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Aspartate 171 Is the Major Primate-specific Determinant of Human Growth Hormone

1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês

10.1074/jbc.272.43.27077

1083-351X

Stuart N. Behncken, Scott W. Rowlinson, Jennifer Rowland, Becky Conway-Campbell, Thea Monks, Michael J. Waters,

Diet and metabolism studies

It has been known for more than 4 decades that only primate growth hormones are effective in primate species, but it is only with the availability of the 2.8 Å structure of the human growth hormone (hGH)·hGH-binding protein (hGHBP)2complex that Souza and co-workers (Souza, S. C., Frick, G. P., Wang, X., Kopchick, J. J., Lobo, R. B., and Goodman, H. M. (1995)Proc. Natl. Acad. Sci. U. S. A. 92, 959–963) were able to provide evidence that Arg-43 on the primate receptor is responsible. Here we have examined systematically the interaction between Arg-43 (primate receptor) or Leu-43 (non-primate receptors) and their complementary hormone residues Asp-171 (primate GH) and His-170 (non-primate hormones) in a four-way comparison involving exchanges of histidine and aspartate and exchanges of arginine and leucine. BAF/B03 lines were created and characterized which stably expressed hGH receptor, R43L hGH receptor, rabbit GH receptor, and L43R rabbit GH receptor. These were examined for site 1 affinity, for the ability to bind intact cells, and for proliferative biopotency using hGH, D171H hGH, porcine GH, or H170D porcine GH. We find that the single interaction between Arg-43 and His-170/171 is sufficient to explain virtually all of the primate species specificity, and this is congruent with the crystal structure. Accordingly, for the first time we have been able to engineer a non-primate hormone to bind to and activate the human GH receptor. It has been known for more than 4 decades that only primate growth hormones are effective in primate species, but it is only with the availability of the 2.8 Å structure of the human growth hormone (hGH)·hGH-binding protein (hGHBP)2complex that Souza and co-workers (Souza, S. C., Frick, G. P., Wang, X., Kopchick, J. J., Lobo, R. B., and Goodman, H. M. (1995)Proc. Natl. Acad. Sci. U. S. A. 92, 959–963) were able to provide evidence that Arg-43 on the primate receptor is responsible. Here we have examined systematically the interaction between Arg-43 (primate receptor) or Leu-43 (non-primate receptors) and their complementary hormone residues Asp-171 (primate GH) and His-170 (non-primate hormones) in a four-way comparison involving exchanges of histidine and aspartate and exchanges of arginine and leucine. BAF/B03 lines were created and characterized which stably expressed hGH receptor, R43L hGH receptor, rabbit GH receptor, and L43R rabbit GH receptor. These were examined for site 1 affinity, for the ability to bind intact cells, and for proliferative biopotency using hGH, D171H hGH, porcine GH, or H170D porcine GH. We find that the single interaction between Arg-43 and His-170/171 is sufficient to explain virtually all of the primate species specificity, and this is congruent with the crystal structure. Accordingly, for the first time we have been able to engineer a non-primate hormone to bind to and activate the human GH receptor. The growth hormone receptor (GHR) 1The abbreviations used are: GHR, growth hormone receptor; GH, growth hormone; (GHBP)2, GH-binding protein; h, rb, b, and p prefixes indicate human, rabbit, bovine, and porcine, respectively; MAb, monoclonal antibody; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. is a member of the hematopoietic cytokine receptor family, sharing common structural and functional features with receptors for prolactin, erythropoietin, granulocyte and granulocyte-macrophage colony-stimulating factors, several interleukins, thrombopoietin, ciliary neurotrophic factor, oncostatin M, and leptin (for review, see Refs. 1Wells J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1-6Crossref PubMed Scopus (362) Google Scholar and 2Takahashi Y. Kaji H. Okimura Y. Goji K. Abe H. Chihara K. N. Engl. J. Med. 1996; 334: 432-436Crossref PubMed Scopus (135) Google Scholar). Of this family, the interaction of GH with its receptor is the best characterized, primarily because of the extensive