©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
G120R, a Human Growth Hormone Antagonist, Shows Zinc-dependent Agonist and Antagonist Activity on Nb2 Cells (*)

Mehul T. Dattani , Peter C. Hindmarsh (§) , Charles G. D. Brook , Iain C. A. F. Robinson (2), John J. Kopchick (3), Nicholas J. Marshall (1)(¶)

From the (1) Endocrine Unit and Division of Molecular Pathology, Middlesex Hospital, London W1N 8AA, United Kingdom, the (2) National Institute of Medical Research, Mill Hill, London NW7 1AA, United Kingdom, and the (3) Edison Biotechnology Institute, Ohio University, Athens, Ohio 45701

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Substitution of arginine for glycine at position 120 in native 22-kDa human growth hormone (hGH) results in an analogue, G120R, which is unable to dimerize the GH receptor and is widely used to probe the molecular mechanism of action of hGH. When acting on human GH receptors, G120R antagonizes several biological effects of hGH, but is itself inactive as an agonist. It has been reported that this mutant also antagonizes hGH activation of the rat or human prolactin (PRL) receptor in cell-based assays, with no agonist activity. We have now tested this mutant in a sensitive MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-ESTA (eluted stain assay) bioassay using rat PRL receptors in the Nb2 cell line. We confirm that G120R acts as an efficient antagonist of native hGH, but show that it can also act as an agonist to generate intracellular signals leading to metabolic activation and proliferation of Nb2 cells. We have demonstrated an unusual sensitivity to the presence of zinc (Zn). In the absence of added Zn, G120R shows weak but full agonist activity in the bioassay, and this can be blocked by co-incubation with recombinant hGH-binding protein. G120R can therefore be utilized to discriminate between the molecular mechanisms of hGH interactions with its somatogenic and lactogenic receptors. Future studies with G120R in the rat may need to take account of its significant agonist effects on PRL receptors.


INTRODUCTION

The third -helix of human growth hormone (hGH)() appears to be especially important for its biological activity (1) . Changes in amino acid residues in this region have particularly marked effects upon the potencies of the resulting hormone analogues (2, 3, 4) . Kopchick and co-workers (5) have demonstrated that a glycine residue at position 120 in hGH is critical in this respect. This amino acid, with its minimal, nonpolar side chain, is thought to create a ``cleft'' in the middle of the third -helix. As a consequence, its substitution by an amino acid which has a bulky resonance-stabilized basic side chain, such as arginine, generates an analogue (G120R) with greatly altered biological effectiveness. For example, G120R fails to stimulate when tested in systems as diverse as hGH receptor down-regulation and the induction of the tyrosine phosphorylation of proteins in IM-9 lymphocytes (5, 6, 7) and lipogenesis in rat adipocytes (7) . In all of these systems, which function via a GH receptor, G120R consistently antagonizes stimulation by wild-type hGH. In addition, it leads to stunted growth in transgenic mice which express G120R (4) .

High resolution functional (8, 9) and structural (10) analyses have shown that there are two receptor-binding sites on hGH, called sites 1 and 2. Site 1 governs the initial binding of hGH to its receptor. This is followed by recruitment of a second growth hormone receptor molecule from the lipid matrix of the plasma membrane, which binds to site 2 on hGH. The consequent formation of receptor homodimers then initiates intracellular signaling. The third -helix and the N terminus of hGH have been demonstrated to form binding site 2 (9, 10) . As a consequence, the analogue G120R has compromised site 2, but not site 1, function. Although G120R can bind normally via site 1 to the first receptor, it is thought that recruitment of the second receptor, and hence receptor dimerization, is blocked by the arginine substitution at the critical position in site 2. This would account for both the failure of G120R to act as an agonist and also its ability to antagonize wild-type hGH. The general validity of this scheme has been established by a number of other mutations based on this two-site hypothesis. Furthermore, prolactin receptor antagonism has also been shown for G120R in cell lines expressing hPRL receptors and in Nb2 cells expressing a rat PRL receptor.

