The Binding between the Stem Regions of Human Growth Hormone (GH) Receptor Compensates for the Weaker Site 1 Binding of 20-kDa Human GH (hGH) than That of 22-kDa hGH
2000; Elsevier BV; Volume: 275; Issue: 21 Linguagem: Inglês
10.1074/jbc.m001236200
ISSN1083-351X
AutoresBunkichi Tsunekawa, Mitsufumi Wada, Miwa Ikeda, Shinichi Banba, Hironori Kamachi, Eishi Tanaka, Masaru Honjo,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoDespite the lower site 1 affinity of the 20-kDa human growh hormone (20K-hGH) for the hGH receptor (hGHR), 20K-hGH has the same hGHR-mediated activity as 22-kDa human GH (22K-hGH) at low hGH concentration and even higher activity at high hGH concentration. This study was performed to elucidate the reason why 20K-hGH can activate hGHR to the same level as 22K-hGH. To answer the question, we hypothesized that the binding between the stem regions of hGHR could compensate for the weaker site 1 binding of 20K-hGH than that of 22K-hGH in the sequential binding with hGHR. To demonstrate it, we prepared 15 types of alanine-substituted hGHR gene at the stem region and stably transfected them into Ba/F3 cells. Using these cells, we measured and compared the cell proliferation activities between 20K- and 22K-hGH. As a result, the activity of 20K-hGH was markedly reduced than that of 22K-hGH in three types of mutant hGHR (T147A, H150A, and Y200A). Regarding these mutants, the dissociation constant of hGH at the first and second step (KD1 and KD2) in the sequential binding with two hGHRs was predicted based on the mathematical cell proliferation model and computational simulation. Consequently, it was revealed that the reduction of the activity in 20K-hGH was attributed to the change of not KD1 but KD2. In conclusion, these findings support our hypothesis, which can account for the same potencies for activating hGHR between 20K- and 22K-hGH, although the site 1 affinity of 20K-hGH is lower than that of 22K-hGH. Despite the lower site 1 affinity of the 20-kDa human growh hormone (20K-hGH) for the hGH receptor (hGHR), 20K-hGH has the same hGHR-mediated activity as 22-kDa human GH (22K-hGH) at low hGH concentration and even higher activity at high hGH concentration. This study was performed to elucidate the reason why 20K-hGH can activate hGHR to the same level as 22K-hGH. To answer the question, we hypothesized that the binding between the stem regions of hGHR could compensate for the weaker site 1 binding of 20K-hGH than that of 22K-hGH in the sequential binding with hGHR. To demonstrate it, we prepared 15 types of alanine-substituted hGHR gene at the stem region and stably transfected them into Ba/F3 cells. Using these cells, we measured and compared the cell proliferation activities between 20K- and 22K-hGH. As a result, the activity of 20K-hGH was markedly reduced than that of 22K-hGH in three types of mutant hGHR (T147A, H150A, and Y200A). Regarding these mutants, the dissociation constant of hGH at the first and second step (KD1 and KD2) in the sequential binding with two hGHRs was predicted based on the mathematical cell proliferation model and computational simulation. Consequently, it was revealed that the reduction of the activity in 20K-hGH was attributed to the change of not KD1 but KD2. In conclusion, these findings support our hypothesis, which can account for the same potencies for activating hGHR between 20K- and 22K-hGH, although the site 1 affinity of 20K-hGH is lower than that of 22K-hGH. 20-kDa human growth hormone 22-kDa human GH interleukin human GH receptor extracellular domain dissociation constant The 20-kilodalton (kDa) human GH (20K-hGH)1 is known to be naturally secreted from the pituitary gland besides 22-kDa human GH (22K-hGH), which is a major component composed of 191 amino acids (1.Lewis U.J. Dunn J.T. Bonewald L.F. Seavey B.K. VanderLaan W.P. J. Biol. Chem. 1978; 253: 2679-2687Abstract Full Text PDF PubMed Google Scholar,2.Baumann G. Endocr. Rev. 1991; 12: 424-449Crossref PubMed Scopus (405) Google Scholar). The 20K-hGH is encoded by the same GH-N gene as 22K-hGH but lacking 15 amino acids (residues 32–46 in 22K-hGH) by an alternative messenger RNA (mRNA) splicing (3.DeNoto F.M. Moore D.D. Goodman H.M. Nucleic Acids Res. 1981; 9: 3719-3730Crossref PubMed Scopus (302) Google Scholar, 4.Lewis U.J. Bonewald L.F. Lewis L.J. Biochem. Biophys. Res. Commun. 