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Proc.
Natl.
Acad.
Sci.
USA
Vol.
86,
pp.
8083-8087,
October
1989
Medical
Sciences
Characterization
of
the
human
growth
hormone
receptor
gene
and
demonstration
of
a
partial
gene
deletion
in
two
patients
with
Laron-type
dwarfism
(exon
structure/DNA
sequence/gene
defects)
PAUL
J.
GODOWSKI*,
DAVID
W.
LEUNG*,
LILLIAN
R.
MEACHAMt, JOHN
P.
GALGANIt,
RENATE
HELLMISS*,
RUTH
KERET§,
PETER
S.
ROTWEIN¶,
JOHN
S.
PARKSt,
ZVI
LARON§,
AND
WILLIAM
I.
WOOD*
*Departmenlts
of
Developmental
Biology
and
Molecular
Biology,
Genentech,
Inc.,
460
Point
San
Bruno
Boulevard,
South
San
Francisco,
CA
94080;
tDepartment
of
Pediatrics,
Emory
University
School
of
Medicine,
2030
Ridgewood
Drive,
Northeast,
Atlanta,
GA
30322;
Departments
of
tPediatrics
and
1lnternal
Medicine
and
Genetics,
Washington
University
School
of
Medicine,
660
South
Euclid
Avenue,
Saint
Louis,
MO
63110;
and
Institute
of
Pediatric
and
Adolescent
Endocrinology,
Tel
Aviv
University,
Beilinson
Medical
Center,
49100
Petah
Tikva,
Israel
Communicated
by
William
H.
Daughaday,
July
24,
1989
(received
for
review
June
8,
1989)
ABSTRACT
Laron-type
dwarfism
is
an
autosomal
reces-
sive
genetic
disorder
that
is
characterized
by
high
levels
of
growth
hormone
and
low
levels
of
insulin-like
growth
factor
I
in
the
circulation.
Several
lines
of
evidence
suggest
that
this
disease
is
caused
by
a
defect
in
the
growth
hormone
receptor.
In
order
to
analyze
the
receptor
gene
in
patients
with
Laron-
type
dwarfism
and
with
other
growth
disorders,
we
have
first
determined
the
gene
structure
in
normal
individuals.
There
are
nine
exons
that
encode
the
receptor
and
several
additional
exons
in
the
5'
untranslated
region.
The
coding
exons
span
at
least
87
kilobase
pairs
of
chromosome
5.
Characterization
of
the
growth
hormone
receptor
gene
from
nine
patients
with
Laron-type
dwarfism
shows
that
two
individuals
have
a
dele-
tion
of
a
large
portion
of
the
extracellular,
hormone
binding
domain
of
the
receptor
gene.
Interestingly,
this
deletion
in-
cludes
nonconsecutive
exons,
suggesting
that
an
unusual
rear-
rangement
may
have
occurred.
Thus,
we
provide
direct
evi-
dence
that
Laron-type
dwarfism
can
result
from
a
defect
in
the
structural
gene
for
the
growth
hormone
receptor.
Growth
hormone
(GH)
is
secreted
from
the
pituitary
and
has
direct
and
indirect
actions
on
various
tissues,
causing
effects
on
growth
and
metabolism
(1,
2).
Though
high-affinity
bind-
ing
sites
for
GH
have
been
identified
in
a
number
of
tissues
(1,
3),
the
highest
concentration
of
GH
receptors
is
found
in
the
liver
(4)
where
GH
induces
the
expression
and
secretion
of
insulin-like
growth
factor
I
(IGF-I)
(3).
The
GH
receptor
recently
has
been
purified
and
characterized
from
rabbit
liver
(5),
and
clones
for
the
rabbit
and
human
GH
receptor
have
been
isolated
from
liver
cDNA
libraries
(6).
These
clones
encode
a
protein
of
620
amino
acids
with
a
single,
centrally
located
transmembrane
domain.
Comparison
of
the
primary
sequences
of
the
GH
receptor
and
of
the
related
prolactin
receptor
(7)
suggests
that
they
may
be
members
of
a
new
family
of
membrane-bound
receptors.
A
high-affinity
GH
binding
protein
has
been
demonstrated
in
the
plasma
of
a
number
of
mammals,
including
man
(8-10).
Characterization
of
this
binding
protein
(11,
12),
including
direct
amino
acid
sequence
data
(5,
6),
shows
that
the
GH
binding
protein
is
the
extracellular,
hormone
binding
domain
of
the
GH
receptor.
The
role
of
this
protein
in
the
regulation
of
growth
is
not
known.
Laron-type
dwarfism
(LTD)
is
a
rare,
autosomal
recessive
disorder
that
is
characterized
by
high
circulating
levels
of
biologically
active
GH
accompanied
by
low
levels
of
IGF-I
(13-16)
and
a
failure
to
respond
to
GH
therapy
(15).
Some
individuals
with
LTD
have
been
shown
to
lack
GH
binding
in
liver
biopsy
samples
(17),
to
lack
GH
binding
activity
in
their
serum
(18-20),
or
to
lack
an
IGF-I
response
in
transformed
T
lymphoblasts
(21).
These
studies
suggest
that
LTD
results
from
a
defect
in
the
GH
receptor.
In
theory,
LTD
could
result
from
defects
in
the
gene
that
encodes
the
GH
receptor
itself
as
well
as
in
other
genes
required
for
its
expression
or
function.