structure/function studies that have been carried out on both GH and the GHR (for review, see Ref. 1Wells J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1-6Crossref PubMed Scopus (362) Google Scholar) and because it is the only member for which the crystal structure of the hormone-receptor complex is known (3De Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2029) Google Scholar). Both crystal structure and solution studies support the concept that two identical receptor subunits bind the helix bundle hormone through similar loop determinants on the receptor ॆ-sandwich structures. The hormone is captured by receptor 1 through binding to determinants located in a 900-Å2 patch encompassing helices 1 and 4 and the unstructured loop between helices 1 and 2. Eight key residues account for 857 of the binding energy, with electrostatic interactions governing the approach of hormone to the receptor binding site (4Cunningham B.C. Wells J.A. J. Mol. Biol. 1993; 234: 554-563Crossref PubMed Scopus (487) Google Scholar, 5Clackson T. Wells J.A. Science. 1995; 267: 383-386Crossref PubMed Scopus (1793) Google Scholar). Electrostatic interactions are also important specificity determinants because 5 of the 7 residues that were modified to enable prolactin to bind to the GH receptor with high affinity involved charged residues (6Cunningham B.C. Wells J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3407-3411Crossref PubMed Scopus (202) Google Scholar). GHs from humans and monkeys (primate GHs) are unique in that they are able to bind with and activate non-primate GHRs as well as primate GHRs, whereas the GHs from non-primates are ineffective in primates (for review, see Ref. 7Lauterio T.J. Trivedi B. Kapadia M. Daughaday W.H. Comp. Biochem. Physiol. 1988; 91: 15-19Crossref Scopus (21) Google Scholar). To elucidate which residues on the receptor are responsible for this species specificity of binding, sequence alignment analysis was performed on human GHR (hGHR) and a number of non-primate GHRs in conjunction with an examination of the crystal structure of the GH⋅(GHBP)2 complex. Of residues within the five major loops involved in hormone binding (2Takahashi Y. Kaji H. Okimura Y. Goji K. Abe H. Chihara K. N. Engl. J. Med. 1996; 334: 432-436Crossref PubMed Scopus (135) Google Scholar), the interaction between Arg-43 of the human receptor and Asp-171 of the human hormone is striking because in non-primate receptors this position is replaced by leucine, and histidine occupies the place of aspartate in non-primate hormones (the equivalent position in porcine and bovine GH is 170). An unfavorable charge repulsion/steric hindrance between hormone His-170 and receptor Arg-43, rather than a favorable salt bridge between this arginine and primate hormone Asp-171, could be an important element in the inability of non-primate hormones to bind to the human receptor. Accordingly, we previously undertook site-directed mutagenesis at position 43, converting Leu-43 of the rabbit receptor to arginine (L43R rbGHR), and compared the ability of wild type and mutant receptors to discriminate between binding of hGH and bovine GH (8Gobius K.S. Rowlinson S.W. Barnard R. Mattick J.S. Waters M.J. J. Mol. Endocrinol. 1992; 9: 213-220Crossref PubMed Scopus (24) Google Scholar). We were disappointed to find that the L43R rbGHR mutant was able to bind human GH (hGH) and bovine GH (bGH) with almost identical affinity, indicating that it was not the species specificity determinant (8Gobius K.S. Rowlinson S.W. Barnard R. Mattick J.S. Waters M.J. J. Mol. Endocrinol. 1992; 9: 213-220Crossref PubMed Scopus (24) Google Scholar). However, a more recent study on bovine and rat GHRs by Souza et al. (9Souza S.C. Frick G.P. Wang X. Kopchick J.J. Lobo R.B. Goodman H.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 959-963Crossref PubMed Scopus (71) Google Scholar) has revealed that conversion of leucine to arginine at position 43 of these receptors severely abrogated the binding ability of bGH and also reduced its signaling ability. Based on the crystal structure of the GH·(GHBP)2 complex, Souzaet al. (9Souza S.C. Frick G.P. Wang X. Kopchick J.J. Lobo R.B. Goodman H.