It has been suggested that activation of PRL receptors by hGH is also due to a two-step sequential dimerization process (9, 11) . Using alanine-scanning mutagenesis, Cunningham and Wells (12) demonstrated that hGH contains a set of functional determinants for binding to the hPRL receptor which overlapped those of site 1 for binding to the hGH receptor. These sites are similar, but not identical. Intriguingly, the site for binding to the hPRL receptor contains three ligands for coordinating zinc (Zn) which are important for the binding of hGH to the hPRL receptor but not for its binding to the hGH receptor (13) . The existence and nature of a second hPRL receptor-binding site on hGH has yet to be established.

Fuh et al. (11) compared the effects of G120R and wild-type hGH on two different cell systems, which expressed different forms of the prolactin receptor. One was the Nb2 rat lymphoma cell which expressed an intermediate length lactogenic receptor. As might have been anticipated from work with the somatogenic growth hormone receptor, they reported that G120R failed to stimulate [H]thymidine uptake by the Nb2 cells and that high concentrations of the analogue antagonized the stimulation achieved with wild-type hGH.

In recent in vivo studies() it has proved difficult to demonstrate the antagonist activity of G120R on growth in the rat and the data was compatible with a weak but significant agonist activity in the absence of GH, via rat PRL receptors. This would seem to be at variance with the data of Fuh et al. (11) , who reported only antagonist activity on rat PRL receptors. To resolve this inconsistency, we took advantage of a recently developed highly sensitive and precise bioassay based on Nb2 cells. This colorimetric bioassay, which is both sensitive and precise, utilizes as its end point the bioreduction of the tetrazolium salt MTT to its intensely colored formazan by hormonally activated cells. This integrated response reflects both increases in cell numbers and, also, metabolic activation of individual cells by the lactogenic hormone (14, 15, 16) . We now report that G120R stimulates Nb2 cells in the MTT-ESTA bioassay.() We have characterized its actions, both as an agonist and an antagonist, and shown that discrepancies with previous reports may be explained by the unexpected sensitivity of the G120R/rPRL receptor interaction to zinc (Zn).


EXPERIMENTAL PROCEDURES

Materials

Recombinant G120R hGH was prepared as described previously (5) . Recombinant human growth hormone-binding protein (rhGHBP), which represents the extracellular hormone-binding domain (17) was a gift from Genentech, Inc. (South San Francisco, CA). The rhGHBP is nonglycosylated and has an affinity for 22-kDa hGH comparable with that of the naturally occurring high affinity hGHBP (18) . Human pituitary hGH (IS 80/505) was provided by the National Institute of Biological Standards and Controls (Blanche Lane, South Mimms, Herts, United Kingdom). The suppliers of 80/505 state that analysis on polyacrylamide gel (pH 7.8) indicated a predominant band of 22-kDa hGH, about 5% 20-kDa and small amounts of ``slow,'' dimer and desamido hGH, as well as trace amounts of several other proteins (19) . Zinc chloride (ZnCl) was purchased from Sigma Ltd. (Poole, Dorset, UK), as was 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). RPMI 1640, L-glutamine, penicillin, and streptomycin were purchased from Life Technologies, Inc. UK Ltd. Horse serum and fetal calf serum were purchased from Tissue Culture Services UK Ltd.

The concentration of zinc in the ``zinc-free'' assay medium was 2 µ M, as previously determined by atomic absorption spectroscopy (20) .

Cell Culture

Rat Nb2 lymphoma cells were routinely grown in suspension culture in RPMI medium containing 50 units penicillin/ml, 50 µg streptomycin/ml, 2 10 M L-glutamine, 10% fetal calf serum, and 10% horse serum. Before commitment to bioassay, the Nb2 cells were maintained as stock cultures for 24 h in 1% as opposed to 10% fetal calf serum. All cultures were incubated at 37 °C in an atmosphere of 5% CO, 95% air throughout this study.

