In
the
absence
of
antibody,
total
specific
receptor-bound
'25I-insulin
was
2483
c.p.m.
(NIH
3T3
HIR3.5),
2521
c.p.m.
(HIRc-B)
and
1564
c.p.m.
(IGF-I-R/3T3),
and
total
receptor-bound
1251I-IGF-I
was
1492
c.p.m.
(NIH
3T3
HIR3.5),
1673
c.p.m.
(HIRc-B)
and
2370
c.p.m.
(IGF-I-R/3T3).
Values
are
the
means
of
duplicate
incubations
within
representative
experiments.
None
of
the
anti-
bodies
precipitated
receptors
from
control
cells
(NIH
3T3,
Rat
1)
which
do
not
express
human
insulin
or
IGF-I
receptors.
N.D.,
not
determined.
Immunoreactive
receptors
(%)
NIH
3T3
HIR3.5
HIRc-B
IGF-I-R/3T3
Antibody
Insulin
IGF-I
Insulin
IGF-I
Insulin
IGF-I
83-7
96
82
70
67
0
0
83-14
94
100
86
76
0
1
18-44
102
83
93
71
0
1
aoIR-3
0
0
0
2
44
85
16-13
0
N.D.
0
2
50
77
receptors
from
untransfected
cells
or
with
human
IGF-I
receptors
in
IGF-1-R/3T3
cells.
As
expected,
antibodies
acIR-3
and
16-13,
specific
for
distinct
epitopes
on
the
human
IGF-I
receptor,
did
not
react
with
rodent
IGF-I
receptors
in
either
untragsfected
or
transfected
cells
(Table
2).
It
was
concluded
that
a
substantial
fraction
of
rodent
IGF-I
receptors
in
transfected
cells
was
incorporated
into
hybrid
structures
which
were
then
reactive
with
the
whole
panel
of
antibodies
specific
for
human
insulin
receptors.
Binding
studies
with
solubilized
receptors
from
ceUs
overexpressing
human
IGF-I
receptors
The
level
of
specific
"25I-insulin
binding
to
intact
IGF-I-R/3T3
cells
was
too
low
to
permit
quantitative
studies.
Moreover,
a
significant
fraction
of
this
binding
appeared
to
reflect
sites
of
low
affinity,
in
terims
of
competition
by
unlabelled
insulin.
Solubilized
receptor
preparations
from
IGF-I-R/3T3
cells
could
be
studied
Vol.
270
100
C
0
0
0
4-
c
0.
m
C
80.
60
40
20
L4L---
-
-
I
387
10-11
10-10
10-9
10-8
10-7
10-6
M.
A.
Soos
and
others
more
readily
than
receptors
in
intact
cells
because
it
was
possible
to
achieve
greater
specific
binding
of
insulin,
both
in
absolute
terms
and
relative
to
non-specific
background.
Binding
of
'251-insulin
to
solubilized
receptors
was
inhibited
by
low
concentrations
of
unlabelled
insulin
(IC50
approx.
1
nM;
Fig.
4b),
whereas
binding
of
1251I-IGF-I
was
inhibited
only
at
much
higher
concentrations
(IC50
approx.
500
nM;
results
not
shown).
It
was
concluded
that
binding
of
1251-insulin
reflected
a
high-affinity
interaction
with
rodent
insulin
receptors,
and
not
low-affinity
binding
to
human
IGF-I
receptors.
Nevertheless,
binding
of
1251_
insulin
was
strikingly
inhibited
by
low
concentrations
of
IGF-I,
almost
identical
with
those
which
inhibited
1251I-IGF-I
binding
(IC50
0.1-0.2
nM,
Fig.
4b).
High-affinity
insulin
binding
in
extracts
of
mock-transfected
NIH
3T3
NEO
cells
was
inhibited
only
by
high
concentrations
of
IGF-1
(IC50>
100
nM;
results
not
shown).
The
immunoreactivity
of
receptors
solubilized
from
IGF-I-R/
3T3
cells
was
investigated
with
two
antibodies
specific
for
human
IGF-I
receptors:
aIR-3,
which
inhibits
insulin
binding
(Kull
et
al.,
1983),
and
16-13,
which
does
not
inhibit
binding.
Both
antibodies
precipitated
the
bulk
of
IGF-I
receptors
in
IGF-1-R/3T3
extracts,
as
expected.
However,
these
antibodies
also
precipitated
approx.
50
%
of
the
rodent
insulin
receptors
in
these
extracts
(Table
2),
although
they
were
unreactive
with
insulin
receptors
in
untransfected
cells.
