Structural Analysis of Human Growth Hormone with Respect to the Dominant Expression of Growth Hormone (GH) Mutations in Isolated GH Deficien
http://www.100md.com
内分泌学杂志 2005年第3期
Pediatric Endocrinology Section, University Children’s Hospital, D-72076 Tuebingen Germany
Address all correspondence and requests for reprints to: Dr. Gerhard Binder, University Children’s Hospital, Pediatric Endocrinology Section, Hoppe-Seyler-Strasse 1, D-72076 Tuebingen Germany. E-mail: gerhard.binder@med.uni-tuebingen.de.
Abstract
Human GH protein consists of four -helices and contains two disulfide bridges. Isolated GH deficiency type II (IGHD II) is mainly caused by heterozygous splice site mutations of GH-1 leading to the disruption of one disulfide bridge (Cys53-Cys165) and to the loss of amino acids (aa) 32–71, which comprise the complete loop between -helices 1 and 2. The mutant GH protein exerts a dominant negative effect on wild-type (wt) GH secretion by unclear mechanisms. For study of the structure-function relationship of GH mutants concerning the dominant negative effect, expression vectors harboring mutated GH cDNAs were transiently cotransfected with a vector encoding wtGH (pwtGH) into GH4C1 cells. Plasmids encoding ?-galactosidase, luciferase, or IGF-binding protein-2 were cotransfected with pwtGH and either of the GH mutants. Compared with the control transfection with pwtGH, GH secretion was mildly decreased by coexpressing wtGH and different GH point mutants with isolated disruption of the disulfide bridge Cys53-Cys165. Similar results were observed with GH mutants deleted in aa 32–46 or 32–52. Deletion of more aa (32–53, 32–63, 32–69, 32–71) ascendingly decreased GH secretion and content in parallel with the increasing length of the deleted stretch. An inhibitory dose-dependent effect of del32–69GH and del32–71GH on the activity/amount of coexpressed ?-galactosidase, luciferase, and IGF-binding protein-2 was found, whereas mRNA levels were unaffected. Hence, the extent of deletion played the major role in expression of the dominant negative effect. The inhibitory effect of GH mutants on heterologously expressed, non-GH proteins suggests that the dominant negative effect is not limited to GH or to proteins of the regulated secretory pathway, but may depend on expression levels.
Introduction
MONOGENETIC GH DEFICIENCY is subclassified into three groups: autosomal recessive [isolated GH deficiency type I (IGHD I)], autosomal dominant [isolated GH deficiency type II (IGHD II)], and X-chromosomal [isolated GH deficiency type III (IGHD III)] (1). The group with IGHD I is further subdivided into patients with total absence of GH (IGHD IA) and those with severe GH deprivation (IGHD IB). Southern blot analysis demonstrated that most of the children with IGHD IA have a homozygous GH-1 gene deletion and, as a consequence, no genetic information for GH (2). The healthy parents of these children are hemizygous for GH-1, showing that the presence of a single GH-1 allele is sufficient for GH production and secretion, resulting in normal GH levels in serum (3). The genetic basis for the most common type of inheritance IGHD IB is unclear in most cases. In a few patients, splice site mutations in intron IV of GH-1 were described, resulting in skipping the nucleotides coding for amino acids (aa) 103–126 and subsequently in a frame shift in exon 5 (4). Inactivating mutations of the gene encoding the GHRH receptor were found as an alternative genetic basis of IGHD IB (5).
IGHD II has mainly been described in patients harboring mutations in the intron III donor splice site of one GH-1 allele, which cause skipping of exon 3 (6, 7). The resulting gene product lacks aa 32–71 and, therefore, the entire loop connecting the first -helix of the GH molecule to the second one (8) (Fig. 1). The mutant GH protein is thus subject to serious structural changes. In patients harboring this kind of mutation, GH concentrations are extremely low even though one intact GH-1 allele is present. Therefore, the presence of del32–71GH in the somatotrophs causes a blockade of wild-type (wt) GH secretion by an as yet unknown mechanism. In contrast, the splicing variant del32–46GH (the 20-kDa GH variant), which also lacks part of the loop connecting -helices 1 and 2 (8), accounts for approximately 10% of the GH found in serum of normal individuals and evidently does not have any adverse effect on the secretion of wtGH (9).