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 959-963Crossref PubMed Scopus (71) Google Scholar) again proposed that an unfavorable interaction between His-170 on non-primate GH and Arg-43 on the human receptor accounts for primate specificity of binding. In this study, we have sought to resolve this conflict. We have examined the binding and signaling characteristics of rabbit and human GHRs with human, porcine, and bovine GHs as well as the effect of exchanging both the receptor leucine for arginine and the hormone histidine for aspartate. This has allowed us to bind a non-primate GH to the human receptor for the first time and to resolve the conflict between our previous study and that of Souza et al. (9Souza S.C. Frick G.P. Wang X. Kopchick J.J. Lobo R.B. Goodman H.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 959-963Crossref PubMed Scopus (71) Google Scholar). Preparation of recombinant pGH and H170D pGH has been described previously (10Rowlinson S.W. Barnard R. Bastiras S. Robins A.J. Senn C. Wells J.R.E. Brinkworth R. Waters M.J. Biochemistry. 1994; 33: 11724-11733Crossref PubMed Scopus (13) Google Scholar, 11Rowlinson S.W. Barnard R. Bastiras S. Robins A.J. Brinkworth R. Waters M.J. J. Biol. Chem. 1995; 270: 16833-16839Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Recombinant bGH was a gift from Cyanamid (Princeton NJ). Human GH and the D171H hGH analog were expressed in Escherichia coli and purified to greater than 987 homogeneity (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the manner described previously (10Rowlinson S.W. Barnard R. Bastiras S. Robins A.J. Senn C. Wells J.R.E. Brinkworth R. Waters M.J. Biochemistry. 1994; 33: 11724-11733Crossref PubMed Scopus (13) Google Scholar), except that the pH of the refolding buffer and Q-Sepharose loading buffer was 8.2. The D171H mutation was introduced into the coding sequence of the hGH cDNA using the Altered Sites mutagenesis procedure (Promega). In this procedure, an oligonucleotide encoding the desired mutation is annealed to single strand hGH-pSelect, and the complex is incubated with T4 DNA polymerase and T4 DNA ligase (Promega) to produce double stranded circular plasmid according to instructions from Promega. This was then subcloned into the pC611 vector (kindly provided by BresaGen, Adelaide, Australia) for expression, as described before (10Rowlinson S.W. Barnard R. Bastiras S. Robins A.J. Senn C. Wells J.R.E. Brinkworth R. Waters M.J. Biochemistry. 1994; 33: 11724-11733Crossref PubMed Scopus (13) Google Scholar). The mutation was confirmed by dideoxy sequencing (U. S. Biochemical Sequenase kit) of the complete D171H coding region. Hormone concentrations were determined spectrophotometrically using an extinction coefficient of ε280 = 18,200 m−1cm−1. Site-directed mutagenesis was undertaken on the rabbit GHR (rbGHR) and hGHR cDNA using the Altered Sites mutagenesis procedure as described previously (8Gobius K.S. Rowlinson S.W. Barnard R. Mattick J.S. Waters M.J. J. Mol. Endocrinol. 1992; 9: 213-220Crossref PubMed Scopus (24) Google Scholar). Mutant sequences were then subcloned into the pECE expression vector. Creation of rabbit L43R and human R43L GHR mutants and the absence of other coding sequence changes were verified by dideoxy sequencing. The interleukin-3-dependent cell line BAF/B03 was a gift from Dr. Tom Gonda (Institute for Medical and Veterinary Sciences, Adelaide). Cells were routinely passaged in 57 CO2, at 37 °C, in RPMI 1640 medium supplemented with 5 ॖg/ml gentamicin, 107 serum supreme (a fetal bovine serum alternative supplied by Biowhittaker through Edward Keller Australia Ltd., Underwood Queensland) and 50 units/ml interleukin-3 (generous gift from Dr A. Hapel, John Curtin School of Medical Research, Australian Capital Territory, Australia). Mid confluent BAF/B03 cells (200 ॖl of 2.5 × 107/ml growth medium) were transfected by electroporation with GHR cDNA and pNeo plasmid at a ratio of 20:1 in a manner identical to that described in Ref. 11Rowlinson S.W. Barnard R. Bastiras S. Robins A.J. Brinkworth R. Waters M.J. J. Biol. Chem. 1995; 270: 16833-16839Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar. After electroporation, transfectants were selected by treatment with G418 at 1.2 mg/ml followed by selection of single cells and clonal expansion. Equilibrium binding constants for all GHs were determined with the appropriate cell line in physiological binding buffer (8Gobius K.S. Rowlinson S.W. Barnard R. Mattick J.S. Waters M.J. J. Mol. Endocrinol. 