1980; 92: 511-516Crossref PubMed Scopus (140) Google Scholar, 5.Masuda N. Watahiki M. Tanaka M. Yamakawa M. Shimizu K. Nagai J. Nakashima K. Biochim. Biophys. Acta. 1988; 949: 125-131Crossref PubMed Scopus (33) Google Scholar). Because of the difficulty in preparing large enough amounts of highly purified 20K-hGH with authentic structure, its biological properties were unclear and controversial. Recently, we established an Escherichia coli secretion system for 20K-hGH with an authentic structure (6.Uchida H. Naito N. Asada N. Wada M. Ikeda M. Kobayashi H. Asanagi M. Mori K. Fujita Y. Konda K. Kusuhara N. Kamioka T. Nakashima K. Honjo M. J. Biotechnol. 1997; 55: 101-112Crossref PubMed Scopus (48) Google Scholar) and reported several properties of 20K-hGH (7.Tsunekawa B. Wada M. Ikeda M. Uchida H. Naito N. Honjo M. Endocrinology. 1999; 140: 3909-3918Crossref PubMed Scopus (25) Google Scholar, 8.Tsushima T. Katoh Y. Miyachi Y. Chihara K. Teramoto A. Irie M. Hashimoto Y. J. Clin. Endocrinol. Metab. 1999; 84: 317-322PubMed Google Scholar, 9.Uchida H. Banba S. Wada M. Matsumoto K. Ikeda M. Naito N. Tanaka E. Honjo M. J. Mol. Endocrinol. 1999; 23: 347-353Crossref PubMed Scopus (24) Google Scholar, 10.Wada M. Ikeda M. Takahashi Y. Asada N. Chang K.T. Takahashi M. Honjo M. Mol. Cell. Endocrinol. 1997; 133: 99-107Crossref PubMed Scopus (35) Google Scholar, 11.Wada M. Uchida H. Ikeda M. Tsunekawa B. Naito N. Banba S. Tanaka E. Hashimoto Y. Honjo M. Mol. Endocrinol. 1998; 12: 146-156Crossref PubMed Scopus (66) Google Scholar).Previous analyses showed that 20K-hGH formed an active 1:2 (hGH:hGHR) complex in a sequential manner as well as 22K-hGH, that is, the first hGHR binds to site 1 on hGH (this is designated “STEP1”) and next the second one binds to site 2 (STEP2) (9.Uchida H. Banba S. Wada M. Matsumoto K. Ikeda M. Naito N. Tanaka E. Honjo M. J. Mol. Endocrinol. 1999; 23: 347-353Crossref PubMed Scopus (24) Google Scholar, 11.Wada M. Uchida H. Ikeda M. Tsunekawa B. Naito N. Banba S. Tanaka E. Hashimoto Y. Honjo M. Mol. Endocrinol. 1998; 12: 146-156Crossref PubMed Scopus (66) Google Scholar, 12.Cunningham B.C. Ultsch M. DeVos A.M. Mulkerrin M.G. Clauser K.R. Wells J.A. Science. 1991; 254: 821-825Crossref PubMed Scopus (782) Google Scholar, 13.DeVos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2016) Google Scholar, 14.Fuh G. Cunningham B.C. Fukunaga R. Nagata S. Goeddel D.V. Wells J.A. Science. 1992; 256: 1677-1680Crossref PubMed Scopus (571) Google Scholar). An active 1:2 (hGH:hGHR) complex formation is followed by tyrosine phosphorylation of hGHR and activation of intracellular signal transducer molecules (15.Argetsinger L.S. Campbell G.S. Yang X. Witthuhn B.A. Silvennoinen O. Ihle J.N. Carter-Su C. Cell. 1993; 74: 237-244Abstract Full Text PDF PubMed Scopus (818) Google Scholar, 16.Meyer D.J. Campbell G.S. Cochran B.H. Argetsinger L.S. Larner A.C. Finbloom D.S. Carter-Su C. Schwartz J. J. Biol. Chem. 1994; 269: 4701-4704Abstract Full Text PDF PubMed Google Scholar, 17.Gouilleux F. Pallard C. Dusanter-Fourt I. Wakao H. Haldosen L.A. Norstedt G. Levy D. Groner B. EMBO J. 1995; 14: 2005-2013Crossref PubMed Scopus (332) Google Scholar). Fig. 1 shows the schematic illustration of the mode of receptor dimerization of both hGH isoforms. Here we tentatively designate the stem region of hGHR, which is considered to be involved in receptor dimerization, “siteBP.” Concerning the binding affinity between hGH and hGHR, we reported that KD1(22K) was smaller than KD1(20K) in the biosensor analysis (9.Uchida H. Banba S. Wada M. Matsumoto K. Ikeda M. Naito N. Tanaka E. Honjo M. J. Mol. Endocrinol. 1999; 23: 347-353Crossref PubMed Scopus (24) Google Scholar) and that KDsite2(20K) was considered to be almost the same as KDsite2(22K), because the site 2 region of 20K-hGH was conformationally similar to 22K-hGH (11.Wada M. Uchida H. Ikeda M. Tsunekawa B. Naito N. Banba S. Tanaka E. Hashimoto Y. Honjo M. Mol. Endocrinol. 1998; 12: 146-156Crossref PubMed Scopus (66) Google Scholar). On the other hand, the activity of 20K-hGH via hGHR is the same as that of 22K-hGH at low hGH concentration and even higher at high hGH concentration in cell proliferation assay (11.Wada M. Uchida H. Ikeda M. Tsunekawa B. Naito N. Banba S. Tanaka E. Hashimoto Y. Honjo M. Mol. Endocrinol. 