- Email: [email protected]
Regulation of human growth hormone receptor gene transcription by human growth hormone binding protein
Molecular and Cellular Endocrinology 131 (1997) 89 – 96
Regulation of human growth hormone receptor gene transcription by human growth hormone binding protein Primus E. Mullis a,*, Johannes K. Wagner a, Andre´e Eble´ a, Jean-Marc Nuoffer a, Marie-Catherine Postel-Vinay b a
Department of Paediatrics, Di6ision of Paediatric Endocrinology, Uni6ersity Childrens Hospital, Inselspital, CH-3010 Bern, Switzerland b INSERM U-344, Endocrinologie Mole´culaire, Necker-Enfants Malades, 75730 Paris, France Received 14 March 1997; accepted 28 April 1997
Abstract The hypothesis that growth hormone binding protein (GHBP) has an effect on its own on the regulation of the GH-receptor/ GHBP transcription was tested. Three different forms of human GHBP (recombinant non-glycosylated GHBP, recombinant glycosylated GHBP and GHBP purified and extracted from serum) were added in different concentrations determined by LIFA [0 pmol/l; 50 pmol/l (low level), 200 pmol/
90
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
ment of GH action through prolonging GH-bioavailability. Furthermore, recent work has shown that the first step in GH action is the formation of a complex consisting of one hormone molecule bound to two receptors and, thereafter, dimerization of the receptors is crucial for signal transduction and receptor-mediated nuclear translocation of GH (for review: Wells, 1996; Lobie et al., 1994a; Kelly et al., 1994). Importantly, nuclear translocation of GH was observed in GH-receptor but not GHBP-transfected cells (Lobie et al., 1994b). Thus, nuclear translocation of GH is receptor dependent. However, whether the receptor is required only for internalization of GH and not for intracellular movement from the cell surface to the nucleus remains unknown. It is possible that once internalized the hormone-receptor complex could dissociate and the hormone translocate by a receptor-independent pathway, possibly complexed to the GHBP. Interestingly, GHBP has been found in a soluble state in nucleoplasma as well as attached to inner and outer nuclear membranes (Lobie et al., 1994c). Although the intracellular and specifically the intranuclear role of GHBP is unknown it can be concluded from all these data that GHBP is more than simply a shed or secreted product with extracellular destination and function. The aim of this study was to test the hypothesis that GHBP might have some direct effect on the regulation of the GH-receptor/GHBP transcription and, therefore, a major impact on the regulation of GH availability to the GH-receptor within the cells, especially in the nucleus.
2. Material and methods
2.1. Cell culture and growth hormone binding protein HuH7 is a human hepatoma cell line that is reported to retain differentiated functions in culture (Nakabayashi et al., 1982) and, therefore, allows functional studies of GH-receptor gene expression. HuH 7 cells were maintained in monolayer culture as previously described (Mullis et al., 1991). When they had reached approximately 70% confluency the medium was aspirated, the cells were washed twice with phosphatebuffered saline (PBS, pH 7.4; Sigma, St. Louis, MO) and 0.5 ml of a serum-free hormonally-defined medium was added. Briefly, serum-free hormonally-defined medium contains as previously reported neither growth hormone nor IGF-I, IGF-II: 0.4 mM ornithine; 2.25 mg/ml L-lactic acid; 500 ng/ml glucagon, 2.5 × 10 − 8 M selenium; 5× 10 − 8
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
was calculated using spectrophotometric absorbance at 260 nm, the molecular weight of cRNA molecule (216 600 g/mol) and Avogadro’s number.
2.4. Oligonucleotide primers used for amplification Oligonucleotide primers were purchased from Mycrosynth, Balgach, Switzerland. The forward primer was 5%CCC TAT ATT GAC AAC ATC AGT TCC-3%; nucleotide 624–647 (exon 7; Leung et al. (1987)) and the antisense primer was 5%-TTT CCT TCC TTG AGG AGA TCT GG-3% nucleotide 931 – 954 (exon 9; Leung et al. (1987)).