Total
(PEG-precipitable)
1251-insulin
binding
under
the
conditions
of
these
experiments
was
decreased
by
antibody
aIR-3
and
increased
by
antibody
16-13
(700
c.p.m.
and
2339
c.p.m.
respectively
compared
with
the
control
value
of
1564
c.p.m.).
This
suggests
that.
aIR-3
accelerates
the
dissociation
of
receptor-bound
insulin,
whereas
16-13
increases
the
binding
affinity.
It
is
concluded
that
rodent
insulin
receptors
in
IGF-I-R/3T3
cells
were
substantially
incorporated
into
hybrid
structures
with
human
IGF-I
receptors.
These
hybrids
differed
from
normal
insulin
receptors
in
that
binding
of
1251-insulin
was
inhibited
by
low
concentrations
of
both
insulin
and
IGF-I.
DISCUSSION
Receptors
for
insulin
(/3aal/)
and
IGF-I
(/J'x'a'/J')
have
gen-
erally
been
considered
to
be
distinct
but
structurally
similar
symmetrical
heterotetramers
(Czech,
1985;
Rechler
&
Nissley,
1985;
Yarden
&
Ullrich,
1988).
Various
observations
have
indicated
that
there
may
be
subtypes
of
both
receptors
(Jonas,
1988),
but
the
structural
basis
and
functional
significance
of
this
heterogeneity
is
unclear.
Two
distinct
hypotheses
have
been
advanced.
We
have
proposed
that
a
significant
fraction
of
IGF-I
receptors
in
human
placenta
occurs
as
hybrids
with
insulin
receptors
(/aa'/J'),
based
on
reactivity
with
a
panel
of
anti-receptor
antibodies
(Soos
&
Siddle,
1989).
The
existence
of
hybrid
structures
was
suggested
independently
as
a
result
of
studies
of
IGF-I-induced
receptor
phosphorylation
in
Hep
G2
cells
(Moxham
et
al.,
1989).
However,
similar
autophosphoryl-
ation
woik
in
brain
(Garofalo
&
Rosen,
1989)
and
muscle
(Alexandrides
&
Smith,
1989)
was
interpreted
as
evidence
for
two
distinct
IGF-I
receptor
polypeptides
differing
in
primary
sequence
and/or
glycosylation
(and
therefore
in
immunological
recognition),
as well
in
developmental
regulation.
We
now
provide
conclusive
evidence
for
the
formation
in
intact
cells
of
hybrids
which
incorporate
subunits
of
receptors
for
both
insulin
and
IGF-I.
When
human
insulin
receptors
(hIR)
were
overexpressed
in
rat
or
mouse
fibroblasts
by
transfection
with
cloned
cDNA,
rodent
IGF-I
receptors
(rIGFR)
were
largely
incorporated
into
hybrid
structures
which
were
detected
by
their
reaction
with
multiple
anti-(human
insulin
receptor)
specific
monoclonal
antibodies
(Figs.
2
and
3;
Table
2).
Conversely,
when
human
IGF-I
receptors
(hIGFR)
were
overexpressed
in
similar
cells,
rodent
insulin
receptors
were
incorporated
into
hybrids
which
then
reacted
with
human
IGF-I-receptor-specific
antibodies
(Table
2).
In
NIH
3T3
HIR3.5
and
HIRc-B
cells,
hybrids
were
clearly
demonstrated
in
intact
as
well
as
solubilized
cells
by
the
criterion
of
inhibition
of
high-affinity
IGF-I
binding
by
monoclonal
antibodies
specific
for
insulin
receptors
(Figs.
2
and
3).
In
IGF-I-R/3T3
cells,
hybrids
were
demonstrated
by
immuno-
precipitation
of
solubilized
preparations
(Table
2),
although
little
high-affinity
insulin
binding
was
detected
in
intact
cells.
Hybrid
receptors
clearly
pre-exist
in
intact
cells
but
the
possibility
cannot
be
ruled
out
that
the
binding
specificity,
or
even
the
formation,
of
hybrids
is
influenced
by
solubilization.
In
previous
studies
with
placental
microsomal
membranes,
the
proportion
of
IGF-
I
receptors
appearing
as
hybrids
appeared
higher
in
solubilized
than
in
particulate
preparations
(Soos
&
Siddle,
1989).
It
has
recently
been
demonstrated
that
hybrid
insulin/IGF-I
receptors
can
be
assembled
in
vitro
from
the
respective
a,z
receptor
halves,
although
this
required
the
presence
of
the
respective
ligands
or
of
Mg
ATP
(Treadway
et
al.,
1989).
Hybrid
receptors
displayed
a
high
affinity
for
both
insulin
and
IGF-I,
as
studied
with
the
rIR/hIGFR
and
rIGFR/hIR
hybrids
respectively
(Fig.
4).