1998; 12: 146-156Crossref PubMed Scopus (66) Google Scholar). Therefore, we hypothesized that binding of 20K-hGH to the first hGHR could result in the transient 1:1 complex, which has a more favorable conformation for forming an active 1:2 complex especially at the siteBP region of hGHR.In this study, to elucidate the difference of siteBP contribution to the 1:2 complex formation with hGHR between 20K- and 22K-hGH, we made an hGHR expression plasmid with conversion of several amino acids at the siteBP region to alanine. Using mouse pro B cell line (Ba/F3) stably expressing the mutant or wild type hGHR, cell proliferation assay of both hGH isoforms was performed. As a result, in three mutants (T147A, H150A, and Y200A), 20K-hGH had markedly reduced activity than 22K-hGH. In addition, we estimated KD1 and KD2 of both hGH isoforms by theoretically simulating the cell proliferation curve, and it was highly speculated that alanine substitution at siteBP could mainly affect the affinity of 20K-hGH at STEP2 (KD2(20K)). By lacking 15 amino acids, 20K-hGH partly lost the site 1 affinity but the binding between siteBPs possibly compensates for the loss.DISCUSSIONIn this study, we have revealed that alanine substitution at Thr-147, Ile-149, His-150, and Tyr-200 in the siteBP region of hGHR reduced the cell proliferation activity of 20K-hGH as compared with that of 22K-hGH. The involvement of the siteBP region in the complex formation of 22K-hGH and hGHR has been studied by several groups. DeVoset al. (13.DeVos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2016) Google Scholar) first reported that there was a substantial contact surface between the C-terminal domains of hGHR-ECD, namely siteBP regions, when 22K-hGH formed a 1:2 complex with hGHR-ECD. Crystallographic study showed the eight residues (Asn-143, Ser-145, Leu-146, Thr-147, His-150, Asp-152, Tyr-200, and Ser-201) were involved in this domain. Furthermore, Clackson et al. (24.Clackson T. Ultsch M.H. Wells J.A. DeVos A.M. J. Mol. Biol. 1998; 277: 1111-1128Crossref PubMed Scopus (246) Google Scholar) showed that hGHR dimerization stabilized loop structure from Val-144 to Gly-148 at siteBP region. Chen et al. (25.Chen C. Brinkworth R. Waters M.J. J. Biol. Chem. 1997; 272: 5133-5140Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) converted several amino acids at siteBP of rabbit GHR to alanine, aspartate, lysine, or cysteine and presented that residues Ser-145, His-150, Asp-152, Tyr-200, and Ser-201 were required for effective signal transduction through the dimerization domain. These previous studies are well consistent with our finding except for Ile-149, because the involvement of Ile-149 in siteBP interaction has been demonstrated for the first time in this study.In patients with Laron syndrome (familial GH resistance characterized by severe dwarfism and metabolic dysfunction), a point mutation in the siteBP region was identified resulting in the substitution of a highly conserved aspartate residue by histidine at position 152 (D152H) of hGHR (26.Duquesnoy P. Sobrier M.L. Duriez B. Dastot F. Buchanan C.R. Savage M.O. Preece M.A. Craescu C.T. Blouquit Y. Goossens M. Amselem S. EMBO J. 1994; 13: 1386-1395Crossref PubMed Scopus (152) Google Scholar). Duquesnoy et al. (26.Duquesnoy P. Sobrier M.L. Duriez B. Dastot F. Buchanan C.R. Savage M.O. Preece M.A. Craescu C.T. Blouquit Y. Goossens M. Amselem S. EMBO J. 1994; 13: 1386-1395Crossref PubMed Scopus (152) Google Scholar) reported that the hGHR with mutation D152H displayed subnormal GH binding activity but hGHR-ECD with D152H substitution was unable to dimerize. In this report, not only 22K-hGH but also 20K-GH had no cell proliferation activity in mutation D152A nor D152H (data not shown). These results mean that Asp-152 in hGHR plays a quite important role in the binding between siteBP regions.Especially, the mutation T147A, H150A, and Y200A of hGHR resulted in a drastic decrease of cell proliferation activity of 20K-hGH compared with that of 22K-hGH. Similar results were observed also in the rat serine protease inhibitor 2.1 gene promoter activation assay in CHO-K1 cells transiently expressing each three mutants (data not shown). These data indicate that Thr-147, His-150, and Tyr-200 considerably contribute to the same hGHR-mediated activity of 20K-hGH as 22K-hGH regardless of its reduced site 1 affinity. To clarify the mechanism of how Thr-147, His-150, and Tyr-200 enable 20K-hGH to form an active 1:2 complex to the same degree as 22K-hGH, we are now under investigation of the x-ray crystal structure of the complex of 20K-hGH and hGHR-ECD.To elucidate the binding affinity at STEP1 and STEP2 resulting from alanine substitution, we estimated the dissociation constant KD1 and KD2 of 20K- and 22K-hGH by modeling the cell proliferation curve mathematically. By fitting the calculative proliferation curve to the experimental data of the wild type hGHR, KD1(20K) and KD1(22K) were predicted to be 13 and 5.1 nm, respectively, and the binding affinity of 20K-hGH at site 1 was weaker than that of 22K-hGH. These estimates are reasonable compared with the results experimentally obtained by biosensor analysis using hGHR-ECD, where KD1(20K) (16 nm) is lager than KD1(22K) (2 nm) (9.Uchida H. Banba S. Wada M. Matsumoto K. Ikeda M. Naito N. Tanaka E. Honjo M. J. Mol. Endocrinol. 