In
order
to
define
the
role
of
the
GH
receptor
in
human
growth
disorders,
we
present
here
the
characterization
of
the
gene
for
the
human
GH
receptor.
11
We
use
these
data
to
demonstrate
the
deletion
of
a
large
portion
of
the
receptor
gene
in
two
LTD
patients.
A
portion
of
this
work
has
been
presented
previously.**
HUMAN
SUBJECTS
Patient
147
is
a
male
with
LTD
born
to
related
parents
of
Jewish
Iraqi
origin.
Birth
length
(49
cm)
and
weight
(3.0
kg)
were
normal.
He
showed
slow
physical
and
mental
develop-
ment
and had
clinical
features
of
GH
deficiency
but
with
high
GH
levels
(>40
ng/ml)
and
low
levels
of
IGF-I.
At
age
35
his
height
was
128.3
cm
(7.3
SD
below
expected)
and
his
weight
was
41
kg.
No
serum
GH
binding
protein
activity
was
detected.
No
251I-labeled
GH
binding
was
detected
in
liver
microsomal
membranes
from
this
patient
after
liver
biopsy
[case
2
(17)].
Patient
D1
is
a
girl
with
LTD
born
to
related
parents
of
Jewish
Iraqi
origin.
Birth
length
(45
cm)
and
weight
(2.85
kg)
were
normal.
At
the
age
of
6
months
she
showed
typical
features
of
GH
deficiency
but
with
high
plasma
levels
of
GH
(20-100
ng/ml).
After
5
days
of
GH
therapy,
there
was
no
increase
in
the
plasma
IGF-I
level.
At
age
8
years,
10
months,
her
length
was
104
cm
(4.2
SD
below
expected)
and
her
weight
was
22.5
kg.
No
serum
GH
binding
protein
activity
was
detected
(20).
Patient
1
is
a
boy
with
LTD
born
to
unrelated
parents
in
the
United
States.
He
lacks
serum
GH
binding
protein
activity
[see
patient
1(18)
for
a
full
clinical
description].
DNA
was
obtained
from
fibroblasts.
The
other
six
LTD
patients,
who
had
no
alteration
in
their
genomic
DNA
pattern,
include
three
of
oriental
Jewish
background,
two
from
the
United
States,
and
one
from
Abbreviations:
GH,
growth
hormone;
IGF-I,
insulin-like
growth
factor
I;
LTD,
Laron-type
dwarfism.
"The
sequence
reported
in
this
paper
has
been
deposited
in
the
GenBank
data
base
(accession
no.
M26401).
**Meacham,
L.
R.,
Parks,
J.
S.,
McKean,
M.
C.,
Keret,
R.
&
Laron,
Z.,
71st
Annual
Meeting
of
the
Endocrine
Society,
Seattle,
June
21-24,
1989,
abstr.
1651.
8083
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.
8084
Medical
Sciences:
Godowski
et
al.
Poland
(fibroblasts
provided
by
Maria
Malinowska
and
Thom-
asz
Romer,
Child
Health
Center,
Warsaw,
Poland).
The
three
oriental
Jewish
patients
[described
previously
as
patients
8,
9,
and
10
(14)]
are
from
one
clan
and
have
clinical
features
of
GH
deficiency
but
with
high
plasma
levels
of
GH,
low
levels
of
IGF-I,
and
undetectable
GH
serum
binding
protein
activity
(20).
All
three
showed
no
response
to
GH
therapy.
The
two
U.S.
patients
lack
serum
binding
protein
activity
and
showed
no
response
to
GH
therapy
[described
previously
as
patients
2
and
3
(18)
or
as
patients
6
and
8,
respectively
(22)].
METHODS
Genomic
clones
for
the
GH
receptor
were
isolated
from
A
libraries
either
described
previously,
A4X
(23)
(AGG.9,
-19,
and
-20),
or
kindly
provided
by
John
McLean
(Genentech)
(AGG.33,
-47,
and
-48).
The
libraries
were
screened
with
32P-labeled
(24)
cDNA
probes
from
the
human
GH
receptor
(6).
The
hybridization
was
performed
at
420C
in
50%
form-
amide/0.75
M
NaCl/0.075
M
trisodium
citrate,
and
the
filters
were
washed
at
420C
in
0.03
M
NaCl/3
mM
trisodium
citrate/0.1%
SDS.
DNA
sequencing
was
performed
by
the
dideoxy
chain-termination
method
(25)
using
genomic
frag-
ments
subcloned
into
plasmid
vectors
(26).
Genomic
DNA
was
isolated
from
normal
or
LTD
blood
or
from
fibroblasts.
Six
micrograms
of
DNA
was
digested
with
HindIII
or
EcoRI,
electrophoresed
on
a
0.7%
agarose
gel,
transferred
to
nitrocellulose,
hybridized
as
above,
and
washed
at
500C
as
above
(24).
Probes
for
the
genomic
hybridization
were
labeled
with
32p
(24).
The
full-length
probe
was
a
1928-base-pair
(bp)
Xho
I
to
SnaBI
fragment
(bp
-11
to
+1917)
of
the
human
GH
receptor
cDNA
(6).
