Hammonds, R. Glenn

Henzel, William J.

Rodriguez, Henry

Spencer, Steven A.

Waters, Michael J.

Wood, William I.

University of Queensland eSpace

0 1988 by The American Society for Biochemistry and Molecular Biology, Inc 
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263, No. 16, Issue of June 5, pp. 7862-7867,1988 
Printed in U.S.A. 
Rabbit Liver Growth  Hormone  Receptor  and  Serum Binding Protein 
PURIFICATION, CHARACTERIZATION, AND SEQUENCE* 
(Received for publication, November 25, 1987) 
Steven A. Spencer, R. Glenn Hammonds, William J. Henzel, Henry Rodriguez, 
Michael J. Waters$, and William I. Wood 
From the Department of Developmental Bwlogy, Genentech, Inc., South San Francisco, California 94080 and the $Department 
of Physiology and  Pharmacology, University of Queensland, St. Lucia,  Queensland, 4067 Australia 
A putative  growth  hormone  receptor  from  detergent- 
solubilized rabbit liver membranes and the growth 
hormone  binding  protein  from  rabbit  serum  have  been 
purified 59,000- and 400,000-fold, respectively, pri- 
marily by affinity chromatography.  Both  purified  pro- 
teins exhibit high affinity binding  for human growth 
hormone; K ,  = 9-30 X los M” for  the liver receptor 
and K,  = 6 X los M” for the binding protein. The 
apparent molecular weight of the liver receptor is 
130,000 by  reduced  sodium  dodecyl sulfate gel electro- 
phoresis, while that  of  the  binding  protein is  51,000. 
Both  contain N-linked carbohydrate.  The  amino-ter- 
minal  sequences  of  the liver growth  hormone  receptor 
and the serum binding protein were found to be the 
same,  indicating  that  the  binding  protein  corresponds 
to the extracellular domain of the liver receptor.  Ubi- 
quitin was found  covalently  linked  to  the liver receptor 
but  not  to  the  serum binding  protein.  The  amino  acid 
sequences  of several peptides  from  the liver receptor 
were also determined after tryptic and V8 protease 
digestion. 
Growth hormone (GH)’  has been used clinically for many 
years to  treat GH-deficient children, but  the mechanism or 
mechanisms by which it promotes normal growth are  not well 
established. According to the somatomedin hypothesis ( l) ,  
GH released from the pituitary  acts on the liver to induce the 
synthesis and release of insulin-like growth factor I. This 
systemic insulin-like growth factor  I  is  then responsible for 
skeletal growth. However, the discovery of high affinity GH 
receptors in tissues other than liver (2, 3), as well as the 
demonstration of direct effects of growth hormone on cartilage 
in  vivo (3-5) and recently in  vitro (6), suggest that  the direct 
action of GH  on peripheral  tissues may also be important. 
One way to address the relative contributions of the sys- 
temic and direct actions of GH is to characterize the GH 
receptor and determine its tissue distribution, to establish 
whether more than one type of receptor exists, and  to deter- 
mine whether the same receptor exerts different actions  in 
different cell types. To date such studies have not been 
* Portions of this work  were supported by NHMRC Grant 322 (to 
M. J. W.). The costs of publication of this article were defrayed in 
part by the payment of page charges. This article  must therefore be 
hereby marked “advertisement” in accordance with 18 U.S.C. Section 
1734 solely to indicate this fact. 
* The abbreviations used are: GH, growth hormone; hGH,  human 
growth hormone; bGH, bovine growth hormone; oPrl, ovine prolactin; 
KIU, Kallikrein inhibitor  units;  PMSF, phenylmethylsulfonyl fluo- 
ride; Tris-Triton, 50 mM Tris-HC1, pH 7.4, containing 1 ml/liter 
Triton X-100; SDS, sodium dodecyl sulfate; Mab, monoclonal anti- 
body; HPLC, high performance liquid chromatography. 
possible because the receptor has  not been isolated and  char- 
acterized and because the intracellular signaling mechanism 
for the receptor has  not been identified (3). In fact, only a few 
cell types have been found which produce a biological response 
in  vitro to GH binding (2, 3). 
The lack of a biological assay for GH receptor function has 
forced most workers to rely on the specific high-affinity 
