0021-972X/91/7306-1175I03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright (c) 1991 by The Endocrine Society

Vol. 73, No. 6 Printed in U.S.A.

Binding of Human Growth Hormone (GH)-Variant (Placental GH) to GH-Binding Proteins in Human Plasma* GERHARD BAUMANN, NORMA DAVILAj, MELISSA A. SHAW, JHARNA RAY, STEPHEN A. LIEBHABER$, AND NANCY E. COOKE Center for Endocrinology, Metabolism, and Nutrition, Department of Medicine, Northwestern University Medical School (G.B., N.D., M.A.S.), Chicago, Illinois 60611; and the Howard Hughes Medical Institute, Departments of Human Genetics (J.R., S.A.L.) and Medicine (S.A.L., N.E.C.), University of Pennsylvania, Philadelphia, Pennsylvania 19104

in receptor binding) were exchanged, also bound with similarly high affinities. A corresponding hGH-N/rat PRL chimeric protein had 25-fold reduced affinity for the GH-BP. We conclude that hGH-V is a potent somatogen in man, and that some of the manifestations of late pregnancy, such as increased insulin-like growth factor-I levels and coarsening of features, are probably related to the high circulating levels of hGH-V. GH-BP measurements in pregnancy must take into account BP saturation by endogenous hGH-V. (J Clin Endocrinol Metab 73: 11751179,1991)

ABSTRACT. Human GH-variant (hGH-V) is a natural GH analog arising from the hGH-V gene. It is expressed in the placenta and secreted into the maternal circulation during the second half of pregnancy. To gain information about its bioactivity in man, we examined the interaction of hGH-V with the high affinity GH-binding protein/receptor (GH-BP) in human plasma. hGH-V was equipotent with pituitary hGH (hGH-N) as a ligand for the GH-BP. hGH-N/hGH-V chimeric proteins, where the sequences encoded by exon 3 (amino acid residues 3271, thought to be exposed on the molecule's surface and involved

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HE GH gene cluster on human chromosome 17 contains five GH-related genes. Two of these code for 2 closely related hGH isoforms, hGH-N and hGH-V; 2 others encode placental lactogen (PL), and a third PLrelated gene is of undefined function (1, 2). hGH-N (N for normal) is expressed in the pituitary gland, whereas hGH-V (V for variant) is expressed in the syncytiotrophoblast of the placenta (3-5). The 2 proteins are similar in structure; their amino acid sequences differ at 13 of 191 positions, with differences distributed throughout the molecule (6). hGH-V has also been termed placental GH (7, 8); it is coexpressed with PL and becomes the dominant GH in the maternal circulation during the second half of pregnancy (9). Concomitantly, pituitary GH (hGH-N) is suppressed in the mother in late pregnancy (9). Because hGH-V has only recently been recognized as

Received February 26,1991. Address all correspondence and requests for reprints to: G. Baumann, M.D., 303 East Chicago Avenue, Chicago, Illinois 60611. * This work was supported by NIH Grant DK-38128 (to G.B.), a grant from the Northwestern Memorial Foundation (to G.B.), NIH Grant HD-25147 (to N.E.C. and S.E.L.), and March of Dimes Grant 1-1015 (to N.E.C). t Supported by the Fondo de Investigation de la Seguridad Social Espanola. $ Associate Investigator of the Howard Hughes Medical Institute.

a distinct entity, its biological role is largely unknown. In the quest for such a role, the study of hormone binding to its specific receptor is of key importance. In initial studies, the interaction of hGH-V with heterologous GH receptors has been examined using microsomal preparations from rat and rabbit liver (10). hGH-V was found to differ from hGH-N in these systems by binding preferentially to somatogenic compared to lactogenic receptors (10). hGH-V has also been shown to possess growthpromoting activity in the hypophysectomized rat (11). However, to draw ultimately valid conclusions for a species-specific hormone, such as hGH, its activity must be tested in the homologous species. The present work examines hGH-V binding to the homologous, i.e. human, receptor/GH-binding protein (GH-BP).1 Human plasma contains two specific GH-BPs, one of which is related to the hepatic GH receptor (13-16). This GH-BP represents a soluble truncated receptor encompassing the extracellular portion, which contains the hormone-binding domain (17). As judged by GH binding assay, levels of this GH-BP appear unchanged in preg1

Although earlier work had examined the interaction of hGH-V with receptors on cultured human IM-9 lymphocytes (12), estimation of hGH-V concentrations, and hence of its affinity for the receptor, was not possible due to its poor reactivity in the RIA used.