2.5. cDNA synthesis and PCR amplification Four micrograms of total RNA and 2.0 × 106 molecules of internal control cRNA were reverse transcribed with 200 U Moloney murine leukemia virus reverse transcriptase (RT-M-MLV; Gibco-BRL, Life Technologies, Basel, Switzerland) primed with 1 mg oligo (deoxythymidine)12 – 18 primer (BoehringerMannheim; Rotkreuz, Switzerland). The RT reaction was carried out in 20 ml (total volume) RT-buffer (50 mM KCl, 2 mM Mg2Cl, and 20 mM Tris – HCl, pH 8.3), 1 mM of each deoxy-NTP, 1 mM dithiothreitol, and 20 U RNasin (Promega, Catalys, Wallisellen, Switzerland). Total RNA, cRNA and Oligo [dT]12 – 18 was heated to 70°C for 10 min and then chilled on ice, thereafter RT-M-MLV, RNasin, dNTP and RT-buffer was added and the mix was incubated for 60 min at 37°C and chilled on ice, subsequently. The PCR amplification was carried out using 8 ml of each diluted RT-mixture in PCR-buffer (50 mM KCl, 2 mM Mg2Cl, and 20 mM Tris–HCl, pH 8.3), 200 mM deoxy-NTPs, 25 pmol forward and reverse primers, 5 ml of 50% formamide, 1×106 cpm 32P-end-labelled sense primer
91
92
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
Table 1 Number of GH-receptor mRNA molecules in HuH7 cells using quantitative PCR GH-receptor mRNA (×106 molecules/mg total RNA) Incubation time (h) 0
P-valuea
1
P-valueb
2
Non-glycosylated [rhGHBP] 0 pmol/l 50 pmol/l 200 pmol/l 500 pmol/l
2.7390.13 2.059 0.07 2.0190.12 2.129 0.04
0.08 B0.001 0.61 B0.001
2.58 9 0.04 2.49 90.06 2.05 90.08 0.899 0.05
0.79 B0.001 0.19 B0.001
2.57 9 0.09 2.89 90.03 1.97 90.08 0.59 90.04
Glycosylated [rhGHBP] 0 pmol/l 50 pmol/l 200 pmol/l 500 pmol/l
2.389 0.10 2.64 9 0.06 2.50 9 0.12 2.49 9 0.11
0.21 0.001 0.27 B0.001
2.31 90.05 2.90 90.10 2.58 90.07 1.12 90.16
0.6 0.016 0.1 B0.001
2.29 90.08 3.14 90.14 2.46 9 0.12 0.56 90.08
Serum extracted [hGHBP] 0 pmol/l 50 pmol/l 200 pmol/l 500 pmol/l
2.459 0.11 2.309 0.10 2.35 9 0.09 2.61 9 0.06
0.06 0.006 0.93 B0.001
2.39 90.07 2.49 90.05 2.35 90.12 1.15 90.09
0.3 0.001 0.59 B0.001
2.33 90.06 2.87 90.14 2.39 90.08 0.48 90.10
Results are are expressed as the mean9 S.E.M. Four different experiments in each set (different GHBPs) were performed to express GH-receptor mRNA concentrations by quantitative PCR as described in Section 2. a Statistical difference between incubation times 0 and 1. b Statistical difference between incubation times 1 and 2.
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
was extracted 2 h after the addition of different concentrations of the GHBP which was extracted and purified from serum.
3.1.1. After addition of recombinant human non-glycosylated GHBP (non-glycosylated r-hGHBP) Treatment with a concentration of 50 pmol/l non-glycosylated r-hGHBP resulted in a significant increase of GH-receptor mRNA molecules given as number of molecules ×106/mg total RNA. The increase was obvious 1 h after addition of the non-glycosylated r-hGHBP to the culture medium (Table 1) and became more significant following the second hour. The concentration of 500 pmol/l, representing a high value of normally occurring GHBP concentration in circulation, showed a significant decrease of GH-receptor mRNA molecules, whereas 200 pmol/l of non-glycosylated rhGHBP produced a GH-receptor gene expression during the 2 h studied which was in between the values of the experiments with 50 and 500 pmol/l of GHBP. In the experiments without any non-glycosylated rhGHBP added the GH-receptor expression presented a constant but not significant decrease of GH-receptor transcripts during the duration of the experiments. 3.1.2. After addition of recombinant human glycosylated GHBP (glycosylated r-hGHBP) Although the increase of GH-receptor expression in the 50 pmol/l glycosylated r-hGHBP experiments was not as significant as in the experiments in which nonglycosylated r-hGHBP were used, the results obtained using glycosylated r-hGHBP were similar to the data obtained in the experiments with the non-glycosylated r-hGHBP form (Table 1). In addition, glycosylated r-hGHBP in a concentration of 500 pmol/l resulted in a decrease of GH-receptor transcripts and 200 pmol/l of glycosylated r-hGHBP did not alter the GH-receptor expression (Table 1). 3.1.3. After addition of GHBP purified and extracted from human serum (serum extracted GHBP) The data obtained were similar to results already described following the addition of either non-glycosylated r-hGHBP or glycosylated r-hGHBP. Therefore, there were no differences whenever the data from the experiments with the different GHBP forms were compared among each other (Table 1). As in the two other experiments the ‘blank’ data (without any GHBP added) showed a slight decrease of GH-receptor transcripts which never reached any statistical significance. 3.2. Run-on assay Furthermore, we assessed by performing nuclear runon experiments the question whether the changes in GH-receptor gene transcription levels were real and,