binding of GH to identify the receptor. Using GH affinity- 
labeling techniques, several groups have partially  character- 
ized GH receptors in rat hepatocytes (7), rat adipocytes (€9, 
mouse liver (9), and  human  IM-9 lymphocytes (10,ll). Others 
have used this binding assay to partially purify the GH 
receptor from rabbit liver (12-14), which is a particularly 
abundant source of GH receptors (15). One complication in 
the choice of rabbit liver as  a source for the GH receptor is 
the observation that  this tissue contains  both lactogenic and 
somatogenic receptors (12, 15). While hGH binds with high 
affinity to both types of receptor, they  can be distinguished 
because bovine growth hormone binds only to  the somatogenic 
receptor while ovine prolactin shows preferential binding for 
the lactogenic receptor (15). 
In addition to  the membrane-bound GH receptor found in 
liver, a  GH binding protein  has recently been identified in 
rabbit (16) and human (17, 18) serum. It is a soluble protein 
which  was not detected earlier because it does not precipitate 
in  the binding assay commonly used to measure the mem- 
brane-bound receptor (19). Antibody data show that this 
binding protein  shares several epitopes with the liver receptor 
(20), indicating the two proteins  are related structurally as 
well as functionally. 
In  this report we describe the isolation of the putative  GH 
receptor from rabbit liver and  the GH binding protein from 
rabbit serum, as well as  the characterization and  partial amino 
acid sequence analysis of the purified proteins. Based on these 
data, full-length cDNA clones of the rabbit  and  human GH 
receptor have been isolated (21). 
EXPERIMENTAL PROCEDURES AND RESULTS~ 
DISCUSSION 
The putative  GH receptor that we have isolated from rabbit 
liver is a glycoprotein with an apparent size of 130 kDa by 
reduced sodium dodecyl sulfate gel electrophoresis. This is 
consistent with the apparent 110-kDa size reported for affin- 
ity-labeled GH receptor from rat hepatocytes (7), rat adipo- 
cytes (8), mouse liver (9),  and  human IM-9 lymphocytes (10, 
Portions of this paper (including “Experimental Procedures,” 
“Results,” Figs. 1-8, and Tables 1-111) are presented in  miniprint at 
the end of this paper. Miniprint is easily read with the aid of a 
standard magnifying glass. Full size photocopies are included in  the 
microfilm edition of the  Journal  that is available from Waverly Press. 
7862 
e r o w ~ h  Hormone  Receptor and Serum Binding Protein 7863 
11) but larger than  the 50-80-kDa size reported previously 
for rabbit liver receptor (12-14). The latter size probably 
reflects the sensitivity of the receptor to proteolysis, which 
can reduce the 130-kDa receptor to 50-60 kDa without sig- 
nificantly affecting its ability to bind hGH. 
The sensitivity of the liver receptor to proteolysis may also 
explain the mechanism by  which the serum binding is pro- 
duced.  We have shown that  the amino-terminal 37 residues 
of the binding protein are identical to  the amino terminus of 
the liver receptor, and preliminary analysis of a mixture of 
cyanogen bromide fragments from the binding protein (21) 
indicates that  this identity continues through much, if not 
all, of the binding protein sequence. Thus, a plausible mech- 
anism for generating the binding protein is proteolytic cleav- 
age of the liver receptor near the t r ~ s ~ e m b r a n e  region, 
thereby releasing the soluble hormone binding domain. 
The presence of ubiquitin covalently linked to the GH 
receptor is unusual but  not unique for cell surface receptors, 
since both the lymphocyte homing receptor (39) and the 
platelet-derived growth factor receptor (40) are ubiquitinated. 
While the function for this ubiquitin is unknown, it has been 
suggested that  it may regulate receptor number or produce an 
intracellular signal upon ligand binding (41). In  the case of 
the GH receptor, ubiquitin could be involved in the rapid 
receptor turnover observed in uiuo (42, 43). Alternatively, 
ubiquitin-mediated cleavage  may  release the binding protein, 