To
generate
the
exon-specific
probes,
pairs
of
60-mer
oligonu-
cleotides
were
synthesized;
each
pair
contains
a
10-bp
com-
plementary
region
at
the
3'
end.
Complementary
oligonucle-
otides
were
annealed
and
extended
with
the
large
fragment
of
DNA
polymerase
I
in
the
presence
of
32P-labeled
nucleotides
(24).
The
marker
lanes
contained
32P-labeled
A
HindIII
DNA
(24).
RESULTS
Characterization
of
the
GH
Receptor
Gene.
In
order
to
analyze
human
gene
defects
in
the
GH
receptor,
we
first
characterized
the
structure
of
the
normal
gene.
This
analysis
also
would
allow
us
to
determine
whether
the
multiple
bands
observed
in
genomic
blots
of
the
receptor
(see
below)
were
due
to
a
family
of
closely related
genes
or
to
the
several
exons
of
a
single-copy
receptor
gene.
Six
clones
were
isolated
from
human
genomic
libraries
by
screening
with
the
cloned
human
GH
receptor
cDNA
(Fig.
1).
These
clones
were
mapped,
and
the
hybridizing
regions
were
sequenced
in
order
to
determine
precisely
the
extent
of
each
exon
(Fig.
2).
With
the
exception
of
a
single
base
difference
in
exon
10
(Fig.
2,
legend),
the
sequence
of
the
coding
region
determined
for
the
genomic
clones
matched
that
determined
previously
from
cDNA
clones
(6).
The
coding
and
3'
untranslated
regions
of
the
receptor
are
encoded
by
9
exons,
numbered
2-10
(Fig.
1).
Exons
2-9
range
in
size
from
66
to
179
bp;
exon
10,
which
encodes
nearly
all
of
the
cytoplasmic
domain
as
well
as
the
3'
untranslated
region,
is
about
3400
bp.
Exons
2
and
8
are
nearly
coincident
with
the
putative
secretion
signal
sequence
and
the
trans-
membrane
domain,
respectively.
The
extracellular,
GH
bind-
ing
region
of
the
receptor
is
encoded
by
exons
3-7.
Most
of
the
5'
untranslated
region
appears
to
be
present
on
a
series
of
alternatively
spliced
exons
that
have
not
yet
been
localized
(see
below).
The
gene
for
the
GH
receptor
has
previously
been
localized
to
chromosome
5p13.1-p12
(27)
and
spans
at
least
87
kbp.
Gaps
of
unknown
length
occur
between
the
genomic
clones
in
two
places.
Thus,
the
2/3
and
6/7
introns
are
at
least
14
and
24
kbp;
the
3/4
intron
is
probably
27
kbp
(Fig.
1).
We
have
used
the
genomic
map
and
hybridization
data
(see
below)
to
assign
exons
2-10
to
the
bands
observed
in
genomic
blots
probed
for
the
receptor
(Fig.
3).
Analysis
of
LTD
Patient
DNA.
The
GH
receptor
gene
was
analyzed
in
DNA
samples
from
patients
with
LTD.
A
cDNA
fragment
encompassing
the
complete
protein
coding
region
of
the
receptor
was
used
to
probe
genomic
blots
of
DNA
from
control
and
nine
LTD
individuals.
No
obvious
alterations
in
the
receptor
gene
were
observed
in
six
LTD
DNA
samples
(data
not
shown).
Patient
1
was
shown
to
be
heterozygous
for
a
200-bp
insertion
in
the
3'
untranslated
region
(data
not
shown).
Since
it
is
unclear
whether
this
insertion
would
affect
the
expression
of
the
GH
receptor
gene,
we
have
not
ana-
lyzed
it
further.
It
is
possible
that
this
insertion
is
due
to
the
expansion
of
the
Alu
repeat
that
is
in
the
3'
untranslated
region
of
the
gene
(Fig.
1).
The
restriction
pattern
of
HindIII-digested
DNA
from
two
patients,
147
and
D1,
appeared
abnormal,
with
the
absence
of
the
16-kbp
band
assigned
to
exons
4
and
5
and
the
3.5-kbp
band
for
exon
6
(Fig.
3).
In
addition,
the
EcoRI
band
assigned
A
kbp
-
Amino
Acids
FeYnncq
0
2
3
4
-181
200
400
600620
11
1
1
11
234
5
6
7
89
10
5'
L
!xtracella
Cytoplasmic
3'
_j
K
A
Signal
Transmembrane
Alu
Repeat
0
,,
20
40
60
,
80
100
I
A/
I
I
/'
I
I
I
2
e,
3
4
5
6
,
7
8
910
X
14Z
1
1
I
-r
7,/>f
---
I_
I
-
..........
I
l
II I
I
1-I-II
I
I
I
11111
I
I
II
I
I
*
*
XGG.48
XGG.20
XGG.9
XGG.47
XGG.19
FIG.
1.
(A)
Schematic
representation
of
the
GH
receptor
mRNA
and
protein.
Shown
are
scales
of
nucleotides
and
amino
acids,
the
loca-
tion
of
the
exon
boundaries,
and
the
major
features
of
the
protein
and
mRNA.
(B)
Map
of
the
GH
hormone
receptor
gene.
Shown
are
a
scale
of
nucleotides,
location
of
the
exons,
re-
striction
map
for
four
enzymes,
and
the
location
of
six
genomic
clones.