93
therefore, resulted from a changed rate of transcription. HuH7 cells were cultured fo
94
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
Fig. 2. Effect of different forms and concentrations of GHBP following 2 h incubation time on transcription of GH-receptor gene in HuH7 cells analysed by run-on experiments. The autoradiographic signals were quantified by liquid scintillation counting of filter pieces as described in Section 2. The ratio of signal obtained with GH-receptor target DNA to that obtained with b-tubulin target DNA was calculated for each set of culture conditions. This ratio was arbitrarily set at 1 U for untreated HuH7 cells, and other values were adjusted accordingly and plotted. Values plotted are means of values obtained in four parallel experiments from four individual cultures. The lines above the bar indicate S.E.M.; ** represents PB 0.005, whereas *PB 0.001 compared with controls; white boxes, without cycloheximide; hatched boxes, with cycloheximide.
longitudinal values for a given individual (Martha et al., 1993). This all together implies that the GHBP levels are regulated in an individual within a characteristic range and, furthermore, it provides evidence that the specific level of serum GHBP is biologically relevant for the growth rate in an individual child in combination and correlation to the active GH. While mapping the somatic distribution of GH-receptor by immunohistochemistry, nuclear localization of GH-receptor and, additionally, GH and GHBP soluble in nucleoplasma as well as attached to nuclear membranes were found (Lobie et al., 1994a,b,c). This lead us to test the
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
GHBP forms used did not differ in their effects on GH-receptor gene expression. After 2 h of
95
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
96
Lobie, P.E., Wood, T.J.J., Chen, C.M., Waters, M.J., Norstedt, G., Mullis, P.E., Lund, T., Patel, M.S., Brook, C.G.D., Brickell, P.M., 1994c. Nuclear translocation and anchorage of the growth hor1991. Regulation of growth geúe exmone receptor. J. Biol. Chem. 269, 31735–31746. pression by human growth a human cell Mannor, D.A., Winer, L.M., Shaw, M.A., Baumann, G., 1991. line. Mol. Cell. Endocrinol. 76, 125 – 133. Plasma growth GH)-binding proteins: effect on Mullis, GH P.E., Holl, R.W., Lund, T., Eble´, A., Brickell, P.M., 1995. binding to receptors and GH action. J. Clin. Endocrinol. Metab. Regulation of human growth protein 73, 30 – 34. tion Martha Jr, P.M., Rogol, A.D., Blizzard, R.M., Shaw, M.A., BauCell. Endocrinol. 111, 181 – 190. mann, G., 1991. Growth activity Nakabayashi, N., Taketa, K., Miyano, K., Yamane, T., Sato, K., inversely related to 24-hour growth 1982. Growth of human hepatoma cell lines with differentiated boys. J. Clin. Endocrinol. Metab. 73, 175–181. functions chemically medium. Cancer Res. Martha Jr, P.M., Rogol, A.D. Jr., Carlsson, L.M.S., Gesundheit, N., 3863. Blizzard, R.M., 1993. A longitudinal assessment of 747.onal and Súedecor, G.W., Cochran, W.G. 1980. Statistical Methods, 7th ed. physical alterations during normal puberty in boys. I. Serum Iowa State University Press, Ames, IA, pp. 59 – 61 growth protein. J. Clin. Metab. Sotiropoulos, A., Goujon, L., Si.07úin, G., Kelly, P.A., Postel-Vinay, 452 – 457. M.C., Finidori, Martini, J.F., Villares, S.M., Nagano, M., Delehaye-Zervas, M.C., growth 747.oúe binding protein through proteolysis of the Eymard, B., Kelly, P.A., Postel-Vinay, M.C., 1995. Quantitative growth membrane receptor. Endocrinology analysis by polymerase chain reaction of growth 747.oúe recep1865. tor geúe expression in human liver an muscle. Endocrinology 136, Wells, J.A., 1996. Binding in the growth receptor 1355 – 1360. Natl. Acad. Sci. USA 93, 1 – 6.