Bioassay

The ESTA is based upon the colorimetric reduction of a yellow tetrazolium salt, MTT, to a purple formazan by Nb2 cells (14) . Dilution of the appropriate hormone standard and the zinc chloride were prepared in assay medium consisting of RPMI 1640, 50 units/ml penicillin, 50 µg/ml streptomycin, 2 10 M L-glutamine, and 10% horse serum. The Nb2 cells were suspended in this assay medium at a density of 4 10cells/ml. The cell suspension (50 µl/well) was transferred to a 96-well microtiter plate (Nunc code 1 67008A; Kamstrup, Denmark (supplied by Life Technologies, Inc.)). The hormone (hGH (IS 80/505) or G120R) was added to the wells at the required concentrations as an additional 50 µl prepared in assay medium. Zinc chloride or the rhGHBP were added at the appropriate concentrations in a volume of 20 µl, as was G120R in the experiment to assess its antagonistic properties on IS 80/505. Experimental controls replaced the hormones, rhGHBP, or ZnClwith assay medium. All concentrations were expressed as final concentrations of added zinc, rhGHBP, or hormone in the wells.

The cells were incubated for 96 h under the conditions described above. Their reducing activity was then assayed by adding 10 µl of MTT solution (5 mg/ml in phosphate-buffered saline). After a further 40-min incubation period, during which time activated cells reduced the yellow MTT salt to its purple formazan, the stain was eluted into the medium by the addition of 50 µl of acidified Triton X-100 (10% Triton X-100 in 0.09 M HCl). Bioassay responses of the 96 wells were quantified with a Bio-Rad microtiter plate reader (scanning in 30 s) reading optical densities at a test wavelength of 595 nm and a reference wavelength of 655 nm. The determinations for all experiments were made on triplicate microcultures and the results expressed as means and standard deviations.

Conventional Nb2 Cell Bioassay

The bioassay was performed as described above. However, instead of the addition of MTT to determine response, cell number was determined using a Coulter counter.

Units of Concentration

All concentrations are expressed in molar terms. These have been calculated using the following molecular weights: pituitary-derived hGH (IS 80/505) and recombinant G120R, 22 kDa; rhGHBP, 28 kDa. The specific bioactivity of the human pituitary GH preparation (IS 80/505) was 2.6 IU/mg.


RESULTS

The Effect of the hGH Analogue G120R on Nb2 Cells

In the absence of added Zn, dose-related stimulation of Nb2 cells by G120R was observed over the concentration range 0.28-142 n M, with an ECof 1.27 n M (Fig. 1). A plateau maximal response was observed, but at the highest concentrations, self-inhibition was observed (self-IC= 523 n M). Within the same experiment, IS 80/505 stimulated Nb2 cells over the concentration range 0.9-8.6 10p M, with an ECof 10 p M. Although the potency of G120R as an agonist was 100-fold less than that of IS 80/505 as measured at the ECthe ascending limbs of the two dose-response relationships were parallel, and the same maximal response was obtained. At high concentrations of IS 80/505 (>17.3 µ M), self-inhibition was also observed, as demonstrated previously by Fuh et al. (11) . The self-ICwas 25.5 µ M. Hence, the potency of G120R as a self-antagonist was 49-fold greater than that of IS 80/505. The plateau in response to IS 80/505 extended over concentration ranges of 5 orders of magnitude, whereas that for G120R was more restricted, approximating to 2 orders of magnitude (Fig. 1).


Figure 1: Stimulation of Nb2 cells by the hGH analogue, G120R, and pituitary-derived hGH (IS 80/505) compared in the same ESTA. Increasing doses of G120R (0.068-1136 n M []) and pituitary-derived hGH (IS 80/505: 0.9 p M to 35 µ M ()) were added to Nb2 cells. The bioassay was performed as described under ``Experimental Procedures,'' and the results are expressed as optical density 100 and are the means ± S.D. obtained from triplicate wells. Both preparations were tested on the same microtiter plate. The horizontal line represents the control optical density in the presence of assay medium ( AM) only.