1999; 23: 347-353Crossref PubMed Scopus (24) Google Scholar).Concerning the mutation T147A, H150A, and Y200A, KD2(20K) increased 10-, 122-, and 68-fold, respectively, compared with KD2(22K) (1.8-, 17-, and 5.3-fold). Generally alanine substitution is minimally perturbing for the secondary and tertiary structure, and three alanine mutations are located only at the siteBP region of hGHR. Therefore, mutations T147A, H150A, and Y200A are considered to have no direct influence on the contact surface structure between the site 2 region on hGH and the second hGHR in sequential binding, that is, the binding affinity KDsite2(20K) in Fig. 1. This suggests that the drastic change of KD2(20K) results from the change of KDsiteBP(20K).In our calculation, the change of hGH bioactivity was in good concordance with the change of the binding affinity (KD2(20K)). However, Rowlinson et al. (27.Rowlinson 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) reported that GH with mutation at site 1 markedly lost its bioactivity without loss of site 1 binding affinity. Recently, Pearce et al. (23.Pearce Jr., K.H. Cunningham B.C. Fuh G. Teeri T. Wells J.A. Biochemistry. 1999; 38: 81-89Crossref PubMed Scopus (97) Google Scholar) reported that the EC50 of cell proliferation via wild type hGHR was unaffected until site 1 affinity of 22K-hGH was reduced about 30-fold from that of wild type 22K-hGH. Under the condition used in our sequential binding and cell proliferation model, it is predicted that an ∼30-fold reduction in site 1 affinity should yield about 30-fold higher EC50 for cell proliferation, which means the experimental data of Pearce et al. (23.Pearce Jr., K.H. Cunningham B.C. Fuh G. Teeri T. Wells J.A. Biochemistry. 1999; 38: 81-89Crossref PubMed Scopus (97) Google Scholar) cannot be simulated by our model. Unfortunately, at present we cannot explain such disagreement, and some modifications might be necessary for more accurate simulation by adding some new parameters to the sequential binding model or the cell proliferation model or by altering some prerequisites.In conclusion, we have shown that some alanine substitutions at the siteBP region of hGHR caused the markedly decreased activity of 20K-hGH compared with 22K-hGH. This means the siteBP region is involved in 1:2 complex formation in a different manner between 20K- and 22K-hGH, and the binding between the siteBP regions compensates for the weaker site 1 binding of 20K-hGH than that of 22K-hGH. The 20-kilodalton (kDa) human GH (20K-hGH)1 is known to be naturally secreted from the pituitary gland besides 22-kDa human GH (22K-hGH), which is a major component composed of 191 amino acids (1.Lewis U.J. Dunn J.T. Bonewald L.F. Seavey B.K. VanderLaan W.P. J. Biol. Chem. 1978; 253: 2679-2687Abstract Full Text PDF PubMed Google Scholar,2.Baumann G. Endocr. Rev. 1991; 12: 424-449Crossref PubMed Scopus (405) Google Scholar). The 20K-hGH is encoded by the same GH-N gene as 22K-hGH but lacking 15 amino acids (residues 32–46 in 22K-hGH) by an alternative messenger RNA (mRNA) splicing (3.DeNoto F.M. Moore D.D. Goodman H.M. Nucleic Acids Res. 1981; 9: 3719-3730Crossref PubMed Scopus (302) Google Scholar, 4.Lewis U.J. Bonewald L.F. Lewis L.J. Biochem. Biophys. Res. Commun. 1980; 92: 511-516Crossref PubMed Scopus (140) Google Scholar, 5.Masuda N. Watahiki M. Tanaka M. Yamakawa M. Shimizu K. Nagai J. Nakashima K. Biochim. Biophys. Acta. 1988; 949: 125-131Crossref PubMed Scopus (33) Google Scholar). Because of the difficulty in preparing large enough amounts of highly purified 20K-hGH with authentic structure, its biological properties were unclear and controversial. Recently, we established an Escherichia coli secretion system for 20K-hGH with an authentic structure (6.Uchida H. Naito N. Asada N. Wada M. Ikeda M. Kobayashi H. Asanagi M. Mori K. Fujita Y. Konda K. Kusuhara N. Kamioka T. Nakashima K. Honjo M. J. Biotechnol. 1997; 55: 101-112Crossref PubMed Scopus (48) Google Scholar) and reported several properties of 20K-hGH (7.Tsunekawa B. Wada M. Ikeda M. Uchida H. Naito N. Honjo M. Endocrinology. 