.
.
Regulation of human growth hormone receptor gene transcription by human growth hormone binding protein Primus E. Mullis a,*, Johannes K. Wagner a, Andre´e Eble´ a, Jean-Marc Nuoffer a, Marie-Catherine Postel-Vinay b a
Department of Paediatrics, Di6ision of Paediatric Endocrinology, Uni6ersity Childrens Hospital, Inselspital, CH-3010 Bern, Switzerland b INSERM U-344, Endocrinologie Mole´culaire, Necker-Enfants Malades, 75730 Paris, France Received 14 March 1997; accepted 28 April 1997
Abstract The hypothesis that growth hormone binding protein (GHBP) has an effect on its own on the regulation of the GH-receptor/ GHBP transcription was tested. Three different forms of human GHBP (recombinant non-glycosylated GHBP, recombinant glycosylated GHBP and GHBP purified and extracted from serum) were added in different concentrations determined by LIFA [0 pmol/l; 50 pmol/l (low level), 200 pmol/
90
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
ment of GH action through prolonging GH-bioavailability. Furthermore, recent work has shown that the first step in GH action is the formation of a complex consisting of one hormone molecule bound to two receptors and, thereafter, dimerization of the receptors is crucial for signal transduction and receptor-mediated nuclear translocation of GH (for review: Wells, 1996; Lobie et al., 1994a; Kelly et al., 1994). Importantly, nuclear translocation of GH was observed in GH-receptor but not GHBP-transfected cells (Lobie et al., 1994b). Thus, nuclear translocation of GH is receptor dependent. However, whether the receptor is required only for internalization of GH and not for intracellular movement from the cell surface to the nucleus remains unknown. It is possible that once internalized the hormone-receptor complex could dissociate and the hormone translocate by a receptor-independent pathway, possibly complexed to the GHBP. Interestingly, GHBP has been found in a soluble state in nucleoplasma as well as attached to inner and outer nuclear membranes (Lobie et al., 1994c). Although the intracellular and specifically the intranuclear role of GHBP is unknown it can be concluded from all these data that GHBP is more than simply a shed or secreted product with extracellular destination and function. The aim of this study was to test the hypothesis that GHBP might have some direct effect on the regulation of the GH-receptor/GHBP transcription and, therefore, a major impact on the regulation of GH availability to the GH-receptor within the cells, especially in the nucleus.
2. Material and methods
2.1. Cell culture and growth hormone binding protein HuH7 is a human hepatoma cell line that is reported to retain differentiated functions in culture (Nakabayashi et al., 1982) and, therefore, allows functional studies of GH-receptor gene expression. HuH 7 cells were maintained in monolayer culture as previously described (Mullis et al., 1991). When they had reached approximately 70% confluency the medium was aspirated, the cells were washed twice with phosphatebuffered saline (PBS, pH 7.4; Sigma, St. Louis, MO) and 0.5 ml of a serum-free hormonally-defined medium was added. Briefly, serum-free hormonally-defined medium contains as previously reported neither growth hormone nor IGF-I, IGF-II: 0.4 mM ornithine; 2.25 mg/ml L-lactic acid; 500 ng/ml glucagon, 2.5 × 10 − 8 M selenium; 5× 10 − 8
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
was calculated using spectrophotometric absorbance at 260 nm, the molecular weight of cRNA molecule (216 600 g/mol) and Avogadro’s number.
2.4. Oligonucleotide primers used for amplification Oligonucleotide primers were purchased from Mycrosynth, Balgach, Switzerland. The forward primer was 5%CCC TAT ATT GAC AAC ATC AGT TCC-3%; nucleotide 624–647 (exon 7; Leung et al. (1987)) and the antisense primer was 5%-TTT CCT TCC TTG AGG AGA TCT GG-3% nucleotide 931 – 954 (exon 9; Leung et al. (1987)).