1992; 9: 213-220Crossref PubMed Scopus (24) Google Scholar) using 125I-hGH as described in Ref. 10Rowlinson S.W. Barnard R. Bastiras S. Robins A.J. Senn C. Wells J.R.E. Brinkworth R. Waters M.J. Biochemistry. 1994; 33: 11724-11733Crossref PubMed Scopus (13) Google Scholar. Assays were done at 4 °C to block receptor internalization, with equilibrium being reached by 18 h. The number of surface-expressed receptors and the hormone binding affinity for each cell line were determined using cells grown in GH-free RPMI 1640 medium containing 1 ॖg/ml gentamicin, 57 serum supreme, and 50 units/ml of interleukin-3. Binding assays were also performed on solubilized cell extracts as follows. Cells were lysed with 0.37 (v/v) Triton X-100 in physiological binding buffer in the presence of 5 ॖg/ml MAb 5 and microcentrifuged at 15,000 rpm for 15 min to recover the solubilized receptors. Equilibrium binding assays (in the presence of MAb 5 (AGEN Ltd., Acacia Ridge, Queensland) at 5 ॖg/ml) were then undertaken on solubilized cell extracts for 18 h at 4 °C, with bound/free separation by polyethylene glycol precipitation, as in Ref. 12Waters M.J. Friesen H.G. J. Biol. Chem. 1979; 254: 6815-6825Abstract Full Text PDF PubMed Google Scholar. MAb 5 has been shown to block receptor dimer formation through binding to the dimerization domain residues (13Fuh G. Cunningham B.C. Fukunaga R. Nagata S. Goeddel D.V. Wells J.A. Science. 1992; 256: 1677-1680Crossref PubMed Scopus (573) Google Scholar) so that this binding assay measures binding to receptor 1 only. Binding data were processed by Scatchard analysis with the LIGAND program (Elsevier Biosoft). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) cell viability assays were performed in the manner described in Ref. 11Rowlinson S.W. Barnard R. Bastiras S. Robins A.J. Brinkworth R. Waters M.J. J. Biol. Chem. 1995; 270: 16833-16839Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholarusing the GHR-expressing BAF/B03 cells described above. Because the magnitude of GH responsiveness of these GHR-expressing cell lines is related to the level of receptor expression, 2S. W. Rowlinson, S. N. Behncken, and M. J. Waters, unpublished observations. it was necessary to plate clonal cell lines at different concentrations to obtain comparable response. BAF/B03-rbGHR cells (expressing wild type rabbit GHR), BAF/B03-hGHR cells (expressing wild type human GHR), and BAF/B03-L43R rbGHR cells (expressing L43R rbGHR) were plated out at 2.5 × 104 cells/well, whereas BAF/B03-R43L hGHR cells (expressing R43L hGHR) were plated at 6 × 104cells/well. The complete coordinates of the hGH·(hGHBP)2 complex were obtained from the PDB data base (accession number 3hhr). The homology-modeled pGH·(rbGHBP)2 complex was obtained with the Homology program (Biosym Technologies, San Diego) with the assistance of Ross Brinkworth, Drug Design Center, University of Queensland (10Rowlinson S.W. Barnard R. Bastiras S. Robins A.J. Senn C. Wells J.R.E. Brinkworth R. Waters M.J. Biochemistry. 1994; 33: 11724-11733Crossref PubMed Scopus (13) Google Scholar). Crystal structure measurements were made with the Insight II program (Biosym Technologies), and molecular dynamics and energy minimizations were done using the Discover module. BAF/B03 cells were preferred over FDC-P1 cells in this study as we have found that they express GHRs at a higher level than FDC-P1 cells. Whole cell Scatchard analysis (at least three independent assays where125I-hGH was displaced by unlabeled hGH for each cell line) revealed an affinity for hGH of of 2.9 ± 0.2 × 109m−1 with 7,419 ± 777 receptors/cell for BAF/B03-hGHR cells, an affinity of 3.3 ± 0.2 × 109m−1 with 3,164 ± 528 receptors/cell for BAF/B03-R43L hGHR cells, an affinity of 6.0 ± 0.6 × 109m−1 with 7,285 ± 618 receptors/cell for BAF/B03-rGHR cells, and an affinity of 5.0 ± 0.4 × 109m−1 with 2,852 ± 455 receptors/cell for BAF/B03-L43R rGHR cells. No specific125I-hGH binding was seen in nontransfected BAF/B03 cells. The fact that ED50 values for proliferative biopotency are around 0.05 × 10−9m (Tables I, II, III, IV, see below) suggests