Two
gaps
in
the
map
are
shown
at
their
minimum
length.
The
length
of
the
3/4
intron
is
established
only
by
the
coinci-
dence
of
genomic
Sst
I
fragments
in
the
region
and
thus
could
be
greater.
The
BamHI
sites
indicated
by
an
asterisk
may
represent
more
than
one
site
separated
by
an
unknown
distance.
The
order
of
the
bracketed
EcoRI
sites
is
un-
known.
The
restriction
map
is
unknown
in
the
shaded
regions.
B
kbp
Exons
Gene
Hindill
EcoRi
Sstl
BamHI
XGG.
'"
33
Proc.
Natl.
Acad.
Sci.
USA
86
(1989)
1
rAvl
,
Medical
Sciences:
Godowski
et
al.
Proc.
Natl.
Acad.
Sci.
USA
86
(1989)
8085
*
EXON
2
1
TTTCATGATAAT
GGTCTGCTTTTAATT
GCTGGGCTTTACCTT
ACCCTTTTTGTGATT
GCAGGTCCTACAGGT
ATGGATCTCTGGCAG
CTGCTGTTGACCTTG
GCACTGGCAGGATCA
AGTGATGCTTTTTCT
GGAAGTGAGGGTGAG
-18
M
D
L
W
Q
L L
L
T L
A
L
A
G
S
SD
A
F
S
G
S
E
A
148
TTCTGCTTTTCCATT
TCCACCCTCAGTGTT
TTGAAACAACACTaA
ACTGTATTC
.
14
kbp
or
more
.
. .
*
~~~~~~~~~~~~~~~~~~EXON
3*
1
GATGGACTAGATGGT
TTTGCCTTCCTCTTT
CTGTTTCAGCCACAG
CAGCTATCCTTAGCA
GAGCACCCTGGAGTC
TGCAAAGTGTTAATC
CAGGCCTAAAGACAA
GTAAGAATTTCAGTC
CTTTTTCTTCCTTCG
AATGATATTTTCCAT
7
TA
A
I
L
SR
A
P
W
S
L
Q
S
V
N
P
G
L
K
T
N
151
GTTTTAGTGTAATTA
AGCTACTATCCT
27
kbp
.
.
pstI
EXON
4
1
AGGATCACATATGAC
TCACCTGATTTCATG
CCTTGCCTTTTCTTT
TTATTCTGCAGATTC
TTCTAAGGAGCCTAA
ATTCACCAAGTGCCG
TTCACCTGAGCGAGA
GACTTTTTCATGCCA
CTGGACAGATGAGGT
TCATCATGGTACAAA
29
S
S
K
E
P
K
F
T
K
C
R
S
P
E
R
E
T F
S
C
H
W
T
D
E
V
H
H
G
T
K
*
~~~~~~~~~~~~~ncoI
151
GAACCTAGGACCCAT
ACAGCTGTTCTATAC
CAGAAGGTGCCACCA
TCATGCCTTTCTGAT
TTTCCTCTCCATGGA
TGTACCTACTAAAGT
ACACTA
6
kbp
60
N
L
G
P
I
Q
L
F
Y
T
R
R
*
~~~~~~~~~EXON
5
1
ACTTAAGCTACAACA
TGATTTTTGGAACAA
TTAATCTTTTTTTAA
CCCTTCATTTTAGGA
ACACTCAAGAATGGA
CTCAAGAATGGAAAG
AATGCCCTGATTATG
TTTCTGCTGGGGAAA
ACAGCTGTTACTTTA
ATTCATCGTTTACCT
72
N
T
Q
E
W
T
Q
E
W
K
E
C
P
D
Y
V
S
A
G
E
N
S
C
Y
F
N
S
S F
T
S
151
CCATCTGGATACCTT
ATTGTATCAAGCTAA
CTAGCAATGGTGGTA
CAGTGGATGAAAAGT
GTTTCTCTGTTGATG
AAATAGGTAAATCAC
AGGTTTTTGTTTCAT
TTGACATAGTTTTAG
ACTAAATAAATGGGG
AAGC
5
kbp
103
I
N
I
P
Y
C
I
K
L
T
S
N
G
G
T
V
DEK
C
F
S
V
D
E
I
V
EXON
6
ecoRV
1
CCATTAATATTAAAT
TGTGTCTGTCTGTGT
ACTAATGCTCTGTTG
AATTGCACAGTGCAA
CCAGATCCACCCATT
GCCCTCAACTGGACT
TTACTGAACGTCAGT
TTAACTGGGATTCAT
GCACATATCCAAGTG
AGATGGGAAGCACCA
130
Q
P
D
P
P
I
A
L
N
W
TL
L
N V
S
L
T
G
I
H
A
D
I
Q
V
R
W
E
A
P
151
CGCAATGCAGATATT
CAGAAAGGATGGATG
GTTCTGGAGTATGAA
CTTCAATACAAAGAA
GTAAATGAAACTAAA
TGGAAAATGGTAAGA
TGTTGCTACACCTTA
CACTTTGACTTTTCT
TTCTATT
.