2.5. cDNA synthesis and PCR amplification Four micrograms of total RNA and 2.0 × 106 molecules of internal control cRNA were reverse transcribed with 200 U Moloney murine leukemia virus reverse transcriptase (RT-M-MLV; Gibco-BRL, Life Technologies, Basel, Switzerland) primed with 1 mg oligo (deoxythymidine)12 – 18 primer (BoehringerMannheim; Rotkreuz, Switzerland). The RT reaction was carried out in 20 ml (total volume) RT-buffer (50 mM KCl, 2 mM Mg2Cl, and 20 mM Tris – HCl, pH 8.3), 1 mM of each deoxy-NTP, 1 mM dithiothreitol, and 20 U RNasin (Promega, Catalys, Wallisellen, Switzerland). Total RNA, cRNA and Oligo [dT]12 – 18 was heated to 70°C for 10 min and then chilled on ice, thereafter RT-M-MLV, RNasin, dNTP and RT-buffer was added and the mix was incubated for 60 min at 37°C and chilled on ice, subsequently. The PCR amplification was carried out using 8 ml of each diluted RT-mixture in PCR-buffer (50 mM KCl, 2 mM Mg2Cl, and 20 mM Tris–HCl, pH 8.3), 200 mM deoxy-NTPs, 25 pmol forward and reverse primers, 5 ml of 50% formamide, 1×106 cpm 32P-end-labelled sense primer
91
92
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
Table 1 Number of GH-receptor mRNA molecules in HuH7 cells using quantitative PCR GH-receptor mRNA (×106 molecules/mg total RNA) Incubation time (h) 0
P-valuea
1
P-valueb
2
Non-glycosylated [rhGHBP] 0 pmol/l 50 pmol/l 200 pmol/l 500 pmol/l
2.7390.13 2.059 0.07 2.0190.12 2.129 0.04
0.08 B0.001 0.61 B0.001
2.58 9 0.04 2.49 90.06 2.05 90.08 0.899 0.05
0.79 B0.001 0.19 B0.001
2.57 9 0.09 2.89 90.03 1.97 90.08 0.59 90.04
Glycosylated [rhGHBP] 0 pmol/l 50 pmol/l 200 pmol/l 500 pmol/l
2.389 0.10 2.64 9 0.06 2.50 9 0.12 2.49 9 0.11
0.21 0.001 0.27 B0.001
2.31 90.05 2.90 90.10 2.58 90.07 1.12 90.16
0.6 0.016 0.1 B0.001
2.29 90.08 3.14 90.14 2.46 9 0.12 0.56 90.08
Serum extracted [hGHBP] 0 pmol/l 50 pmol/l 200 pmol/l 500 pmol/l
2.459 0.11 2.309 0.10 2.35 9 0.09 2.61 9 0.06
0.06 0.006 0.93 B0.001
2.39 90.07 2.49 90.05 2.35 90.12 1.15 90.09
0.3 0.001 0.59 B0.001
2.33 90.06 2.87 90.14 2.39 90.08 0.48 90.10
Results are are expressed as the mean9 S.E.M. Four different experiments in each set (different GHBPs) were performed to express GH-receptor mRNA concentrations by quantitative PCR as described in Section 2. a Statistical difference between incubation times 0 and 1. b Statistical difference between incubation times 1 and 2.
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
was extracted 2 h after the addition of different concentrations of the GHBP which was extracted and purified from serum.
3.1.1. After addition of recombinant human non-glycosylated GHBP (non-glycosylated r-hGHBP) Treatment with a concentration of 50 pmol/l non-glycosylated r-hGHBP resulted in a significant increase of GH-receptor mRNA molecules given as number of molecules ×106/mg total RNA. The increase was obvious 1 h after addition of the non-glycosylated r-hGHBP to the culture medium (Table 1) and became more significant following the second hour. The concentration of 500 pmol/l, representing a high value of normally occurring GHBP concentration in circulation, showed a significant decrease of GH-receptor mRNA molecules, whereas 200 pmol/l of non-glycosylated rhGHBP produced a GH-receptor gene expression during the 2 h studied which was in between the values of the experiments with 50 and 500 pmol/l of GHBP. In the experiments without any non-glycosylated rhGHBP added the GH-receptor expression presented a constant but not significant decrease of GH-receptor transcripts during the duration of the experiments. 3.1.2. After addition of recombinant human glycosylated GHBP (glycosylated r-hGHBP) Although the increase of GH-receptor expression in the 50 pmol/l glycosylated r-hGHBP experiments was not as significant as in the experiments in which nonglycosylated r-hGHBP were used, the results obtained using glycosylated r-hGHBP were similar to the data obtained in the experiments with the non-glycosylated r-hGHBP form (Table 1). In addition, glycosylated r-hGHBP in a concentration of 500 pmol/l resulted in a decrease of GH-receptor transcripts and 200 pmol/l of glycosylated r-hGHBP did not alter the GH-receptor expression (Table 1). 3.1.3. After addition of GHBP purified and extracted from human serum (serum extracted GHBP) The data obtained were similar to results already described following the addition of either non-glycosylated r-hGHBP or glycosylated r-hGHBP. Therefore, there were no differences whenever the data from the experiments with the different GHBP forms were compared among each other (Table 1). As in the two other experiments the ‘blank’ data (without any GHBP added) showed a slight decrease of GH-receptor transcripts which never reached any statistical significance. 3.2. Run-on assay Furthermore, we assessed by performing nuclear runon experiments the question whether the changes in GH-receptor gene transcription levels were real and,
93
therefore, resulted from a changed rate of transcription. HuH7 cells were cultured fo
94
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
Fig. 2. Effect of different forms and concentrations of GHBP following 2 h incubation time on transcription of GH-receptor gene in HuH7 cells analysed by run-on experiments. The autoradiographic signals were quantified by liquid scintillation counting of filter pieces as described in Section 2. The ratio of signal obtained with GH-receptor target DNA to that obtained with b-tubulin target DNA was calculated for each set of culture conditions. This ratio was arbitrarily set at 1 U for untreated HuH7 cells, and other values were adjusted accordingly and plotted. Values plotted are means of values obtained in four parallel experiments from four individual cultures. The lines above the bar indicate S.E.M.; ** represents PB 0.005, whereas *PB 0.001 compared with controls; white boxes, without cycloheximide; hatched boxes, with cycloheximide.