FIG. 1. Schematic representation of the wtGH protein tertiary structure. The two intramolecular disulfide bridges and distinct aa positions corresponding to end points of deleted aa stretches are outlined.
When human wtGH and del32–71GH were coexpressed in different cell types, the dominant negative effect was observed only in neuroendocrine cells, whereas no impairment of wtGH secretion was found in other cell types (e.g. COS, Chinese hamster ovary, and Epstein-Barr virus-transformed lymphocytes) (10, 11, 12). Neuroendocrine cells differ from these cells by having specific modes of protein transport, sorting, storage, and release (13). In transient transfection studies involving rat GH4C1 neuroendocrine cells, it was observed that del32–71GH suppresses both intracellular accumulation and secretion of wtGH without itself being accumulated or secreted, whereas wtGH and del32–71GH mRNAs were shown to be present in approximately equal quantities (12). Studies with COS cells revealed an uneven distribution of wtGH and del32–71GH, the former being localized in the Golgi apparatus and the latter retained in the endoplasmic reticulum. It was suggested that the presence of the misfolded protein causes Golgi apparatus fragmentation, thus disrupting transport from the endoplasmic reticulum to the Golgi apparatus also involving other membrane and secretory proteins (14). These findings in nonneuroendocrine cells were not observed in GH4C1 neuroendocrine cells, showing no deleterious effect of human del32–71GH on the production and secretion of heterologously expressed human prolactin, suggesting that the effects of mutant GH are cell type specific (12). In transgenic mice expressing human del32–71GH, a marked decrease in GH levels was found in pituitary extracts, and the affected animals developed short stature (15). Moreover, multiple anterior pituitary deficiencies, pituitary hypoplasia, morphological abnormalities of somatotrophs with few secretory vesicles, and macrophage hypophyseal invasion were detected. In contrast, the pituitary abnormalities observed in patients with IGHD II are not as severe as those evident in the in vivo mouse model, because a small to normal size pituitary gland was confirmed by magnetic resonance tomographic analysis (16).
Several hypotheses have been discussed in the literature to explain the basic mechanisms of the interference of some mutant GH forms with wtGH. These include 1) accumulation of toxic aggregates of mutant proteins, 2) decrease in intracellular stability of wtGH due to cellular responses induced by unfolded proteins (overload response, unfolded protein response, and induction of cell type-specific degradation systems), 3) specific blockade of GH aggregation and/or sorting into secretory granules, and 4) impaired maturation of secretory granules.
The aim of this in vitro study using GH4C1 cells was to elucidate the importance of specific aa or stretches of aa in the context of the GH tertiary structure for exhibition of the dominant negative effect.
