Monoclonal Antibodies to the Rabbit Liver Growth Hormone Receptor: Production and Characterization*

Barnard, Ross; Bundesen, Peter; Rylatt, Dennis B.; Waters, Michael J.

doi:10.1210/endo-115-5-1805

Cited by 75 publications

(76 citation statements)

References 17 publications

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“…A monoclonal antibody to the hGHbp (mAb5, obtained from Agen, Parsippany, NJ; ref. 16) was used to precipitate the 1251-hGHIhGHbp complex. Zinc-dependent binding of the hGH mutants to the hPRLbp was measured by competitive displacement of 1251-hGH as described for the hGHbp (6,9) except that assays included 50 AM ZnCl2 and 10 mM MgCl2 (10).…”

Section: Methodsmentioning

confidence: 99%

Rational design of receptor-specific variants of human growth hormone.

Cunningham¹,

Wells²

1991

Proc. Natl. Acad. Sci. U.S.A.

194

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Human growth hormone (hGH) binds to both the growth hormone (GH) and the prolactin (PRL) receptors. Competition experiments followed by mutational analysis show that the epitope on hGH for hPRL receptor consists of strong determinants in the middle of helix 1 (comprising residues His-18, His-21, and Phe-25), a loop region (including , and the central portion of helix 4 (containing residues Arg-167, Lys-168, Lys-172, Glu-174, . When these residues are mapped on a structural model of hGH, they form a patch that overlaps but is not identical to that previously determined for the hGH receptor. Three of these side chains (His-18, His-21, and Glu-174) are ligands for binding Zn2+, which is required for high-affinity hGH-hPRL receptor complex formation. By introducing pairs of mutations into hGH that exploit the zinc dependency for hPRL receptor binding and remove side chains in the nonoverlapping regions, we have shifted the binding preference toward the hGH receptor by a factor of 34,000 or toward the hPRL receptor by a factor of 150 without substantial loss in binding affinity for the preferred receptor. The energetic effects of the individual mutations are additive within the double mutants, suggesting that each functions independently and does not introduce gross perturbations in structure. Such receptorselective variants of hGH should be useful molecular probes to link specific receptor binding events to the various biological activities of hGH.Human growth hormone (hGH) elicits a myriad of biological effects including linear growth, lactation, nitrogen retention, diabetogenic and insulin-like effects, and macrophage activation (1-4). Each of these effects begins with the interaction of hGH with specific cellular receptors (5). Thus far, the only cloned human genes whose products bind hGH are the hGH receptor from liver (6) and the human prolactin (hPRL) receptor from mammary gland (7). However, there may be other receptors that bind hGH, including the placental lactogen (PL) receptor (8).The hGH and hPRL receptors (6, 7) contain an extracellular hormone binding domain (consisting of 246 and 211 residues, respectively) that share 32% sequence identity, a single transmembrane domain, and a cytoplasmic domain that differs widely in sequence and length. Nonglycosylated forms of the extracellular binding domains of the hGH and hPRL receptors (hGHbp and hPRLbp, respectively) can be expressed in large quantities in Escherichia coli. These highly purified recombinant binding proteins retain the same affinity for hGH and specificity as their natural glycosylated counterparts (9, 10) and are extremely useful reagents for efficient assessment of the binding affinity for mutants of hGH (9-13).It is not known whether the binding sites on hGH for the hGH and hPRL receptors are identical or which receptor (or combination of receptors) is responsible for which pharmacological effect. To address these issues, we have mapped binding determinants on hGH for the hGH and hPRL receptors and showed that they overlap but are no...

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Section: Methodsmentioning

confidence: 99%

Rational design of receptor-specific variants of human growth hormone.

Cunningham¹,

Wells²

1991

Proc. Natl. Acad. Sci. U.S.A.

194

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“…These mAbs and mAb 1 have previously been shown to be poor inhibitors of 125 -hGH binding to the rbGHR (12)(13)(14). We have recently shown (46) that high affinity binding of non-primate GHs to the GHR is dependent on receptor dimerization, so the loss in binding of 125 I-bGH observed in the presence of these mAbs may be a result of the inhibition of trimeric complex formation rather than direct competition for the GH binding domain.…”

Section: Receptor Expression In Clonal Lines-fdc-p1 Cells Contain-mentioning

confidence: 96%

“…Preparation of mAbs-mAbs 1, 5, 7, 43, 44, and 263 have been extensively characterized (12)(13)(14) and were supplied by Agen Ltd. (Acacia Ridge, Brisbane, Australia). mAb 1C9 was an anti-idiotype mAb, raised against a rabbit anti-hGH Fab (15), while all other antihuman receptor mAbs used in this study were raised against full-length recombinant nonglycosylated hGH receptor (residues 1-246) but known to cross-react with native hGH-binding protein (16).…”

Section: Establishment and Characterization Of Cell Lines Expressing Thementioning

confidence: 99%

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Activation of Chimeric and Full-length Growth Hormone Receptors by Growth Hormone Receptor Monoclonal Antibodies