1999; 140: 3909-3918Crossref PubMed Scopus (25) Google Scholar, 8.Tsushima T. Katoh Y. Miyachi Y. Chihara K. Teramoto A. Irie M. Hashimoto Y. J. Clin. Endocrinol. Metab. 1999; 84: 317-322PubMed Google Scholar, 9.Uchida H. Banba S. Wada M. Matsumoto K. Ikeda M. Naito N. Tanaka E. Honjo M. J. Mol. Endocrinol. 1999; 23: 347-353Crossref PubMed Scopus (24) Google Scholar, 10.Wada M. Ikeda M. Takahashi Y. Asada N. Chang K.T. Takahashi M. Honjo M. Mol. Cell. Endocrinol. 1997; 133: 99-107Crossref PubMed Scopus (35) Google Scholar, 11.Wada M. Uchida H. Ikeda M. Tsunekawa B. Naito N. Banba S. Tanaka E. Hashimoto Y. Honjo M. Mol. Endocrinol. 1998; 12: 146-156Crossref PubMed Scopus (66) Google Scholar). Previous analyses showed that 20K-hGH formed an active 1:2 (hGH:hGHR) complex in a sequential manner as well as 22K-hGH, that is, the first hGHR binds to site 1 on hGH (this is designated “STEP1”) and next the second one binds to site 2 (STEP2) (9.Uchida H. Banba S. Wada M. Matsumoto K. Ikeda M. Naito N. Tanaka E. Honjo M. J. Mol. Endocrinol. 1999; 23: 347-353Crossref PubMed Scopus (24) Google Scholar, 11.Wada M. Uchida H. Ikeda M. Tsunekawa B. Naito N. Banba S. Tanaka E. Hashimoto Y. Honjo M. Mol. Endocrinol. 1998; 12: 146-156Crossref PubMed Scopus (66) Google Scholar, 12.Cunningham B.C. Ultsch M. DeVos A.M. Mulkerrin M.G. Clauser K.R. Wells J.A. Science. 1991; 254: 821-825Crossref PubMed Scopus (782) Google Scholar, 13.DeVos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2016) Google Scholar, 14.Fuh G. Cunningham B.C. Fukunaga R. Nagata S. Goeddel D.V. Wells J.A. Science. 1992; 256: 1677-1680Crossref PubMed Scopus (571) Google Scholar). An active 1:2 (hGH:hGHR) complex formation is followed by tyrosine phosphorylation of hGHR and activation of intracellular signal transducer molecules (15.Argetsinger L.S. Campbell G.S. Yang X. Witthuhn B.A. Silvennoinen O. Ihle J.N. Carter-Su C. Cell. 1993; 74: 237-244Abstract Full Text PDF PubMed Scopus (818) Google Scholar, 16.Meyer D.J. Campbell G.S. Cochran B.H. Argetsinger L.S. Larner A.C. Finbloom D.S. Carter-Su C. Schwartz J. J. Biol. Chem. 1994; 269: 4701-4704Abstract Full Text PDF PubMed Google Scholar, 17.Gouilleux F. Pallard C. Dusanter-Fourt I. Wakao H. Haldosen L.A. Norstedt G. Levy D. Groner B. EMBO J. 1995; 14: 2005-2013Crossref PubMed Scopus (332) Google Scholar). Fig. 1 shows the schematic illustration of the mode of receptor dimerization of both hGH isoforms. Here we tentatively designate the stem region of hGHR, which is considered to be involved in receptor dimerization, “siteBP.” Concerning the binding affinity between hGH and hGHR, we reported that KD1(22K) was smaller than KD1(20K) in the biosensor analysis (9.Uchida H. Banba S. Wada M. Matsumoto K. Ikeda M. Naito N. Tanaka E. Honjo M. J. Mol. Endocrinol. 1999; 23: 347-353Crossref PubMed Scopus (24) Google Scholar) and that KDsite2(20K) was considered to be almost the same as KDsite2(22K), because the site 2 region of 20K-hGH was conformationally similar to 22K-hGH (11.Wada M. Uchida H. Ikeda M. Tsunekawa B. Naito N. Banba S. Tanaka E. Hashimoto Y. Honjo M. Mol. Endocrinol. 1998; 12: 146-156Crossref PubMed Scopus (66) Google Scholar). On the other hand, the activity of 20K-hGH via hGHR is the same as that of 22K-hGH at low hGH concentration and even higher at high hGH concentration in cell proliferation assay (11.Wada M. Uchida H. Ikeda M. Tsunekawa B. Naito N. Banba S. Tanaka E. Hashimoto Y. Honjo M. Mol. Endocrinol. 1998; 12: 146-156Crossref PubMed Scopus (66) Google Scholar). Therefore, we hypothesized that binding of 20K-hGH to the first hGHR could result in the transient 1:1 complex, which has a more favorable conformation for forming an active 1:2 complex especially at the siteBP region of hGHR. In this study, to elucidate the difference of siteBP contribution to the 1:2 complex formation with hGHR between 20K- and 22K-hGH, we made an hGHR expression plasmid with conversion of several amino acids at the siteBP region to alanine. Using mouse pro B cell line (Ba/F3) stably expressing the mutant or wild type hGHR, cell proliferation assay of both hGH isoforms was performed. As a result, in three mutants (T147A, H150A, and Y200A), 20K-hGH had markedly reduced activity than 22K-hGH. In addition, we estimated KD1 and KD2 of both hGH isoforms by theoretically simulating the cell proliferation curve, and it was highly speculated that alanine substitution at siteBP could mainly affect the affinity of 20K-hGH at STEP2 (KD2(20K)). By lacking 15 amino acids, 20K-hGH