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nancy (18). However, the latter observation did not take into account potential interference in the assay by endogenous hGH-V, which could influence GH-BP measurements by partially saturating the GH-BP. Knowledge about the quantitative aspects of the interaction between the GH-BP and hGH-V is required to address this question. The present study was undertaken to 1) examine the interaction of hGH-V with the homologous GH-BP/ receptor, 2) to better define the structural domains of hGH-N and hGH-V important in homologous receptor binding, and 3) to gain more precise information on the regulation of the GH-BP in pregnancy.

Materials and Methods Materials Natural pituitary hGH-N (22-kDa form; lot 306-13-3) was a gift from Dr. U. J. Lewis (La Jolla, CA). It was radioiodinated with 125I by a lactoperoxidase method, as previously described (13). A normal human plasma pool (endogenous GH, ^ D \ft

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FIG. 1. Displacement curves for hGH-N, hGH-V, and chimeric hGH proteins bound to the high affinity GH-BP in whole human plasma. [128I]hGH-N was incubated with plasma and conditioned medium containing the recombinant hGH proteins indicated, followed by separation of GH-BP complexes and free GH by size exclusion chromatography. Radioactivity associated with peak II (high affinity GH-BP complex; 85 kDa) was quantitated (see text for details). Total binding of [v25I]hGH was 18.5%. The curves for hGH-N and hGH-V represent averages of two independently prepared batches; those for the hGH chimeras represent a single production batch. vc

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Protein Concentration (pg/L)

FlG. 2. Displacement curves for hGH-N, hGH-V, and chimeric hGH proteins bound to purified high affinity GH-BP/receptor. [125I]hGH-N was incubated with purified GH-BP and analyzed as stated in Fig. 1. The total binding of [126I]hGH was 19.7%. Conditions and experiment numbers are as in Fig. 1. TABLE 1. Binding constants of recombinant hGH-related proteins with human high affinity GH-BP/receptor

hGH-N hGH-V hGH-NV3 hGH-VN3 hGH-NrP3

1177

IC50 (Mg/L)

Ka (L/M)

230 260 320 500 6000

2.38 X 108 2.09 x 108 1.06 X 108 0.52 x 108 8.90 x 106

IC50 denotes the concentration required for half-displacement; Ka is the equilibrium association constant.

The present study demonstrates that hGH-V is equipotent with hGH-N in its interaction with the human high affinity GH-BP in plasma. Since this GH-BP represents a truncated GH receptor, it can be assumed that hGH-V is also equipotent with hGH-N as a ligand for the human GH receptor. It is, therefore, likely that hGHV is biologically active as a somatogenic hormone in man. The findings reported here are consistent with previous data on the interaction of hGH-V with heterologous GH receptors. In rabbit liver, hGH-V bound with an affinity similar to that of hGH-N to somatogenic receptors (10). The chimeric proteins hGH-NV3 and hGH-VN3 also bound with similar affinities, although in the rabbit their potency rank was reversed compared to the present results. These differences are relatively minor and may be species related. [It should be noted in this context that the affinity of GH-BP for hGH is somewhat lower than that of the receptor (see Ref. 22 for review).] A natural preparation of placental GH, extracted from placenta and probably representing hGHV, was also shown to interact with GH receptors in rabbit liver membranes (9), although affinity estimates were not possible in that study. The results are also qualitatively consistent with those obtained in cultured human IM-9 lymphocytes (12), where hGH-V was shown to bind to the GH receptor, albeit without quantitative information. We wish to emphasize that information gained in animal models, although informative from a variety of viewpoints, is insufficient to reach physiological conclusions. For a species-specific hormone, such as hGH, testing in homologous systems is crucial for deriving physiologically meaningful concepts. The hGH-PRL chimera (hGH-NrP3) had 25-fold lower affinity for the GH-BP than hGH-N or hGH-V. This is, again, similar to its behavior in rabbit liver microsomes, where it interacted weakly with somatogenic receptors (21). This partial agonist activity of the hGH-PRL chimera differs from PRL itself, which shows no detectable binding to the human GH-BP (13) or the rabbit liver GH receptor (21) up to concentrations of 3 or 1 mg/L, respectively. Thus, although the sequence encoded by exon 3 is critical for efficient hGH binding to somatogenic receptors (21), the rest of the hGH sequence also plays some role in this interaction. Placentally produced hGH-V is released into the maternal circulation during pregnancy. Its plasma level rises progressively during the second and third trimesters and reaches an average level of 18 Mg/L near term (9). In view of the present data, this relatively high concentration of hGH-V has several consequences. First, GH-BP levels measured by the GH binding assay must be cor-