The stimulatory effect of G120R on Nb2 cells in the MTT-ESTA system was accompanied by increases in cell numbers (Fig. 2). Doses of G120R and IS 80/505 which provoked the same maximum responses in the MTT-ESTA bioassay (Fig. 2 A) increased Nb2 cell numbers equivalently (Fig. 2 B). This stimulatory effect was also confirmed using an alternative tetrazolium salt system (Promega cell titer 96 Aqueous kit) and cell proliferation as assessed by increased uptake of [H]thymidine (not shown). These confirmatory experiments were performed using a different batch of Nb2 cells. The Effect of Zinc [Zn] on G120R Stimulation of Nb2 Cells-Stimulation of Nb2 cells by G120R was shown to be unusually sensitive to the addition of Zn(Fig. 3). When added over the concentration range 0.78-400 µ M, Zninhibited the stimulation of Nb2 cells by a dose of G120R which provoked near-maximum stimulation in the MTT-ESTA bioassay in the absence of added Zn(2.4 n M). The stimulation was abolished with 50 µ M added Zn(51.6 µ M, including endogenous Zn), and the ICfor this was 6.25 µ M added Zn(7.9 µ M including endogenous Zn). Within the same experiment, we confirmed our previous report (20) that 50 µ M added Znslightly increases the stimulation by a low dose of IS 80/505 (9 p M) observed in this bioassay. As before, higher doses of Zninhibited the stimulation with 80/505.


Figure 2: Stimulation of Nb2 cells by the hGH analogue, G120R, and pituitary-derived hGH (IS 80/505): a comparison between the ESTA bioassay and direct cell counts. Nb2 cells were stimulated with 2.8 n M G120R and 0.11 n M hGH as for a standard ESTA bioassay, except that parallel triplicate wells were also set up in the same microtiter plate and used to determine changes in cell numbers. The ESTA bioassay was performed as described under ``Experimental Procedures,'' and the results are expressed as optical density 100 for the MTT-ESTA assay ( A) or cell number 10/ml for the parallel wells ( B). Both are expressed as the means ± S.D. obtained from triplicate wells. The results are compared with the assay medium controls in the absence of added lactogen.




Figure 3: Effect of increasing concentrations of Zn on stimulation of Nb2 cells by G120R and the pituitary-derived preparation of hGH (IS 80/505) in the ESTA bioassay. Increasing doses of Zn(0.78-400 µ M) were added to Nb2 cells in the presence of a constant dose of either G120R (2.4 n M (⊡ ) or the pituitary-derived preparation of hGH (IS 80/505 ()). The bioassay was performed as described under ``Experimental Procedures,'' and the results are expressed as optical density 100 and are the means ± S.D. obtained from triplicate wells. The horizontal lines represent the effect of lactogen in the absence of added Zn. 50 µ M Znadded to assay medium in the absence of lactogen had no effect on Nb2 cells (assay medium ( AM) ± Zn). The entire bioasay was performed within a single microtiter plate.



Zninhibition of stimulation of Nb2 cells by G120R was due to a reduced maximum magnitude of response and not due to a shift of the dose-response relationship to the right (Fig. 4). The addition of 20 µ M Znwould be expected to have only a minimal effect upon stimulation of Nb2 cells with IS 80/505 (see Fig. 3 and also Ref. 20), but it reduced the plateau response achieved with G120R by approximately 85%.