partly lost the site 1 affinity but the binding between siteBPs possibly compensates for the loss. DISCUSSIONIn this study, we have revealed that alanine substitution at Thr-147, Ile-149, His-150, and Tyr-200 in the siteBP region of hGHR reduced the cell proliferation activity of 20K-hGH as compared with that of 22K-hGH. The involvement of the siteBP region in the complex formation of 22K-hGH and hGHR has been studied by several groups. DeVoset al. (13.DeVos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2016) Google Scholar) first reported that there was a substantial contact surface between the C-terminal domains of hGHR-ECD, namely siteBP regions, when 22K-hGH formed a 1:2 complex with hGHR-ECD. Crystallographic study showed the eight residues (Asn-143, Ser-145, Leu-146, Thr-147, His-150, Asp-152, Tyr-200, and Ser-201) were involved in this domain. Furthermore, Clackson et al. (24.Clackson T. Ultsch M.H. Wells J.A. DeVos A.M. J. Mol. Biol. 1998; 277: 1111-1128Crossref PubMed Scopus (246) Google Scholar) showed that hGHR dimerization stabilized loop structure from Val-144 to Gly-148 at siteBP region. Chen et al. (25.Chen C. Brinkworth R. Waters M.J. J. Biol. Chem. 1997; 272: 5133-5140Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) converted several amino acids at siteBP of rabbit GHR to alanine, aspartate, lysine, or cysteine and presented that residues Ser-145, His-150, Asp-152, Tyr-200, and Ser-201 were required for effective signal transduction through the dimerization domain. These previous studies are well consistent with our finding except for Ile-149, because the involvement of Ile-149 in siteBP interaction has been demonstrated for the first time in this study.In patients with Laron syndrome (familial GH resistance characterized by severe dwarfism and metabolic dysfunction), a point mutation in the siteBP region was identified resulting in the substitution of a highly conserved aspartate residue by histidine at position 152 (D152H) of hGHR (26.Duquesnoy P. Sobrier M.L. Duriez B. Dastot F. Buchanan C.R. Savage M.O. Preece M.A. Craescu C.T. Blouquit Y. Goossens M. Amselem S. EMBO J. 1994; 13: 1386-1395Crossref PubMed Scopus (152) Google Scholar). Duquesnoy et al. (26.Duquesnoy P. Sobrier M.L. Duriez B. Dastot F. Buchanan C.R. Savage M.O. Preece M.A. Craescu C.T. Blouquit Y. Goossens M. Amselem S. EMBO J. 1994; 13: 1386-1395Crossref PubMed Scopus (152) Google Scholar) reported that the hGHR with mutation D152H displayed subnormal GH binding activity but hGHR-ECD with D152H substitution was unable to dimerize. In this report, not only 22K-hGH but also 20K-GH had no cell proliferation activity in mutation D152A nor D152H (data not shown). These results mean that Asp-152 in hGHR plays a quite important role in the binding between siteBP regions.Especially, the mutation T147A, H150A, and Y200A of hGHR resulted in a drastic decrease of cell proliferation activity of 20K-hGH compared with that of 22K-hGH. Similar results were observed also in the rat serine protease inhibitor 2.1 gene promoter activation assay in CHO-K1 cells transiently expressing each three mutants (data not shown). These data indicate that Thr-147, His-150, and Tyr-200 considerably contribute to the same hGHR-mediated activity of 20K-hGH as 22K-hGH regardless of its reduced site 1 affinity. To clarify the mechanism of how Thr-147, His-150, and Tyr-200 enable 20K-hGH to form an active 1:2 complex to the same degree as 22K-hGH, we are now under investigation of the x-ray crystal structure of the complex of 20K-hGH and hGHR-ECD.To elucidate the binding affinity at STEP1 and STEP2 resulting from alanine substitution, we estimated the dissociation constant KD1 and KD2 of 20K- and 22K-hGH by modeling the cell proliferation curve mathematically. By fitting the calculative proliferation curve to the experimental data of the wild type hGHR, KD1(20K) and KD1(22K) were predicted to be 13 and 5.1 nm, respectively, and the binding affinity of 20K-hGH at site 1 was weaker than that of 22K-hGH. These estimates are reasonable compared with the results experimentally obtained by biosensor analysis using hGHR-ECD, where KD1(20K) (16 nm) is lager than KD1(22K) (2 nm) (9.Uchida H. Banba S. Wada M. Matsumoto K. Ikeda M. Naito N. Tanaka E. Honjo M. J. Mol. Endocrinol. 