However,
comparison
of
ligand
binding
to
these
two
hybrids
revealed
a
striking
asymmetry
in
properties.
The
rIGFR/hIR
hybrids
behaved
similarly
to
homomeric
IGF-I
receptors,
in
that
binding
of
1251-IGF-I
was
inhibited
by
low
concentrations
of
IGF-I
but
only
by
high
concentrations
of
insulin.
In
contrast,
binding
of
251I-insulin
to
rIR/hIGFR
hybrids
was
inhibited
by
low
concentrations
of
both
insulin
and
IGF-I
(Fig.
4).
It
is
unknown
in
either
case
whether
the
unlabelled
ligands
remain
bound
together
with
251I-labelled
ligand
and
therefore
whether
hybrid
receptors
can
bind
both
insulin
and
IGF-I
simultaneously.
It
is
possible
that
the
different
properties
of
rIR/hIGFR
compared
with
hIR/rIGFR
reflects
the
species
asymmetry.
However,
it
is
tempting
to
speculate
that
the
differences
in
the
properties
of
the
two
hybrids
are
a
consequence
of
asymmetry
of
ligand-receptor
interactions
common
to
both
structures.
This
would
imply
that
the
insulin-IR/IGFR
complex
retained
a
high
affinity
for
IGF-I,
whereas
the
IR/IGFR-IGF-I
complex
no
longer
had
a
high
affinity
for
insulin.
It
has
been
shown
that
the
homomeric
insulin
receptor
binds
only
one
molecule
of
insulin
with
high
affinity,
this
interaction
converting
the
unoccupied
site
to
a
low-affinity
state
(Pang
&
Shafer,
1984;
Boni-Schnetzler
et
al.,
1987;
Sweet
et
al.,
1987).
The
homomeric
IGF-I
receptor
may
behave
differently
in
binding
two
molecules
of
ligand
with
high
affinity
(Feltz
et
al.,
1988),
although
this
is
not
certain
(Tollefsen
&
Thompson,
1988).
A
model
can
be
proposed
in
which
the
unoccupied
half
of
a
hybrid
receptor
displays
the
same
kinetic
properties
as
in
the
corresponding
homomeric
receptor
(Fig.
5).
It
therefore
appears
that
the
interaction
between
heterologous
receptor
halves
is
sufficient
not
only
to
allow
their
covalent
association
but
also
to
permit
inter-
subunit
conformational
transitions.
Further
evidence
for
inter-subunit
interactions
is
provided
by
the
observation
that
the
anti-(IGF-I
receptor)
antibody
aIR-3
accelerated
dissociation
of
insulin
from
hybrid
receptors,
whereas
the
anti-(insulin
receptor)
antibody
25-49
accelerated
dissociation
of
IGF-I
(M.
A.
Soos
&
K.
Siddle,
unpublished
work).
Antibody
47-9
consistently
showed
a
lower
affinity
for
hybrid
receptors
than
for
homomeric
receptors
in
transfected
cells
(Figs.
2
and
3),
as
in
placenta
(Soos
&
Siddle,
1989).
This
suggests
that
the
conformation
of
the
insulin
receptor
a-subunit
may
differ
somewhat
in
the
two
types
of
receptor.
It
is
perhaps
surprising
that
three
distinct
insulin
receptor
antibodies
47-9,
25-49
and
(results
not
shown)
83-14
all
inhibited
binding
of
IGF-I
to
hybrids
not
only
as
bivalent
antibodies
but
also
as
Fab
1990
388
Insulin/insulin-like
growth
factor-I
receptor
hybrids
H
H
H
H
H H
H
H
Empty
QOOQW
IR
IR
IGFR
IR
IR
IGFR
IGFR
IGFR
LL
HH
Filled
W
oOlc1
IR
IR
IGFR
IR
IR
IGFR
IGFR
IGFR
Fig.
5.
Schematic
representation
of
the
affinity
of
ligand
binding
sites
Homomeric
(IR.IR,
IGFR.IGFR)
and
hybrid
(IGFR.IR,
IR.IGFR)
receptors
are
represented
in
the
empty
state
and
with
one
site
occupied
by
insulin
(0)
or
IGF-I
(-).
Affinities
of
the
empty
sites
are
represented
as
high
(H)
or
low
(L).
homomeric
human
receptors.
However,
in
Hep
G2
and
NIH
3T3
cells
it
appeared
that
low
concentrations
of
IGF-I
stimulated
autophosphorylation
of
both
fl-subunits
within
putative
hybrids,
although
insulin
was
less
potent
(Moxham
et
al.,
1989).
Interestingly,
NIH
3T3
HIR3.5
cells
show
enhanced
responses
not
only
to
insulin
but
also
to
IGF-I,
concomitant
with
overexpression
of
insulin
receptors
(Hofmann
et
al.,
1989).