24
kbp
or
more
161
R
N
A
D
I
Q
K
G
I
K
V
L
E
Y
E
L
Q
Y
K
E
V
N
E
T K
W
N
M
*
EEXON
7
1
ATACCTGTAGTGTTC
ATTGGCATTGAGTTG
TTGACTCTTTGGCCA
ATATGGCGTTTATAT
TTTTGTCTTGAAAGA
TGGACCCTATATTGA
CAACATCAGTTCCAG
TGTACTCATTGAAAG
TGGATAAGGAATATG
AAGTGCGTGTGAGAT
189
M
D
P
I
L
TT
S
V
P
V
Y S L
K V
D
K
E
Y
E
V
R
V
R
S
151
CCAAACAACGAAACT
CTGGAAATTATGGCG
AGTTCAGTGAGGTGC
TCTATCTAACACTTC
CTCAGATCACCCAAT
TTACATGTCAACAAG
GTAAAAGAAATAAAA
GATTAAAATAGTAGC
TAACCTGGCTTTTGT
CAATATAACAGTTGA
215
K
Q
R
N
S
G
N
Y
G
E
F
SE
V
L
Y
V
T
L
P
0
M
S
Q
F
T
C
E
E
D
ecoRV
301
TTCACCCCTGCACTG
GTAGTGTGTTGTCCA
AATCAAAATATATTA
ACATCAGATATCAGG
AT
3
kbp
ncoI
EXON
8
1
GAAACTGTGCTTCAA
CTAGTCGTAATTCTG
AAAGCGAAATATTCT
TGTGTGTTTGCAGAT
TTCTACTTTCCATGG
CTCTTAATTATTATC
TTTGGAATATTTGGG
CTAACAGTGATGCTA
TTTGTATTCTTATTT
TCTAAACAGCAAAGG
245
F
Y
F
P
W
L
L
I
I
I
F
G
I
F
G
L
T
V
M
LF
V
F L
F S
K
Q
Q
R
*kpn
I
151
TAGGATGTAGGMGG
TAGTATTCTTTGGTA
CCTTCTGTACCAGTT
GTGTTAGACCTTGCC
AT
4
kbp
EXON
9
claI
1
GCTATAATTGAGAAT
ATGTAGCTTTTAAGA
TGTCAAAACCAAAAT
TTTTATATGTTTTCA
AGGATTAAAATGCTG
ATTCTGCCCCCAGTT
CCAGTTCCAAAGATT
AAAGGAATCGATCCA
GATCTCCTCAAGGTA
ACTAATAATTTTATC
275
I
K
M
L
I
L
P P
V
P
V
P
K
I
K
G
I
D
P
D
L
L
K
151
TAAAGTTGTAGCTAG
TACTAATTAACACCT
GAAGACTCCTGTCAT
ATG
. .
0.4
kbp
EXON
10
ecoRI
1
GCTAATTCATTTAAT
TATTATGAGTTTCTT
TTCATAGATCTTCAT
TTTCTTTCTATTTTC
TAGGAAGGAAAATTA
GAGGAGGTGAACACA
ATCTTAGCCATTCAT
GATAGCTATAAACCC
GAATTCCACAGTGAT
GACTCTTGGGTTGAA
298
E
G
K
L E E
V
N
T
I
L
A
I
H
D
S
Y
K
P
E
F
H
S
D
D S
W
V
E
151
TTTATTGAGCTAGAT
ATTGATGAGCCAGAT
GAAAAGACTGAGGAA
TCAGACACAGACAGA
CTTCTAAGCAGTGAC
CATGAGAAATCACAT
AGTAACCTAGGGGTG
AAGGATGGCGACTCT
GGACGTACCAGCTGT
TGTGAACCTGACATT
327
F
I
E
L
D
I
D
E
P
D
E
K
T
E
S
D
T
D
R
L
L
S
S
D
H
E
K
S
H
S
N
L
G
V
K
D
G
D
S
G
R
T
S
C
C
E
P
D
I
kpnI
301
CTGGAGACTGATTTC
AATGCCAATGACATA
CATGAGGGTACCTCA
GAGGTTGCTCAGCCA
CAGAGGTTAAAAGGG
GAAGCAGATCTCTTA
TGCCTTGACCAGAAG
AATCAAAATAACTCA
CCTTATCATGATGCT
TGCCCTGCTACTCAG
377
L
ET
D
F
N
A
N
D
I
H
E
G
T
S
E
V
A
Q
P
H
R
L
K
G
E
A
D
L
L
C
L
D
I
K
N
Q
N
S
P
Y
H
D
A
C
P
A
T
Q
451
CAGCCCAGTGTTATC
CAAGCAGAGAAAAAC
AAACCACAACCACTT
CCTACTGAAGGAGCT
GAGTCAACTCACCAA
GCTGCCCATATTCAG
CTAAGCAATCCAAGT
TCACTGTCAAACATC
GACTTTTATGCCCAG
GTGAGCGACATTACA
427
Q
P
S
V
I
Q
A
E K
N
K
P
Q
P
L
P
T
E
G
A
E
S
T
H
Q
A
A
H
I
Q
L
S
N
P
S
S
L
S
N
I
D
F
Y
A
Q
V
S
D
I
T
sma
I
601
CCAGCAGGTAGTGTG
GTCCTTTCCCCGGGC
CAAAAGAATAAGGCA
GGGATGTCCCAATGT
GACATGCACCCGGAA
ATGGTCTCACTCTGC
CAAGAAAACTTCCTT
ATGGACAATGCCTAC
TTCTGTGAGGCAGAT
GCCAAAWAGTGCCTC
477
P
A
G
S
V
V
L
SP
G
Q
K
N
K
A
G
M
S
Q
C
D
M
H
P
E
M V
S
L
C
Q
E
N
F
L
M
D
N
A
Y
F
C
E
A
D
A
K K
C
L
hindIII
751
CCTGTGGCTCCTCAC
ATCAAGGTTGAATCA
CACATACAGCCAAGC
TTAAACCAAGAGGAC