either by the direct action of ubiquitin itself (41) or through 
the ATP-dependent protease pathway (44). 
Characterization of the hormone binding properties of the 
purified liver receptor confirms previous observations of both 
a somatogenic and a lactogenic binding activity in  the rabbit 
liver (12,15).  The urea fraction from the hGH affinity column 
contained only  somatogenic receptor while the MgCL fraction 
was  composed primarily of somatogenic receptor (over 80% 
of the binding sites)  and a small amount of the lactogenic 
receptor (20%). 
The lactogenic receptor is unusual in its ability to bind I2'I- 
hGH so tightly that it is not readily displaceable by a large 
excess of unlabeled hGH. Its apparent association constant 
for hGH is over 10" M-' by Scatchard analysis, but since this 
binding is  not at equilibrium, the significance of this value is 
unclear. This irreversible binding also tends  to overemphasize 
the effects of the lactogenic receptor in  the binding assay and 
makes it difficult to assay mixtures of the two receptors, since 
receptor concentration, temperature, incubation time, and 
ligand concentration all affect the relative amount of lz5I- 
hGH bound to  the lactogenic receptor. In contrast,  the so- 
matogenic receptor behaves normally with respect to reversi- 
bility of hGH binding and  has  an  apparent K, = 10 X lo9 M-'. 
The serum binding protein has a slightly lower affinity of rC, 
= 6 X lo9 M-'. 
The GH receptor described here is the major protein in 
rabbit liver capable of binding GH, but whether it is the  entire 
receptor or simply the ligand binding subunit  has yet to be 
determined. Proof that  the 130-kDa protein is a functional 
receptor is hampered by the lack of information on the  intra- 
celfular signaling mechanism of the  GH receptor. However, 
evidence that  the GH receptor described here plays an impor- 
tant role in growth  comes  from recent results showing that 
Laron-type dwarfs, who have normal hGH levels but no liver 
GH binding (45), also lack functional serum binding protein 
(46,47). These observations, coupled with our demonstration 
that  the serum binding protein is the extracellular GH binding 
domain of the liver receptor, show that these proteins are 
intimately involved in  the growth  process. In addition, anti- 
body binding data indicate that  the GH receptor (or a struc- 
turally similar protein) is localized to chrondrocytes at  the 
epiphyseal growth plate (48). However, final proof of the role 
of the GH receptor described here in  the transduction of the 
growth signal must await reconstitution of a functional system 
containing the cloned GH receptor or genetic analysis of 
patients with receptor defects. 
Acknowledgments-We thank  Jim McCabe for his help in  obtain- 
ing some of the  starting materials and Dr. Mike Spellman for his 
help with the glycosidase digestions. 
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RABBIT LIVER  GROWTH HORMONE RECEmOR AND SERUM BINDING PROTEIN: 
PURIFICATION, CHARACTUIIZATIONANDSEQUENCE 
Seven A. Spencer. R. Glenn Hammonds, William J. Hemel, 
Heluy Rodnguer. Michael 1. Wa1c.n. ad William 1. Woat 
Serum Bindinc Protein 
ARnrcyIoad 24340 1.ZW" 0.050 1 l W 6  
Urcaduate 
MgSlieinaiare 
0.87 430 494 9,900 36% 
0.15 470 3.130 63.0ix) 396 
0.086 170 20.UW 396.000 14% 
Growth Hormone Receptor and Serum  Binding  Protein 7865 
A 
kDa 
200- 
116- 
92 - 
- 
67 - 
44- 
30 - 
1 2 3 4 5 6  
""C .L B l 2  
kDa 
200 - 
116- 7 
92 - 
67 - 
44- 30- 
21 - 
A 
"- 1 2 3 4 5 6  11m- 
kDa 
200- 
116- 
92 - 
67 - 
4 
44- 
B - +  - 
kDa - "  
200- 
67- e 
44- 
1 2 3  
kDa 
- 200 
0"" 
116- 
92- 
- 116 
- 92 
- 67 
30- - 45 
- 30 
"- 
7866 Growth  Hormone Receptor  and Serum  Binding Protein 
Growth  Hormone Receptor  and Serum  Binding  Protein 
Relentlon Tlme (mmutes) 
0 10 20 30 40 50 
Relenmn Tlme (minutes) 


Rabbit liver growth hormone receptor and serum binding protein: purification, characterization, and sequence

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Rabbit liver growth hormone receptor and serum binding protein: purification, characterization, and sequence

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