This
suggests
that
some
effects
of
IGF-I
might
be
signalled
more
efficiently
via
hybrid
receptors
than
via
homomeric
IGF-I
receptors.
However,
the
full
physiological
implications
of
the
finding
that
insulin
and
IGF-I
receptors
behave
as
isoenzymes
which
can
combine
to
form
hybrid
as
well
as
homomeric
structures
require
further
investigation.
Studies
with
cells
co-
transfected
with
cDNAs
for
both
insulin
and
IGF-I
receptors
may
shed
light
on
this
issue,
and
on
the
relative
efficiency
of
formation
of
hybrid
and
homomeric
structures.
fragments.
It
is
unclear
whether
steric
factors
alone
could
account
for
this
inhibition,
as
the
relationships
of
epitopes
and
potential
ligand
binding
sites
in
the
heterotetrameric
receptor
is
unknown.
However,
this
observation
might
also
reflect
an
ability
of
antibodies
bound
at
one
a-subunit
to
influence
the
ligand
affinity
for
the
other
a-subunit
within
a
hybrid
molecule
by
conformational
effects.
Several
reports
have
previously
described
receptors
with
anomalous
properties,
which
might
now
be
attributed
to
the
presence
of
hybrids.
In
the
rat
L6
myocyte
line
(Burant
et
al.,
1987)
and
in
bovine
neural
retina
(Waldbillig
&
Chader,
1988)
binding
of
1251-insulin
was
observed
which
was
competed
by
IGF-I
with
greater
potency
than
by
insulin
itself.
This
could
be
explained
if
most
of
the
insulin
receptors
exist
as
hybrids
like
the
rIR/hIGFR
species
of
the
IGF-I-R/3T3
cells.
The
converse
situation
of
125I-IGF-II
binding
which
is
competed
by
insulin
at
unexpectedly
high
potency
has
been
described
in
IM-9
cells
and
placenta
(Hintz
et
al.,
1984;
Misra
et
al.,
1986;
Jonas
et
al.,
1986).
These
properties
have
been
attributed
to
'atypical'
insulin
receptors.
However,
we
did
not
observe
such
behaviour
when
using
125I1IGF-I
as
tracer
either
in
previous
studies
with
IM-9
cells
and
placenta
(Soos
&
Siddle,
1989)
or
with
rIGFR/hIR
hybrids
in
the
present
study
(Figs.
2
and
3).
It
is
possible,
though
unlikely,
that
binding
of
insulin
to
hybrid
receptors
differentially
affects
their
affinity
for
IGF-I
and
IGF-II.
However
'atypical'
receptors
in
placenta
reacted
with
anti-(insulin
receptor)
anti-
bodies
but
not
with
the
anti-(IGF-I
receptor)
antibody
aIR-3,
which
would
not
be
consistent
with
a
hybrid
species
(Jonas
et
al.,
1989).
The
presence
of
hybrid
receptors
in
some
tissues
does
not
of
course
preclude
the
existence
of
receptor
heterogeneity,
dependent
perhaps
on
differences
in
primary
sequence
(reflecting
products
of
distinct
genes
or
alternative
RNA
splicing)
and/or
differential
glycosylation.
It
appears
that
hybrids
as
well
as
homomeric
receptors
occur
in
normal
human
tissues
in
which
insulin
and
IGF-I
receptors
are
co-expressed
(Soos
&
Siddle,
1989),
and
this
could
have
important
implications
for
metabolic
regulation
by
the
respective
ligands.
The
tyrosine
kinase
activity
of
these
receptors
appears
to
be
an
essential
component
of
their
signalling
mechanism
(Rosen,
1987;
Zick,
1989).
Ligand-induced
activation
of
the
insulin
receptor
kinase
occurs
through
an
intramolecular
auto-
phosphorylation
reaction
within
intact
heterotetramers
(O'Hare
&
Pilch,
1988;
Morrison
et
al.,
1988;
Wilden
et
al.,
1989).
The
observation
that
both
insulin
and
IGF-I
bind
to
hybrid
receptors
with
high
affinity
raises
the
question
of
whether
both
ligands
can
activate
both
receptor
kinases
within
hybrids.
This
is
difficult
to
answer
in
transfected
cells
because
of
the
very
large
excess
of
We
are
grateful
to
Dr.
Steven
Jacobs
for
the
gift
of
antibody
a.IR-3.
We
thank
the
Wellcome
Trust,
the
Medical
Research
Council
and
Serono
Diagnostics
Ltd.
for
financial
support,
and
Jill
Stigter
for
technical
assistance.
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Received
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1990
390