1999; 23: 347-353Crossref PubMed Scopus (24) Google Scholar).Concerning the mutation T147A, H150A, and Y200A, KD2(20K) increased 10-, 122-, and 68-fold, respectively, compared with KD2(22K) (1.8-, 17-, and 5.3-fold). Generally alanine substitution is minimally perturbing for the secondary and tertiary structure, and three alanine mutations are located only at the siteBP region of hGHR. Therefore, mutations T147A, H150A, and Y200A are considered to have no direct influence on the contact surface structure between the site 2 region on hGH and the second hGHR in sequential binding, that is, the binding affinity KDsite2(20K) in Fig. 1. This suggests that the drastic change of KD2(20K) results from the change of KDsiteBP(20K).In our calculation, the change of hGH bioactivity was in good concordance with the change of the binding affinity (KD2(20K)). However, Rowlinson et al. (27.Rowlinson 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) reported that GH with mutation at site 1 markedly lost its bioactivity without loss of site 1 binding affinity. Recently, Pearce et al. (23.Pearce Jr., K.H. Cunningham B.C. Fuh G. Teeri T. Wells J.A. Biochemistry. 1999; 38: 81-89Crossref PubMed Scopus (97) Google Scholar) reported that the EC50 of cell proliferation via wild type hGHR was unaffected until site 1 affinity of 22K-hGH was reduced about 30-fold from that of wild type 22K-hGH. Under the condition used in our sequential binding and cell proliferation model, it is predicted that an ∼30-fold reduction in site 1 affinity should yield about 30-fold higher EC50 for cell proliferation, which means the experimental data of Pearce et al. (23.Pearce Jr., K.H. Cunningham B.C. Fuh G. Teeri T. Wells J.A. Biochemistry. 1999; 38: 81-89Crossref PubMed Scopus (97) Google Scholar) cannot be simulated by our model. Unfortunately, at present we cannot explain such disagreement, and some modifications might be necessary for more accurate simulation by adding some new parameters to the sequential binding model or the cell proliferation model or by altering some prerequisites.In conclusion, we have shown that some alanine substitutions at the siteBP region of hGHR caused the markedly decreased activity of 20K-hGH compared with 22K-hGH. This means the siteBP region is involved in 1:2 complex formation in a different manner between 20K- and 22K-hGH, and the binding between the siteBP regions compensates for the weaker site 1 binding of 20K-hGH than that of 22K-hGH. In this study, we have revealed that alanine substitution at Thr-147, Ile-149, His-150, and Tyr-200 in the siteBP region of hGHR reduced the cell proliferation activity of 20K-hGH as compared with that of 22K-hGH. The involvement of the siteBP region in the complex formation of 22K-hGH and hGHR has been studied by several groups. DeVoset al. (13.DeVos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2016) Google Scholar) first reported that there was a substantial contact surface between the C-terminal domains of hGHR-ECD, namely siteBP regions, when 22K-hGH formed a 1:2 complex with hGHR-ECD. Crystallographic study showed the eight residues (Asn-143, Ser-145, Leu-146, Thr-147, His-150, Asp-152, Tyr-200, and Ser-201) were involved in this domain. Furthermore, Clackson et al. (24.Clackson T. Ultsch M.H. Wells J.A. DeVos A.M. J. Mol. Biol. 1998; 277: 1111-1128Crossref PubMed Scopus (246) Google Scholar) showed that hGHR dimerization stabilized loop structure from Val-144 to Gly-148 at siteBP region. Chen et al. (25.Chen C. Brinkworth R. Waters M.J. J. Biol. Chem. 1997; 272: 5133-5140Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) converted several amino acids at siteBP of rabbit GHR to alanine, aspartate, lysine, or cysteine and presented that residues Ser-145, His-150, Asp-152, Tyr-200, and Ser-201 were required for effective signal transduction through the dimerization domain. These previous studies are well consistent with our finding except for Ile-149, because the involvement of Ile-149 in siteBP interaction has been demonstrated for the first time in this study. In patients with Laron syndrome (familial GH resistance characterized by severe dwarfism and metabolic dysfunction), a point mutation in the siteBP region was identified resulting in the substitution of a highly conserved aspartate residue by histidine at position 152 (D152H) of hGHR (26.Duquesnoy P. Sobrier M.L. Duriez B. Dastot F. Buchanan C.R. Savage M.O. Preece M.A. Craescu C.T. Blouquit Y. Goossens M. Amselem S. EMBO J. 1994; 13: 1386-1395Crossref PubMed Scopus (152) Google Scholar). Duquesnoy et al. (26.Duquesnoy P. Sobrier M.L. Duriez B. Dastot F. Buchanan C.R. Savage M.O. Preece M.A. Craescu C.T. Blouquit Y. Goossens M. Amselem S. EMBO J. 1994; 13: 1386-1395Crossref