ATTTACATCACCACA
GAAAGCCTTACCACT
GCTGCTGGGAGGCCT
GGGACAGGAGAACAT
GTTCCAGGTTCTGAG
ATGCCTGTCCCAGAC
527
P
V
A
P
H
I
K
V
E S
H
I
Q
P
S
L
N
I
E
D
I
Y
I
T
T
E
S
L
T
T
A
A
G
R
P
G
T
G
E
H
V
P
G
S
E
M
P
V
P
D
901
TATACCTCCATTCAT
ATAGTACAGTCCCCA
CAGGGCCTCATACTC
AATGCGACTGCCTTG
CCCTTGCCTGACAAA
GAGTTTCTCTCATCA
TGTGGCTATGTGAGC
ACAGACCAACTGAAC
AAAATCATGCCTTAG
CCTTTCTTTGGTTTC
577
Y
T
S
I
H
I
V
Q
S
P
Q
G
L
I
L
N
A
T
A
L
P
L
P
D
K
E
F
L
S S
C
G
Y
V
S
T
D
QL
N K
I
M
P
C
1051
CCAAGAGCTACGTAT
TTAATAGCAAAGAAT
TGACTGGGGCAATAA
CGTTTAAGCCAAAAC
AATGTTTAAACCTTT
TTTGGGGGAGTGACA
GGATGGGGTATGGAT TCTAAAATGCCTTTT
CCCAAAATGTTGAAA
TATGATGTTAAAAAA
1201
ATAAGAAGAATGCTT
AATCAGATAGATATT
CCTATTGTGCAATGT
AAATATTTTAAAGAA
TTGTGTCAGACTGTT
TAGTAGCAGTGATTG
TCTTAATATTGTGGG
TGTTAATTTTTGATA
CTAAGCATTGAATGA
CTATGTTTTTAATGT
1351
ATAGTAAATCACGCT
TTTTGAAAAAGCGAA
AAAATCAGGTGGCTT
TTGCGGTTCAGGAAA
ATTGAATGCAAACCA
TAGCACAGGCTAATT
TTTTGTTGTTTCTTA
AATAAGAAACTTTTT
TATTTAAAAAACTAA
AAACTAGAGGTGAGA
ostI
1501
AATTTAAACTATAAG
CAAGAAGGCAAAAAT
AGTTTGGATATGTAA
AACATTTATTTTGAC
ATAAAGTTGATAAAG
ATATTTTTTAATAAT
TTAGACTTCAAGCAT
GGCTATTTTATATTA
CACTACACACTGTGT
ACTGCAGTTGGTATG
1651
ACCCCTCTAAGGAGT
GTAGCAACTACAGTC
TAAAGCTGGTTTAAT
GTTTTGGCCAATGCA
CCTAAAGAAAAACAA
ACTCGTTTTTTACAA
AGCCCTTTTATACCT
CCCCAGACTCCTTCA
ACAATTCTAAAATGA
TTGTAGTAATCTGCA
1801
TTATTGGAATATAAT
TGTTTTATCTGAATT
TTTAAACAAGTATTT
GTTAATTTAGAAAAC
TTTAAAGCGTTTGCA
CAGATC
about
1530
bp
unsequenced
.
.
3411
CCTTCAAAGTTTAAT
AAATTTATTTTCTTG
GATTCCTGATAGTGT
GCTTCTGTTATCAAA
CACCAACATAAAAAT
GATCTAAACCA
FIG.
2.
DNA
sequence
of
the
GH
receptor
genomic
clones.
Exons
2-10
are
overlined,
and
the
encoded
protein
sequence
is
shown.
Asterisks
indicate
the
extent
of
the
exon-specific
oligonucleotide
probes.
Nucleotides
that
differ
from
the
cDNA
sequence
(6)
are
underlined.
to
exon
4
is
absent.
New
bands
not
found
in
these
and
other
pattern,
but
clearly
much
of
the
receptor
gene
is
present.
This
control
DNAs
are
observed
for
patient
147
(HindIII,
4.4
and
result
suggests
that
either
the
two
patients
contain
a
partial
1.8
kbp;
EcoRI,
4.8
and
1.3
kbp)
(Fig.
3).
The
two
large-sized
deletion
in
the
GH
receptor
gene
or,
alternatively,
they
have
HindIII
bands
for
patient
147
(of
about
20
and
11
kbp)
appear
an
unusual
polymorphism.
to
be
a
polymorphic
pattern
found
in
other
control
DNAs
of
In
order
to
reduce
the
complexity
of
the
analysis
and
to
Israeli
origin
(data
not
shown).
The
integrity
of
the
remaining
examine
individual
exons,
replicate
blots
containing
DNA
bands
is
difficult
to
judge
because
of
the
complex
restriction
from
the
two
LTD
patients
were
hybridized
with
synthetic
8086
Medical
Sciences:
Godowski
et
al.