Rowlinson

Behncken

Rowland

et al. 1998

Journal of Biological Chemistry

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Signal transduction by the growth hormone receptor (GHR) occurs through growth hormone (GH)-induced dimerization of two GHRs to form a trimeric complex. It is thought that dimerization alone is sufficient for signaling, since monoclonal antibodies (mAbs) against the extracellular domain of the GHR elicit proliferation of FDC-P1 cells transfected with a chimeric receptor comprising the extracellular domain of the GHR and the fibronectin and cytoplasmic domains of the murine granulocyte colony-stimulating factor receptor. We have screened 14 GHR mAbs for proliferative activity against characterized FDC-P1 and BaF-B03 cell lines stably expressing the full-length human, rabbit, or rat GHR, or the chimeric human GHR/granulocyte colonystimulating factor receptor, and for transactivation of the c-fos promoter and STAT activation. With the chimeric receptor, eight mAbs were able to elicit proliferation, although there was no correlation between inhibition of hormone binding and agonist activity. In contrast, no mAbs were able to act as agonists with the full-length GHR FDC-P1 cell lines, although nine competed with GH for binding. A weak proliferative response was observed in the BaF-B03 cell lines with two of the mAbs (263 and 1C9), and the addition of anti-mouse F(ab) 2 resulted in increased signaling in the hGHR BaF-B03 cell line to a plateau of 28 ؎ 4% of the GH maximum for mAb 263. These data could indicate considerable stringency in the ability of mAbs to correctly dimerize the full-length GHR. However, the ability of mAb 263 to stimulate a mutant hGHR altered in the F-G loop of domain 2 was nearly abolished, concurrent with an increased affinity of this mAb for the receptor. Since the F-G loop undergoes a conformational change on GH binding and is necessary for full proliferative signaling, we propose that in addition to promoting receptor dimerization, mAb 263 may induce specific changes in receptor conformation similar to GH, which are required for the biological response. Growth hormone (GH)1 regulates a wide range of processes including somatic growth, metabolism, and synthesis of specific proteins (1). It does this by triggering multiple second messenger pathways in response to ligand binding to the GHR. The first signaling step after ligand binding is thought to be hormone-induced dimerization of identical receptor subunits. It has been shown that formation of the trimeric complex (GH⅐(GHR) 2 ) is a sequential process involving the binding of the hormone, first to receptor 1 and then to receptor 2, on opposite sides of the four-␣-helical bundle hormone. Receptor binding sites on the hormone are referred to as sites 1 and 2, respectively (2, 3). Evidence supporting a role for hormoneinduced dimerization in signaling was first provided by Fuh et al. (4) using a cell line expressing the extracellular binding domain of the GHR fused to the extracellular fibronectin and cytoplasmic domains of the murine granulocyte colony-stimulating factor (mG-CSF) receptor. These workers reported that a hGH analogue mutated in...

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“…A monoclonal antibody to GHr raised in mice against a human GH affinity purified rat liver GHr (Barnard et al, 1984;1985). This antibody has been used to detect the receptor in rat tissue (Lobie et al, 1990;García-Aragón et al, 1992;Zhang et al, 1992;Symons et al, 1994Symons et al, , 1996aSymons et al, , 1996bSymons et al, , 1998Tengku 2000;Ong et al, 2001).…”

Section: Antibody Productionmentioning

confidence: 99%

Growth Hormone Receptor and Insulin-like Growth Factor-I Immunoreactivity in Osteoclast-like Cells During Tooth Eruption in the Toothless (Osteopetrotic) Rat Following Treatment with Colony-Stimulating Factor-1

Symons

Weerakoon

Marks

2003

Crit Rev Eukaryot Gene Expr

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ABSTRACT:In the toothless (tl/tl) osteopetrotic rat, teeth form but fail to erupt. Treatment of tl/tl rats with colony-stimulating factor-1 (CSF-1) activates bone resorption by osteoclasts, permits tooth eruption, and upregulates the immunoreactivity of bone marrow mononuclear cells to growth hormone receptor (GHr) and insulinlike growth factor (IGF)-I. This study examined the distribution of tartrate-resistant acid phosphatase (TRAP) and immunoreactivity for GHr and IGF-I in osteoclast-like cells located on the alveolar bone margin, adjacent to the lower first molar crown, in 14-day-old normal and tl/tl rats, following treatment with CSF-1. Osteoclast-like cells demonstrated a positive reaction for TRAP, GHr, and IGF-I in all groups. However, in tl/tl tissue, osteoclast-like cells were generally negative for GHr. There was no significant difference in the total number of TRAP-, GHr-, and IGF-I-positive osteoclast-like cells on the adjacent bone margin in normal, normal treated with CSF-1, and tl/tl rats. CSF-1 treatment of the tl/tl rat significantly increased the total number of osteoclast-like cells, which were positive for TRAP (p < 0.001), GHr (p < 0.05) and IGF-I (p < 0.01).

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Monoclonal Antibodies to the Rabbit Liver Growth Hormone Receptor: Production and Characterization*

Cited by 75 publications

References 17 publications

Rational design of receptor-specific variants of human growth hormone.

Rational design of receptor-specific variants of human growth hormone.

Activation of Chimeric and Full-length Growth Hormone Receptors by Growth Hormone Receptor Monoclonal Antibodies

Growth Hormone Receptor and Insulin-like Growth Factor-I Immunoreactivity in Osteoclast-like Cells During Tooth Eruption in the Toothless (Osteopetrotic) Rat Following Treatment with Colony-Stimulating Factor-1

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