that there is a substantial number of spare receptors in these lines.Table IFold changes of various hormones with wild type hGHR (relative to wild type hGH)HormoneAffinityProliferative abilitySolubilized receptorWhole cellhGH1 (K a = 1.18 ± 0.13 × 109 m−1)1 (K a = 2.50 ± 0.15 × 109 m−1)1 (ED50 = 2.09 ± 0.36 × 10−11 m)D171H hGH36.7 ± 1.1↓2.1 ± 0.3↓1.9 ± 0.3↓pGH>10,000↓3,904.5 ± 828.7↓2,342.2 ± 390.3↓H170D pGH32.3 ± 1.9↓24.5 ± 3.9↓14.0 ± 2.2↓bGHND1-aND, not determined.314.1 ± 46.8↓235.8 ± 64.2↓Fold change values were derived from experiments on all five hormones performed simultaneously with the same cell/receptor preparation (see 舠Experimental Procedures舡). Arrows indicate increase or decrease relative to wild type hGH (shown as unity). Whole cell binding affinities were determined by Scatchard analysis, and fold changes were calculated (except for pGH) within each experiment. Because of low receptor level and low affinity for some hormones, values for the solubilized receptors (and whole cell binding to pGH) were determined from ED50 estimates calculated from a specific binding curve. Proliferative ability was determined by comparing ED50 estimates from a dose-response curve (a decrease in proliferative ability is implied from an increase in ED50). Results are the mean of at least three independently calculated fold change values with S.E. shown. The absolute affinity and ED50 values for wild type hGH are shown in parentheses (also determined from at least three independent experiments). The affinity of bGH for the solubilized receptors was not determined.1-a ND, not determined. Open table in a new tab Table IIFold changes of various hormones with R43L hGHR (relative to wild type hGH)HormoneAffinityProliferative abilitySolubilized receptorWhole cellhGH1 (K a = 6.32 ± 1.76 × 109 m−1)1 (K a = 3.33 ± 0.21 × 109 m−1)1 (ED50 = 4.54 ± 0.50 × 10−11 m)D171H hGH1.7 ± 0.3↑1.1 ± 0.2↓1.2 ± 0.3↑pGH27.2 ± 7.9↓14.5 ± 2.1↓13.5 ± 3.0↓H170D pGH4.6 ± 1.1↓20.8 ± 1.4↓15.3 ± 3.9↓bGHND2-aND, not determined.3.5 ± 0.6↓3.6 ± 1.5↓For experimental details, see legend of Table I.2-a ND, not determined. Open table in a new tab Table IIIFold changes of various hormones with wild type rbGHR (relative to wild type hGH)HormoneAffinityProliferative abilitySolubilized receptorWhole cellhGH1 (K a = 5.38 ± 0.97 × 109 m−1)1 (K a = 5.95 ± 0.57 × 109 m−1)1 (ED50 = 5.73 ± 0.50 × 10−11 m)D171H hGH1.5 ± 0.2↓1.2 ± 0.1↑1.6 ± 0.3↓pGH179.2 ± 72.0↓8.3 ± 1.1↓5.4 ± 0.4↓H170D pGH40.4 ± 9.9↓20.6 ± 3.1↓11.6 ± 0.7↓bGH3.5 ± 0.9↓2.0 ± 0.6↓1.3 ± 0.2↓Fold change values were derived from experiments on all five hormones performed simultaneously with the same cell/receptor preparation (see 舠Experimental Procedures舡). Arrows indicate increase or decrease relative to wild type hGH (shown as unity). Whole cell and solubilized receptor binding affinities were determined by Scatchard analysis, and fold changes were calculated within each experiment. Proliferative ability was determined by estimating the ED50 from a dose-response curve (a decrease in proliferative ability is implied from an increase in ED50). Results are the mean of at least three independently calculated fold change values with S.E. shown. The absolute affinity and ED50 values for wild type hGH are shown in parentheses (also determined from at least three independent experiments). The affinity of bGH for the solubilized receptors was not determined. Open table in a new tab Table IVFold changes of various hormones with L43R rbGHR (relative to wild type hGH)HormoneAffinityProliferative abilitySolubilized receptorWhole cellhGH1 (K a = 7.53 ± 0.78 × 109 m−1)1 (K a = 4.99 ± 0.41 × 109 m−1)1 (ED50 = 3.68 ± 0.68 × 10−11 m)D171H hGH52.3 ± 15.1↓1.7 ± 0.2↓1.9 ± 0.3↓pGH>10,000↓125.5 ± 26.9↓256.0 ± 36.3↓H170D pGH65.2 ± 19.5↓20.6 ± 2.1↓12.1 ± 2.7↓bGH690.8 ± 72.4↓2.6 ± 0.1↓37.4 ± 8.6↓For experimental details, see legend of Table III. Open table in a new tab Fold change values were derived from experiments on all five hormones performed simultaneously with the same cell/receptor preparation (see 舠Experimental Procedures舡). Arrows indicate increase or decrease relative to wild type hGH (shown as unity). Whole cell binding affinities were determined by Scatchard analysis, and fold changes were calculated (except for