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rected for partial BP saturation by endogenous hGH-V. We have previously reported that GH-BP levels in late pregnancy are unchanged from those in the nongravid state (18), at a time when the potential impact of hGHV on GH-BP measurement was unknown. We have now recalculated the pregnancy data reported in Ref. 18, taking into account the effect of hGH-V (at an average level of 18 fig/L). This resulted in an upward correction of values by a factor of 1.14, which yielded a mean GHBP level virtually identical to that in nonpregnant women (mean ± SD, 24.4 ± 2.4% us. 24.8 ± 1.0% GH bound). Thus, the original conclusion that the high affinity GH-BP is not affected in pregnancy was corroborated and actually strengthened. Second, since hGH-V can be assumed to exhibit somatogenic activity, a plasma level of 10-20 ixg/L, if sustained, can be expected to result in a condition resembling mild acromegaly. hGH-V appears to be released tonically, and its plasma level is indeed sustained (23, 24). The marked increase in insulin-like growth factor I (IGF-I) levels during pregnancy (25-27) is consistent with hGH-V action, since hGH-N is largely suppressed (9, 24). Finally, the soft tissue swelling and occasional appearance of coarsened acromegaloid features in late pregnancy, previously attributed to high levels of PL, are more likely due to hGH-V than to PL, which is an extremely weak somatogen. This view is consistent with a previous report that showed a correlation between IGF-I and hGH-V, but none between IGFI and hPL (27). In summary, we report that hGH-V (placental GH) has full potency compared to hGH-N as a ligand for the human high affinity GH-BP/receptor. We, therefore, predict that hGH-V also exhibits full potency in binding to tissue GH receptors. Chimeric proteins in which the native sequence 32-71 [encoded by exon 3, exposed as a loop on the molecule's surface, and a region believed to be important for receptor binding (28)] is exchanged with the corresponding sequence of the other hGH isoform (either hGH-V or hGH-N) also bind with high affinity. In contrast to the largely conserved binding for hGH-N/hGH-V chimeras, replacement of the same sequence by the homologous PRL sequence decreases affinity 25-fold. hGH-V is likely to be active as a somatogenic hormone in humans; its high plasma levels in late pregnancy are probably responsible for several of the metabolic changes and clinical manifestations in the third trimester. The potential role of hGH-V as a lactogen in man remains to be elucidated.

References 1. Chen EY, Liao YC, Smith DH, Barrera-Saldana HA, Gelinas RF, Seeburg PH. The growth hormone locus: nucleotide sequence, biology, and evolution. Genomics. 1989;4:479-97. 2. Parks JS. Molecular biology of growth hormone. Acta Paediatr