Figure 4: Stimulation of Nb2 cells by the hGH analogue, G120R, and pituitary-derived hGH (IRP 80/505) in the presence of 20 µ M ZnClcompared in the same ESTA. Increasing doses of G120R (0.068-1136 n M ()) and pituitary-derived hGH (IS 80/505: 0.9 p M to 35 µ M ()) were added to Nb2 cells in the presence of 20 µ M Zn. The bioassay was performed as described under ``Experimental Procedures,'' and the results are expressed as optical density 100 and are the means ± S.D. obtained from triplicate wells. The entire experiment was performed within a single microtiter plate. The horizontal line represents the control optical density in the presence of assay medium, with the only additional component being Zn(20 µ M).



The Effect of G120R on Nb2 Cells Stimulated with IRP 80/505

G120R antagonized IS 80/505 stimulation of Nb2 cells (Fig. 5). Using a dose of IS 80/505 (0.91 n M) which was close to the mid-point of the plateau of the maximum response (Fig. 1), we found that the addition of up to 114 n M G120R did not further increase this magnitude of response. However, higher doses of G120R antagonized hGH in a concentration-dependent manner. The potency of G120R as an antagonist of hGH was enhanced 4-fold by the addition of 10 µ M Zn; a 50% reduction in the response to IS 80/505 was observed with 90 n M G120R. This potentiating effect of Znhas been described previously (11) .

The Effect of rhGHBP on Nb2 Cells Stimulated with G120R

rhGHBP, which represents the extracellular domain of the hGH receptor, interacted with G120R and inhibited stimulation of Nb2 cells by this mutant in a dose-dependent manner (Fig. 6). When stimulated with a dose of G120R (1.2 n M) which provokes a near-maximal response (Fig. 1), a dose-dependent inhibition of this response was first observed with a concentration of 0.23 n M rhGHBP, and concentrations of the binding protein in excess of 3.5 n M abolished the response. The ICfor rhGHBP was 1.1 n M; the molar ratios of rhGHBP:G120R were thus approximately unity (0.9) at this midpoint in the antagonist dose-response relationship.


DISCUSSION

Using the MTT-ESTA bioassay, we have confirmed several of the properties of the G120R analogue of hGH, which have been reported in other systems. For example, we have confirmed its ability to antagonize hGH and that this inhibition is potentiated by Zn. This is consistent with normal binding of the analogue at its unmodified binding site 1 to the first receptor and an inability to induce receptor dimerization by binding to site 2. We have also demonstrated that G120R interacts with the extracellular domain of the hGH receptor at rhGHBP:G120R ratios of unity. However, these molar ratios were much higher (100-300) when rhGHBP inhibited stimulation by IS 80/505 and, also, its isohormone, recombinant methionyl 20-kDa hGH, as reported in separate studies (21) .

In contrast to an earlier report (11) , we found that binding of G120R to the lactogenic receptors located on the surface of Nb2 cells resulted in cell activation. We have characterized this stimulation in detail, using the high throughput MTT-ESTA bioassay system. We have also confirmed the agonist actions of G120R with a conventional system based upon direct cell counts (Fig. 2) and also two other response systems, namely one using an alternative tetrazolium salt and one based upon increased uptake of [H]thymidine. Stimulation required high concentrations of G120R and was unusually sensitive to added Zn. The inhibitory effect of Znprobably reconciles the two separate investigations. Fuh et al. (11) added 10 µ M Znto their medium when they tested for agonist properties, whereas we performed our experiments with a medium which contained no special addition of Znand has a basal concentration of only 2 µ M Zn(20) . In the MTT-ESTA bioassay, the addition of 10 µ M Znreduced the maximum agonist response to G120R by 85%, whereas it had little effect on hGH agonist activity. It is also possible that the lower serum concentration in the medium used by Fuh et al. (11) (0.5% horse serum as opposed to the 10% in the present studies) exacerbated the inhibitory effect of Zn, due to a reduced concentration of zinc-binding protein in the medium, with a corresponding increase in the concentration of free zinc.