pGH) within each experiment. Because of low receptor level and low affinity for some hormones, values for the solubilized receptors (and whole cell binding to pGH) were determined from ED50 estimates calculated from a specific binding curve. Proliferative ability was determined by comparing ED50 estimates from a dose-response curve (a decrease in proliferative ability is implied from an increase in ED50). Results are the mean of at least three independently calculated fold change values with S.E. shown. The absolute affinity and ED50 values for wild type hGH are shown in parentheses (also determined from at least three independent experiments). The affinity of bGH for the solubilized receptors was not determined. For experimental details, see legend of Table I. Fold change values were derived from experiments on all five hormones performed simultaneously with the same cell/receptor preparation (see 舠Experimental Procedures舡). Arrows indicate increase or decrease relative to wild type hGH (shown as unity). Whole cell and solubilized receptor binding affinities were determined by Scatchard analysis, and fold changes were calculated within each experiment. Proliferative ability was determined by estimating the ED50 from a dose-response curve (a decrease in proliferative ability is implied from an increase in ED50). Results are the mean of at least three independently calculated fold change values with S.E. shown. The absolute affinity and ED50 values for wild type hGH are shown in parentheses (also determined from at least three independent experiments). The affinity of bGH for the solubilized receptors was not determined. For experimental details, see legend of Table III. TableI presents the combined data for wild type human GH receptor-expressing cells, and Fig.1, A–C, presents representative data in graphic form. First, it can be seen that substitution of histidine for aspartate at position 171 of hGH has only a small effect (about a 2-fold reduction) on binding in the whole cell assay and on biopotency. However, in the solubilized assay where dimerization cannot occur, this substitution is seen to have a considerable impact on site 1 affinity (about a 40-fold decrease). Porcine GH is seen to bind very poorly in the whole cell assay (about 4,000-fold less than hGH) and to be very poorly effective in the bioassay (about 2,300-fold less). In the solubilized receptor assay, pGH effectively does not bind to the hGHR. However, substitution of aspartate for histidine at position 170 of pGH results in a hormone that is only 24-fold less than hGH in whole cell binding affinity and 14-fold less in biopotency. This is essentially a result of increased site 1 interaction because in the solubilized receptor assay the affinity of H170D pGH for the hGHR is only about 30-fold lower than hGH. The converse study, where Arg-43 in the hGHR is replaced by a leucine, shows that D171H hGH now binds and signals as effectively as wild type hGH (Table II). Against the R43L hGHR, pGH now behaves similarly to the way H170D pGH does against the wild type human receptor, i.e. its whole cell affinity is about 15-fold less than hGH, its biopotency is about 14-fold less than hGH, and in the solubilized receptor assay its affinity is only 30-fold less than hGH. Thus, mutation of either receptor Arg-43 to non-primate leucine or mutation of hormone Asp-171 to non-primate histidine has a very similar effect on magnitude of binding affinity and biopotency. This effect is also seen with another non-primate GH, bGH, which binds and acts poorly against the wild type human receptor but is quite effective (3–4-fold less than hGH) against human receptor with leucine in position 43. TableIII presents the combined affinity and biopotency data for the same GH analogs, but in this case against the rbGHR. It can be seen that replacement of Asp-171 of hGH with histidine has little effect on binding or bioactivity. Again there is a small loss in solubilized receptor affinity, suggesting that aspartate is slightly more effective in this position against receptor Leu-43, but clearly either histidine or aspartate sits well against a leucine at receptor residue 43. Porcine GH is considerably less effective than hGH against the rabbit receptor in accord with our previous