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Scand. 1989;349(Suppl):127-35. 3. Frankenne F, Rentier-Delrue F, Scippo ML, Martial J, Hennen G. Expression of the growth hormone variant gene in human placenta. J Clin Endocrinol Metab. 1987;64:635-7. 4. Cooke NE, Ray J, Emery JG, Liebhaber SA. Two distinct species of human growth hormone-variant mRNA in the human placenta predict the expression of novel growth hormone proteins. J Biol Chem. 1988;263:9001-6. 5. Liebhaber SA, Urbanek M, Ray J, Tuan RS, Cooke NE. Characterization and histological localization of human growth hormonevariant gene expression in the placenta. J Clin Invest. 1989;83:1985-91. 6. Seeburg PH. The human growth hormone gene family: nucleotide sequences show recent divergence and predict a new polypeptide hormone. DNA. 1982;l:239-49. 7. Hennen G, Frankenne F, Pirens G, et al. A chorionic GH-like antigen: increasing levels during second half of pregnancy with pituitary suppression as revealed by monoclonal antibody radioimmunoassays. Int J Fertil. 1985;30:27-33. 8. Frankenne F, Scippo M-L, Van Beeumen J, Igout A, Hennen G. Identification of placental human growth hormone as the growth hormone-V gene expression product. J Clin Endocrinol Metab. 1990;71:15-8. 9. Frankenne F, Closset J, Gomez F, Scippo ML, Hennen G. The physiology of growth hormones (GHs) in pregnant women and partial characterization of the placental GH variant. J Clin Endocrinol Metab. 1988;66:1171-80. 10. Ray J, Okamura H, Kelly PA, Cooke NE, Liebhaber SA. Human growth hormone-variant demonstrates a receptor binding profile distinct from that of normal pituitary growth hormone. J Biol Chem. 1990;265:7939-44. 11. MacLeod JN, Worsley I, Ray J, Friesen HG, Liebhaber SA, Cooke NE. Human growth hormone-variant is a biologically active somatogen and lactogen. Endocrinology. 1991;128:1298-302. 12. Pavlakis GN, Hizuka N, Gorden P, Seeburg P, Hamer DH. Expression of two human growth hormone genes in monkey cells infected by simian virus 40 recombinants. Proc Natl Acad Sci USA. 1981;78:7398-402. 13. Baumann G, Stolar MW, Amburn K, Barsano CP, DeVries BC. A specific growth hormone-binding protein in human plasma: initial characterization. J Clin Endocrinol Metab. 1986;62:134-41. 14. Herington AC, Ymer S, Stevenson J. Identification and characterization of specific binding proteins for growth hormone in normal human sera. J Clin Invest. 1986;77:1817-23. 15. Baumann G, Shaw MA. A second, lower affinity growth hormonebinding protein in human plasma. J Clin Endocrinol Metab. 1990;70:680-6. 16. Baumann G, Shaw MA. Immunochemical similarity of the human plasma growth hormone-binding protein and the rabbit liver growth hormone receptor. Biochem Biophys Res Commun. 1988;152:573-8. 17. Leung DW, Spencer SA, Cachianes G, et al. Growth hormone receptor and serum binding protein: purification, cloning, and expression. Nature. 1987;330:537-43. 18. Baumann G, Shaw MA, Amburn K. Regulation of plasma growth hormone-binding proteins in health and disease. Metabolism. 1989;38:683-9. 19. Baumann G, Amburn KD, Buchanan TA. The effect of circulating growth hormone binding protein on metabolic clearance, distribution, and degradation of human growth hormone. J Clin Endocrinol Metab. 1987;64:657-60. 20. Cooke NE, Ray J, Watson MA, Estes PA, Kuo BA, Liebhaber SA. Human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns. J Clin Invest. 1988;82:270-5. 21. Ray J, Okamura H, Kelly PA, Liebhaber SA, Cooke NE. Alteration in the receptor binding specificity of human growth hormone by genomic exon exchange. Mol Endocrinol. 199O;4:101-7.

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hGH-V BINDING TO GH-BP 22. Mohler M, Cook J, Baumann G. Binding proteins of protein therapeutics. In: Ferraiolo BL, Gloff CA, Mohler MA, eds. Protein pharmacokinetics and metabolism. New York: Plenum Press; In Press. 23. Eriksson L, Frankenne F, Eden S, Hennen G, Von Schoultz B. Growth hormone 24-h serum profiles during pregnancy-lack of pulsatility for the secretion of the placental variant. Br J Obstet Gynaecol. 1989;96:949-53. 24. Beckers A, Stevenaert A, Foidart J-M, Hennen G, Frankenne F. Placental and pituitary growth hormone secretion during pregnancy in acromegalic women. Clin Endocrinol Metab. 1990;71:72531.

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25. Furlanetto RW, Underwood LE, Van Wyk JJ, Handwerger S. Serum immunoreactive somatomedin-C is elevated late in pregnancy. J Clin Endocrinol Metab. 1978;47:695-8. 26. Wilson DM, Bennett A, Adamson GD, et al. Somatomedins in pregnancy: a cross-sectional study of insulin-like growth factors I and II and somatomedin peptide content in normal human pregnancies. J Clin Endocrinol Metab. 1982;55:858-69. 27. Caufriez A, Frankenne F, Englert Y, et al. Placental growth hormone as a potential regulator of maternal IGF-I during human pregnancy. Am J Physiol. 1990;258:El014-9. 28. Cunningham BC, Jhurani P, Ng P, Wells JA. Receptor and antibody epitopes in human growth hormone identified by homologscanning mutagenesis. Science. 1989;243:1330-6.

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Binding of human growth hormone (GH)-variant (placental GH) to GH-binding proteins in human plasma.

Human GH-variant (hGH-V) is a natural GH analog arising from the hGH-V gene. It is expressed in the placenta and secreted into the maternal circulatio...
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