Hind/I
_
EcoRi
1
2 3
4
M
Ml
2
3
4
exon
kbp
23.123.1
4.5
_o
go
7,
go
A
9.4
.4e
8,9,10
2
6.6
-.
2
*.
4.4
6
"'-
4.
10
-
*.,
-w
2.3
4s
-P
.2.
exon
3+1
0
4.
4
Ae2
10
9,10
8
with
full-length
or
other
exon-specific
probes.
Interestingly,
an
abnormal
size
band
was
found
with
the
exon
4
probe
in
digests
from
both
patients
(Fig.
4).
These
results
show
that
a
substantial
portion
of
the
GH
receptor
gene
is
missing
from
both
alleles
of
these
two
patients
yet
indicate
that
more
than
a
simple
deletion
has
occurred.
Blots
hybridized
with
an
exon
10-specific
probe
show
that
for
both
patients
two
EcoRI
bands
of
11
and
16
kbp
are
found
(data
not
shown).
The
larger
band
obscures
the
lack
of
the
exon
5/6
EcoRI
band
for
the
two
patients
(Fig.
3).
0.56
FIG.
3.
Genomic
blot
hybridized
with
the
full-lengt
cDNA
probe.
Blots
contained
DNA
from
two
norn
(adult
male,
United
States)
(lanes
1
and
4)
or
DNA
froi
147
(lanes
2)
or
patient
D1
(lanes
3).
The
sizes
of
the
(lanes
M)
are
shown
between
the
panels.
Assignmei
receptor
exons
to
the
HindIII
or
EcoRI
bands
are
she
and
right
of
the
panels,
respectively.
probes
specific
for
exons
2-7.
A
normal
pattei
ization
was
observed
with
the
exon
2
and
7
prc
DNAs
from
the
two
LTD
patients
failed
to
hybri
exon
3,
5,
and
6
probes.
The
failure
to
detect
h
was
not
due
to
the
lack
of
DNA
in
the
LTD
la
artifact
of
the
transfer
since
similar
results
v
multiple
experiments
and
since
these
same
filte
normal
hybridization
signals
when
stripped
and:
Exon
2
Hind/i
EcoRi
M
1
2
3
4
1
2
3
4
M
-
_
A*
...
Exon
3
Hind/li
EcoRi
M
1
2
3
4
1
2
3
4
M
IN.
VW
..
~~~*
4..4
,-
Exon
5
Hind//i
EcoRl
M
1
2
34
1
2
3
4M
Hindi.
1
4w
aw
:w
Exon
6
Hind/l/
EcoRi
M
1
2
3
4
1
2
3
4
M
-
4.
a
a
-m
4.
4.
4.
qw
4w
Hindt
Ml1
2
4..
4
a
FIG.
4.
Genomic
blots
hybridized
with
exon-sp4
Blots
containing
DNA
from
two
normal
individuals
(lai
LTD
patient
147
(lanes
2)
or
patient
D1
(lanes
3)
were
h3
exon-specific
probes
as
indicated.
Marker
DNA
(lan(
Fig.
3.
DISCUSSION
We
have
determined
the
exon
structure
of
the
human
GH
receptor
gene,
and,
using
this
information,
we
have
examined
the
receptor
gene
in
nine
LTD
patients.
The
data
show
that
two
patients
have
a
deletion
in
both
receptor
alleles
that
removes
a
large
part
of
the
coding
region
for
the
GH
binding
th
GH
receptor
domain.
With
such
a
large
deletion,
we
consider
it
very
nal
individuals
unlikely
that
the
receptor
would
retain
its
normal
function.
m
LTD
patient
Thus,
these
data
provide
direct
evidence
that
LTD
can
result
marker
DNAs
from
defects
in
the
structural
gene
for
the
GH
receptor.
These
nts
of
the
GH
findings
also
provide
convincing
genetic
data
showing
that
)wn
on
the
left
the
gene
identified
by
GH
binding
(1,
5)
and
cloned
as
a
putative
GH
receptor
(6)
is,
in
fact,
required
for
proper
growth.
rn
of
hybrid-
No
obvious
alterations
were
observed
in
the
restriction
)bes
(Fig.
4).
pattern
of
seven
other
LTD
individuals,
clearly
demonstrat-
idize
with
the
ing
that
LTD
is
caused
by
a
heterogeneity
of
gene
defects.
hybridization
Based
on
the
wide
collection
of
gene
defects
found
in
other
Lnes
or
to
an
inherited
diseases
(28-31),
we
expect
that
point
mutations
in
vere
seen
in
the
GH
receptor
gene
also
may
account
for
many
LTD
cases.
zrs
produced
The
detection
of
these
defects
will
require
a
more
detailed
rehybridized
analysis
than
studies
employing
genomic
blots
as
described
here.
In
addition,
mutations
in
other
genes,
such
as
those
Exon
4
required
for
the
expression
of
the
receptor
or
for
transduction
I//
EdoRl
of
the
GH
signal,
also
might
result
in
a
similar
or
identical
3
4
1
2 3
4
M
phenotype.
One
surprising
finding
is
that
noncontiguous
exons
have
been
deleted
in
the
two
LTD
patients.
Such
a
mutation
could
result
from
two
independent
deletion
events
or
from
a
complex
deletion
and
rearrangement.