data (11Rowlinson S.W. Barnard R. Bastiras S. Robins A.J. Brinkworth R. Waters M.J. J. Biol. Chem. 1995; 270: 16833-16839Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). This would appear to be primarily a result of poor site 1 interactions because the affinity of pGH in the solubilized receptor assay is markedly less than hGH. This poor interaction is evidently not a result of hormone residue 170 interactions because substituting aspartate for histidine at this position again improves solubilized receptor affinity by only 2–3-fold, against leucine at position 43. Bovine GH is seen to bind and act considerably more effectively than pGH against the rabbit receptor (Table III). Creation of a 舠humanized舡 rabbit receptor by replacing Arg-43 with leucine provides the final argument for the importance of this interaction in determining primate specificity (TableIV). As with the wild type rabbit receptor, hGH binds more strongly than it does against its own receptor. However, replacing Asp-171 with histidine markedly decreases (about 50-fold) site 1 affinity. Evidently strong site 2 interactions are able to compensate in that the whole cell affinity and biopotency are not markedly (about 2-fold) different from wild type hGH. Porcine GH now binds very poorly in the whole cell assay and effectively does not bind in the solubilized receptor assay. Biopotency is also very markedly reduced. This situation is effectively reversed by substitution of His-170 with aspartate, so that this analog now behaves similarly to pGH binding in its binding to the wild type rabbit receptor. In support of both our previous study (8Gobius K.S. Rowlinson S.W. Barnard R. Mattick J.S. Waters M.J. J. Mol. Endocrinol. 1992; 9: 213-220Crossref PubMed Scopus (24) Google Scholar) and the study of (9Souza S.C. Frick G.P. Wang X. Kopchick J.J. Lobo R.B. Goodman H.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 959-963Crossref PubMed Scopus (71) Google Scholar), bovine GH binding to the L43R rabbit receptor is only about 2.5-fold less than binding to wild type rabbit receptor, yet in the solubilized receptor assay its site 1 binding is very poor (700-fold less), and its biopotency is reduced quite strongly (about 35-fold). MTT assays were used as described previously (11Rowlinson S.W. Barnard R. Bastiras S. Robins A.J. Brinkworth R. Waters M.J. J. Biol. Chem. 1995; 270: 16833-16839Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) to compare the biopotencies of the various hormone preparations against wild type or mutant GHR-expressing cells. Because two GHRs interact with one GH molecule and because they use virtually the same residues to interact with the hormone at either site 1 or site 2 (3De Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2029) Google Scholar, 14Cunningham B.C. Ultsch M. Abraham M. DeVos A.M. Mulkerrin M.G. Clauser K.R. Wells J.A. Science. 1991; 254: 821-825Crossref PubMed Scopus (788) Google Scholar), it is not possible to compare the relative biopotencies between wild type and mutant cell lines because of the uncertainty that the mutation could be affecting the bioactivity through site 2 interactions and not site 1 interactions. For this reason the biopotencies of the GHs with different cell lines are compared relative to wild type hGH. With the hGHR-expressing cell line, the D171H hGH mutant was similar to wild type in its biopotency (see 舠Discussion舡). The non-primate GHs, bGH and pGH, were very poor agonists for this cell line, showing 235- and 2,342-fold lower biopotency relative to hGH. For pGH, the introduction of the H170D mutation improved the biopotency such that it is only 14-fold lower than that of hGH, an improvement of greater than 150-fold. Similar improvements in biopotency were observed with bGH and pGH upon the introduction of the R43L mutation in the hGHR. In this case, the biopotency of these hormones relative to hGH became only 3.6- and 13.5-fold lower than hGH, an improvement of more than 80- and 150-fold, respectively, when compared with the wild type hGHR-expressing cell line. In contrast, the H170D pGH analog exhibited similar biopotencies against the wild type hGHR- and R43L hGHR-expressing cell lines, indicating that aspartate at this location is equally well tolerated against either