Growth Hormone (GH) Stimulates Protein Synthesis in Cells Transfected with GH Receptor Complementary DNA
Morgan Emtner, Lawrence S. Mathews*, and Gunnar Norstedt Center for Biotechnology (L.M., G.N.) Department of Medical Nutrition (G.N.) Karolinska Institute Novum Huddinge University Hospital 141 80 Huddinge, Sweden Department of Biochemistry University of Uppsala Biomedical Center S-751 23 Uppsala, Sweden
An expression vector containing a rat GH receptor cDNA was transfected into Chinese hamster ovary (CHO) cells, and stable cell lines expressing GH receptors were established. In contrast to nontransfected CHO cells, expression of GH receptors in transfected cells resulted in the appearance of high affinity (Kd = 1.53 nivi) specific binding of GH. Crosslinking of [125l]hGH to the receptors and subsequent sodium dodecyl sulfate (SDS)-electrophoresis gave an estimated receptor mol wt of 84,000. GH treatment stimulated protein synthesis 60% over basal levels in GH receptor-expressing CHO cells, but not in the receptor-negative parental cells. The effect was observed only under serum-free conditions and was time and dose dependent. These results show that heterologous expression of the rat GH receptor results in the appearance of specific binding of GH and the acquisition of a functional GH response. (Molecular Endocrinology 4:2014-2020,1990)
gests that the GH and PRL receptors may belong to a separate family of receptors (6), although they are homologous in a part of the extracellular region with a number of other receptors, including the erythropoietin, interleukin-2, interleukin-4, interleukin-6, and granulocyte-macrophage colony-stimulating factor receptors (7). The GH receptor sequence predicts a protein of approximately 620 amino acids, with a single membrane-spanning domain. Transfection of the GH receptor cDNA into mammalian tissue culture cells results in high affinity binding of GH (3,8); however, to fully characterize the GH receptor it is essential to determine whether expression of exogenous receptors affects cellular function. In this study we have transfected the rat GH receptor cDNA into Chinese hamster ovary K1 (CHO) cells and analyzed the regulation of protein synthesis by GH.
RESULTS Expression of Rat GH Receptor cDNA in CHO Cells
INTRODUCTION
A rat GH receptor cDNA clone was ligated into a plasmid containing the SV40 enhancer and the human metallothionein Ha promotor (Fig. 1) and was transfected into CHO cells together with the plasmid pIBP1, which contains a thymidine kinase promotor fused to the bacterial neo gene coding for resistance to the drug G418 (9). G418-resistant cell lines were established from five different clones, all of which expressed GH receptor mRNA. One of the cell lines, CHO-4, expressed high levels of GH receptor mRNA and was chosen for further studies. The level of GH receptor mRNA in CHO-4 cells was 20.3 ± 2.2 amol/^g DNA (n = 4), while in untransfected CHO cells no GH receptor
The initial step of GH action is binding of the hormone to a membrane-associated receptor. Biochemical analysis of the GH receptor, including cross-linking studies, has shown that the receptor is glycosylated and occurs in several different mol wt forms (1, 2). GH receptor cDNA clones have recently been obtained from man and rabbit (3), mouse (4), and rat (5), and show a 7075% homology between species. Comparison of the cDNA sequences and sequences of other proteins sug0888-8809/90/2014-2020$02.00/0 Molecular Endocrinology
2014
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 01 February 2015. at 21:08 For personal use only. No other uses without permission. . All rights reserved.
GH Stimulation of Protein Synthesis
2015
Bam HVBgl U
f
j
200
Spe I/Xba I
92 69
Fig. 1. Construction of GH Receptor Expression Vector A full-length GH receptor cDNA was ligated (see Materials and Methods) into the plasmid pUC 8 containing elements of human metallothionein lla promotor (hMT lla), SV 40 (SV 40 enhancer), and hGH (polyadenylation site and transcription termination derived from a hGH gene).