A
more
detailed
anal-
ysis
of
the
genome
structure
of
these
two
patients
may
suggest
which
mutational
event
is
more
likely.
Both
patients
are
from
families
with
consanguinity
and,
at
the
level
of
resolution
determined
here,
are
homozygous
for
the
muta-
tion.
Although
both
patients
are
from
the
same
ethnic
back-
ground,
their
hybridization
patterns
with
the
full-length
probe
are
not
identical
(Fig.
3),
showing
that
their
GH
receptor
genes
do
have
polymorphic
differences.
Exon
7
Assignment
of
the
GH
receptor
exons
to
the
multiple
bands
found
on
genomic
blots
(Fig.
3)
shows
that
the
complex
/I/
EcoRi
pattern
observed
results
from
the
multiple
exons
of
a
single-
copy
gene
rather
than
from
a
gene
family.
However,
the
many
bands
could
obscure
other
cross-hybridizating
genes,
and
our
data
do
not
exclude
the
existence
of
other
genes
closely
related
to
the
GH
receptor.
Under
our
hybridization
conditions,
we
do
not
detect
the
distantly
related
prolactin
receptor
gene
(7).
g
Ad
When
cDNA
clones
for
the
human
and
rabbit
GH
receptor
were
isolated,
nearly
all
of
these
clones
diverged
12
bp
5'
of
the
initiating
methionine
codon
(6).
We
speculated
that
these
clones
resulted
from
multiple
splicing
5'
of
the
coding
region
(6)
and
perhaps
from
transcription
initiated
from
different
promoters.
The
current
sequence
data
show
that
the
point
of
ecific
probes.
5'
divergence
corresponds
to
the
beginning
of
exon
2.
Thus,
nes
1
and
4)
or
there
is
a
diverse
splicing
pattern
in
the
5'
untranslated
region
ybridized
with
of
the
GH
receptor
gene.
Localization
of
these
5'
exons
and
es
M)
is
as
in
characterization
of
the
promoter(s)
and
their
possible
tissue-
specific
regulation
will
await
further
studies.
Proc.
Natl.
Acad.
Sci.
USA
86
(1989)
qw
a*
4w
40
dw
Proc.
Natl.
Acad.
Sci.
USA
86
(1989)
8087
Human
and
rabbit
GH
receptor
cDNA
clones
also
have
been
isolated
that
diverge
within
the
coding
region
(6).
Most
(six
in
all)
of
these
points
of
divergence
coincide
with
the
exon
boundaries
determined
here
(data
not
shown).
These
clones
represent
a
series
of
unspliced
or
differentially
spliced
mRNAs,
including
two
clones,
ghr.244
and
ghr.438,
that
have
exon
3
or
4
missing,
respectively.
The
biological
significance
of
these
observations
awaits
further
analysis.
Recently,
the
two
prominent
GH
receptor
mRNAs
found
in
mouse
liver
have
been
cloned
(32).
These
mRNAs
encode
two
forms
of
the
mouse
receptor,
a
high
molecular
weight,
membrane-bound
form
and
a
low
molecular
weight
form
that
diverges
prior
to
the
transmembrane
domain
and
appears
likely
to
encode
the
secreted
GH
binding
protein
(33).
The
point
of
divergence
of
these
two
clones
matches
the
exon
7/8
boundary
found
here,
supporting
the
proposal
that
alterna-
tive
splicing
of
the
GH
receptor
mRNA
generates
the
two
receptor
species
(32).
cDNA
clones
encoding
the
prolactin
receptor
also
have
been
isolated
from
rat,
rabbit,
and
mouse
libraries
(7,
34,
35).
Some
of
these
clones
encode
long
and
short
forms
of
the
prolactin
receptor.
The
points
of
divergence
of
these
clones
match
the
9/10
exon
junction
found
here
for
the
GH
receptor.
Thus,
it
would
appear
that
the
prolactin
and
GH
receptor
will
have
at
least
some
similarity
in
their
exon
structures.
This
coupled
with
an
overall
amino
acid
identity
of
the
two
receptors
of
about
25%
shows
that
the
two
receptors
have
evolved
by
duplication
and
divergence
of
a
common
ancestral
gene.
We
thank
Ellen
Heath-Mannig
for
providing
fibroblasts
for
the
three
U.S.
LTD
patients,
Jeroen
Knops
for
the
initial
analysis
of
patient
1
DNA,
and
the
Genentech
organic
synthesis
group
for
oligonucleotide
probes.
Z.L.
is
incumbent
of
the
Irene
and
Nicholas
Marsh
Chair
for
Pediatric
Endocrinology
and
Diabetes
at
Tel
Aviv
University.
L.R.M.
was
supported
by
a
J.
N.
Goddard
Research
Fellowship
and
by
a
National
Institutes
of
Health
Fellowship
(5
T32
DKO
7298-09);
J.P.G.
was
supported
by
a
National
Institutes
of
Health
Training
Grant
(DK
07120).
This
work
was
supported
by
grants
from
the
March
of
Dimes
(Clinical
Research
Grant
6-454,
J.S.P.
and
Z.L.),
from
the
National
Institutes
of
Health
(R01
HD24960,
J.S.P.;
and
P01
HD20805,
P.S.R.),
and
from
the
Emory
Egleston
Children's
Research
Center
(J.S.P.)
and
by
Genentech,
Inc.
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