PubMed Scopus (152) Google Scholar) reported that the hGHR with mutation D152H displayed subnormal GH binding activity but hGHR-ECD with D152H substitution was unable to dimerize. In this report, not only 22K-hGH but also 20K-GH had no cell proliferation activity in mutation D152A nor D152H (data not shown). These results mean that Asp-152 in hGHR plays a quite important role in the binding between siteBP regions. Especially, the mutation T147A, H150A, and Y200A of hGHR resulted in a drastic decrease of cell proliferation activity of 20K-hGH compared with that of 22K-hGH. Similar results were observed also in the rat serine protease inhibitor 2.1 gene promoter activation assay in CHO-K1 cells transiently expressing each three mutants (data not shown). These data indicate that Thr-147, His-150, and Tyr-200 considerably contribute to the same hGHR-mediated activity of 20K-hGH as 22K-hGH regardless of its reduced site 1 affinity. To clarify the mechanism of how Thr-147, His-150, and Tyr-200 enable 20K-hGH to form an active 1:2 complex to the same degree as 22K-hGH, we are now under investigation of the x-ray crystal structure of the complex of 20K-hGH and hGHR-ECD. To elucidate the binding affinity at STEP1 and STEP2 resulting from alanine substitution, we estimated the dissociation constant KD1 and KD2 of 20K- and 22K-hGH by modeling the cell proliferation curve mathematically. By fitting the calculative proliferation curve to the experimental data of the wild type hGHR, KD1(20K) and KD1(22K) were predicted to be 13 and 5.1 nm, respectively, and the binding affinity of 20K-hGH at site 1 was weaker than that of 22K-hGH. These estimates are reasonable compared with the results experimentally obtained by biosensor analysis using hGHR-ECD, where KD1(20K) (16 nm) is lager than KD1(22K) (2 nm) (9.Uchida H. Banba S. Wada M. Matsumoto K. Ikeda M. Naito N. Tanaka E. Honjo M. J. Mol. Endocrinol. 1999; 23: 347-353Crossref PubMed Scopus (24) Google Scholar). Concerning the mutation T147A, H150A, and Y200A, KD2(20K) increased 10-, 122-, and 68-fold, respectively, compared with KD2(22K) (1.8-, 17-, and 5.3-fold). Generally alanine substitution is minimally perturbing for the secondary and tertiary structure, and three alanine mutations are located only at the siteBP region of hGHR. Therefore, mutations T147A, H150A, and Y200A are considered to have no direct influence on the contact surface structure between the site 2 region on hGH and the second hGHR in sequential binding, that is, the binding affinity KDsite2(20K) in Fig. 1. This suggests that the drastic change of KD2(20K) results from the change of KDsiteBP(20K). In our calculation, the change of hGH bioactivity was in good concordance with the change of the binding affinity (KD2(20K)). However, Rowlinson et al. (27.Rowlinson 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) reported that GH with mutation at site 1 markedly lost its bioactivity without loss of site 1 binding affinity. Recently, Pearce et al. (23.Pearce Jr., K.H. Cunningham B.C. Fuh G. Teeri T. Wells J.A. Biochemistry. 1999; 38: 81-89Crossref PubMed Scopus (97) Google Scholar) reported that the EC50 of cell proliferation via wild type hGHR was unaffected until site 1 affinity of 22K-hGH was reduced about 30-fold from that of wild type 22K-hGH. Under the condition used in our sequential binding and cell proliferation model, it is predicted that an ∼30-fold reduction in site 1 affinity should yield about 30-fold higher EC50 for cell proliferation, which means the experimental data of Pearce et al. (23.Pearce Jr., K.H. Cunningham B.C. Fuh G. Teeri T. Wells J.A. Biochemistry. 1999; 38: 81-89Crossref PubMed Scopus (97) Google Scholar) cannot be simulated by our model. Unfortunately, at present we cannot explain such disagreement, and some modifications might be necessary for more accurate simulation by adding some new parameters to the sequential binding model or the cell proliferation model or by altering some prerequisites. In conclusion, we have shown that some alanine substitutions at the siteBP region of hGHR caused the markedly decreased activity of 20K-hGH compared with 22K-hGH. This means the siteBP region is involved in 1:2 complex formation in a different manner between 20K- and 22K-hGH, and the binding between the siteBP regions compensates for the weaker site 1 binding of 20K-hGH than that of 22K-hGH. We thank Professor Junichi Miyazaki (Osaka University) for kindly providing pCXN2 plasmid.
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