100-
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25 50 B(fmol) 1
75
10
^ ^ ^ « " ^
100
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1000
nM hormone Fig. 2. Binding of [125l]hGH to CHO-4 Cells Was Measured in the Presence of Increasing Amounts of Unlabeled hGH (•) and ovine (o) PRL (O). Inset, Scatchard analysis of the data. B/Bo, Bound to free ratio.
mRNA (
Morgan Emtner, Lawrence S. Mathews*, and Gunnar Norstedt Center for Biotechnology (L.M., G.N.) Department of Medical Nutrition (G.N.) Karolinska Institute Novum Huddinge University Hospital 141 80 Huddinge, Sweden Department of Biochemistry University of Uppsala Biomedical Center S-751 23 Uppsala, Sweden
An expression vector containing a rat GH receptor cDNA was transfected into Chinese hamster ovary (CHO) cells, and stable cell lines expressing GH receptors were established. In contrast to nontransfected CHO cells, expression of GH receptors in transfected cells resulted in the appearance of high affinity (Kd = 1.53 nivi) specific binding of GH. Crosslinking of [125l]hGH to the receptors and subsequent sodium dodecyl sulfate (SDS)-electrophoresis gave an estimated receptor mol wt of 84,000. GH treatment stimulated protein synthesis 60% over basal levels in GH receptor-expressing CHO cells, but not in the receptor-negative parental cells. The effect was observed only under serum-free conditions and was time and dose dependent. These results show that heterologous expression of the rat GH receptor results in the appearance of specific binding of GH and the acquisition of a functional GH response. (Molecular Endocrinology 4:2014-2020,1990)
gests that the GH and PRL receptors may belong to a separate family of receptors (6), although they are homologous in a part of the extracellular region with a number of other receptors, including the erythropoietin, interleukin-2, interleukin-4, interleukin-6, and granulocyte-macrophage colony-stimulating factor receptors (7). The GH receptor sequence predicts a protein of approximately 620 amino acids, with a single membrane-spanning domain. Transfection of the GH receptor cDNA into mammalian tissue culture cells results in high affinity binding of GH (3,8); however, to fully characterize the GH receptor it is essential to determine whether expression of exogenous receptors affects cellular function. In this study we have transfected the rat GH receptor cDNA into Chinese hamster ovary K1 (CHO) cells and analyzed the regulation of protein synthesis by GH.
RESULTS Expression of Rat GH Receptor cDNA in CHO Cells
INTRODUCTION
A rat GH receptor cDNA clone was ligated into a plasmid containing the SV40 enhancer and the human metallothionein Ha promotor (Fig. 1) and was transfected into CHO cells together with the plasmid pIBP1, which contains a thymidine kinase promotor fused to the bacterial neo gene coding for resistance to the drug G418 (9). G418-resistant cell lines were established from five different clones, all of which expressed GH receptor mRNA. One of the cell lines, CHO-4, expressed high levels of GH receptor mRNA and was chosen for further studies. The level of GH receptor mRNA in CHO-4 cells was 20.3 ± 2.2 amol/^g DNA (n = 4), while in untransfected CHO cells no GH receptor
The initial step of GH action is binding of the hormone to a membrane-associated receptor. Biochemical analysis of the GH receptor, including cross-linking studies, has shown that the receptor is glycosylated and occurs in several different mol wt forms (1, 2). GH receptor cDNA clones have recently been obtained from man and rabbit (3), mouse (4), and rat (5), and show a 7075% homology between species. Comparison of the cDNA sequences and sequences of other proteins sug0888-8809/90/2014-2020$02.00/0 Molecular Endocrinology
2014
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 01 February 2015. at 21:08 For personal use only. No other uses without permission. . All rights reserved.
GH Stimulation of Protein Synthesis
2015
Bam HVBgl U
f
j
200
Spe I/Xba I
92 69
Fig. 1. Construction of GH Receptor Expression Vector A full-length GH receptor cDNA was ligated (see Materials and Methods) into the plasmid pUC 8 containing elements of human metallothionein lla promotor (hMT lla), SV 40 (SV 40 enhancer), and hGH (polyadenylation site and transcription termination derived from a hGH gene).
100-
• 0.0&]
80-
o
1
1
O.Ofr
60-
Us 0.04-
•
hGH
O
oPRL
40" \ 0.02-
m
200.00" (
o.01
\ \
\
\ "X
25 50 B(fmol) 1
75
10
^ ^ ^ « " ^
100
•
1000
nM hormone Fig. 2. Binding of [125l]hGH to CHO-4 Cells Was Measured in the Presence of Increasing Amounts of Unlabeled hGH (•) and ovine (o) PRL (O). Inset, Scatchard analysis of the data. B/Bo, Bound to free ratio.
mRNA (