Thus, according to the two-site sequential receptor dimerization model, which has been well established for hGH interacting with somatogenic receptors, our findings would indicate that although binding of G120R to site 2 may be compromised, it can interact with lactogenic receptors and stimulate target cells, if the analogue is present at sufficiently high concentrations. Our finding that this results in a bell-shaped dose-response relationship (Fig. 1) would be consistent with the biological responses being linked to dimer formation, as predicted from a computerized simulation of the dimerization model (7) . Moreover, the restricted concentration range of the plateau for G120R, compared with the wild-type hGH (Fig. 1), would be consistent with the formation of less stable dimeric complexes by G120R (11) . However, our findings are at variance with some of the other conclusions which Ilondo et al. (7) made from their mathematical simulation. They predicted that mutagenesis at site 1 would result in a shift of the whole bioactivity curve to the right, but with the maximum magnitude of the response being unaffected. In contrast, they concluded that site 2 mutants would exhibit lowered or absent magnitudes of response. Clearly, G120R, which is a site 2 mutant, gave a result (Fig. 1) which closely resembled that anticipated for the analogues which have been subjected to mutagenesis at site 1. It therefore behaved in a similar manner to the site 1 double mutant, K172A/F176A, when acting on Nb2 cells (11) .

From our own work, and that of Fuh et al. (11) , we conclude that the important difference between K172A/F176A and G120R when acting as lactogenic agonists is their altered sensitivities to added Zn. It is well recognized that this divalent cation is required for site 1 association between hGH and the hPRL receptor. It does not, however, influence the binding of hGH to the hGH receptor. Scanning mutational analysis has identified three residues in hGH (His, His, and Glu) and one residue on human prolactin-binding protein (hPRLBP), which represents the extracellular domain of the hPRL (lactogenic) receptor, as the four probable residues involved in Znbinding (13) . It would appear that Znmay be used as an intriguing discriminator between the different analogues of hGH, since we find a wide spectrum of the effects of this ion, when investigating lactogenic Nb2 cell receptors. These influences range from marked inhibition of the agonist activity of G120R, potentiation of its inhibitory effects, the slight enhancement of native hGH when low doses of the latter are used, which changes to inhibition at higher hormone concentrations, through to striking potentiation of the bioactivity of the alternative splice variant of hGH, 20-kDa hGH (20) .

In conclusion, we have characterized the agonist and antagonist properties of the G120R analogue of hGH when acting on the intermediate length lactogenic receptor as encountered in the Nb2 cells. The finding that this analogue stimulates in this system enhances its importance as a probe for deciphering the molecular mechanism of growth hormone action via different receptors. Our findings are equally important for in vivo studies. It has proved difficult to establish significant hGH antagonism in vivo in the rat, and it was the suspicion of mild PRL agonist activity that prompted this study. The present results confirm that PRL agonist activity occurs at far lower doses than those necessary to antagonize hGH at the rat Nb2 PRL receptor and that in vivo studies in which G120R is used as a specific GH antagonist in the rat may need to be interpreted with caution.


FOOTNOTES

*
This work was supported by the David and Frederick Barclay Foundation and by the Special Trustees of the Middlesex Hospital. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Children Nationwide and Pharmacia, Stockholm, Sweden.

To whom correspondence should be addressed: Division of Molecular Pathology, University College London, The Windeyer Bldg., 46 Cleveland St., London W1P 6DB, UK. Tel.: 44-071-636-8333 (ext. 3362); Fax: 44-071-380-9496.

The abbreviations used are: hGH, human growth hormone; PRL, prolactin; ESTA, eluted stain assay; rhGHBP, recombinant human growth hormone-binding protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; hPRLBP, human prolactin-binding protein.

T. Wells, A. Mode, J. Kopchick, and I. Robinson, manuscript in preparation.

The use of the ESTA bioassay system is the subject of an International Patent Application.


ACKNOWLEDGEMENTS

We are grateful to Genentech Inc. for their kind gift of the recombinant human GHBP.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.