longitudinal values for a given individual (Martha et al., 1993). This all together implies that the GHBP levels are regulated in an individual within a characteristic range and, furthermore, it provides evidence that the specific level of serum GHBP is biologically relevant for the growth rate in an individual child in combination and correlation to the active GH. While mapping the somatic distribution of GH-receptor by immunohistochemistry, nuclear localization of GH-receptor and, additionally, GH and GHBP soluble in nucleoplasma as well as attached to nuclear membranes were found (Lobie et al., 1994a,b,c). This lead us to test the
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
GHBP forms used did not differ in their effects on GH-receptor gene expression. After 2 h of
95
P.E. Mullis et al. / Molecular and Cellular Endocrinology 131 (1997) 89–96
96
Lobie, P.E., Wood, T.J.J., Chen, C.M., Waters, M.J., Norstedt, G., Mullis, P.E., Lund, T., Patel, M.S., Brook, C.G.D., Brickell, P.M., 1994c. Nuclear translocation and anchorage of the growth hor1991. Regulation of growth geúe exmone receptor. J. Biol. Chem. 269, 31735–31746. pression by human growth a human cell Mannor, D.A., Winer, L.M., Shaw, M.A., Baumann, G., 1991. line. Mol. Cell. Endocrinol. 76, 125 – 133. Plasma growth GH)-binding proteins: effect on Mullis, GH P.E., Holl, R.W., Lund, T., Eble´, A., Brickell, P.M., 1995. binding to receptors and GH action. J. Clin. Endocrinol. Metab. Regulation of human growth protein 73, 30 – 34. tion Martha Jr, P.M., Rogol, A.D., Blizzard, R.M., Shaw, M.A., BauCell. Endocrinol. 111, 181 – 190. mann, G., 1991. Growth activity Nakabayashi, N., Taketa, K., Miyano, K., Yamane, T., Sato, K., inversely related to 24-hour growth 1982. Growth of human hepatoma cell lines with differentiated boys. J. Clin. Endocrinol. Metab. 73, 175–181. functions chemically medium. Cancer Res. Martha Jr, P.M., Rogol, A.D. Jr., Carlsson, L.M.S., Gesundheit, N., 3863. Blizzard, R.M., 1993. A longitudinal assessment of 747.onal and Súedecor, G.W., Cochran, W.G. 1980. Statistical Methods, 7th ed. physical alterations during normal puberty in boys. I. Serum Iowa State University Press, Ames, IA, pp. 59 – 61 growth protein. J. Clin. Metab. Sotiropoulos, A., Goujon, L., Si.07úin, G., Kelly, P.A., Postel-Vinay, 452 – 457. M.C., Finidori, Martini, J.F., Villares, S.M., Nagano, M., Delehaye-Zervas, M.C., growth 747.oúe binding protein through proteolysis of the Eymard, B., Kelly, P.A., Postel-Vinay, M.C., 1995. Quantitative growth membrane receptor. Endocrinology analysis by polymerase chain reaction of growth 747.oúe recep1865. tor geúe expression in human liver an muscle. Endocrinology 136, Wells, J.A., 1996. Binding in the growth receptor 1355 – 1360. Natl. Acad. Sci. USA 93, 1 – 6.
.
.