Materials and Methods
DNA vectors
Deletion and point mutations of GH cDNA were performed using overlap extension PCR technology (17). The wtGH cDNA inserted in a pcDNA3-vector (pwtGH; gift from P. Dannies, Yale School of Medicine, New Haven, CT) was used as a template. The oligonucleotides used for site-directed mutation were: C53A forward, 5'-GAC CTC CCT CGC ATT CTC AG-3'; C53A reverse, 5'-CTG AGA ATG CGA GGG AGG TC-3' (for C53A-GH and C53A-C165A-GH); C165A forward, 5'-GCT CTA CGC ATT CAG GAA GGA-3'; C165A reverse, 5'-TCC TTC CTG AAT GCG TAG AGC-3' (for C165A-GH and C53A-C165A-GH); del32–46 forward, 5'-CAG GAG TTT AAC CCC CAG ACC TCC CTC-3'; del32–46 reverse, 5'-CTG GGG GTT AAA CTC CTG GTA GGT GTC-3' (for del32–46-GH); del32–52 forward, 5'-CAG GAG TTT TGT TTC TCA GAG T-3'; del32–52 reverse, 5'-ACT CTG AGA AAC AAA ACT CCT G-3' (for del32–52-GH); del32–53 forward, 5'-CTA CCA GGA GTT TTT CTC AGA G-3'; del32–53 reverse, 5'-GAC TCT GAG AAA AAC TCC TGG T-3' (for del32–53-GH); del32–63 forward, 5'-TAC CAG GAG TTT AGG GAG GAA-3'; del32–63 reverse, 5'-TTC CTC CCT AAA CTC CTG GTA-3' (for del32–63-GH); del32–69 forward, 5'-CCA GGA GTT TAA ATC CAA CCT-3'; del32–69 reverse, 5'-GGT TGG ATT TAA ACT CCT GGT-3' (for del32–69-GH); GH-5'-HindIII, 5'-GTT AAG CTT CCT GTG GAC AGC TCA C-3'; and GH-3'-XhoI, 5'-AGA CTC GAG TAT TAG GAC AAG GCT GGT-3' (homologous to the 5' and 3' noncoding regions of the hGH cDNA, including, in addition, HindIII and XhoI restriction sites, respectively).
In a first round of PCR, amplicons were generated using either GH-5'-HindIII and one of the mutant reverse primers or GH-3'-XhoI primer and one of the mutant forward primers. In a second PCR round, the primers GH-5'-HindIII and GH-3'-XhoI were used with both amplicons of the first PCR round as template. The PCR products were purified using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), cut with restriction enzymes specific for HindIII and XhoI sites, and cloned into the transfection vector pcDNA3.1 (Invitrogen Life Technologies, Inc., Karlsruhe, Germany) opened with HindIII and XhoI. Plasmids were transformed into XL-1 Blue supercompetent Escherichia coli cells (Stratagene, Cedar Creek, CA) and screened for efficient cloning by use of restriction enzyme analysis (HindIII and XhoI). Plasmid DNA was isolated from a suitable colony using EndoFree Plasmid Maxi Kit (Qiagen). The presence of the point or deletion mutation and the integrity of the human GH (hGH) cDNA were verified by sequencing (GENterpise, Mainz, Germany).
Cell culture
GH4C1 cells used for the transfection experiments were purchased from American Type Culture Collection (LGC Promochem, Wesel, Germany). GH4C1 cells were cultured in DMEM/Ham’s F-12 (Invitrogen Life Technologies, Inc., Paisley, UK) supplemented with 15% horse serum (Invitrogen Life Technologies, Inc.) and incubated at 37 C in 5% CO2.
Transfection experiments
The cDNA expression vectors generated, harboring various mutant GH cDNAs, were cotransfected with pwtGH using the Effectene Transfection Reagent Kit (Qiagen). Cotransfection experiments with expression vectors for ?-galactosidase (pcDNA3.1.V5/His-lacZ, Invitrogen Life Technologies, Inc.), firefly luciferase (pGL2LUC, Promega Corp., Mannheim, Germany), or human IGF-binding protein 2 (pcDNA3.1-IGFBP-2) were performed. For transfection, 5 x 105 cells were seeded in 60-mm poly-D-lysine-coated transfection dishes (BD Biosciences, Meylan, France). Transfection was carried out according to the manufacturer’s instructions 24 h after seeding at approximately 80% confluence. A total of 1 μg DNA and a DNA ratio of wtGH to mutant GH of 1:1 were used, if not otherwise stated. If the amounts of mutant constructs were varied, the total amount of the transfected plasmid DNA was adjusted to 1 μg DNA using an empty pcDNA3.1 vector. Forty-eight hours posttransfection, medium and cells were harvested, and cellular proteins were extracted using reporter lysis buffer (400 μl/culture dish) according to the manufacturer’s instructions (Promega Corp., Madison, WI). Shorter or longer incubation periods (18, 24, and 72 h, respectively) were demonstrated to be inappropriate for the RIA used, because the GH values measured were beyond the detection capacity of the system.
RNA extraction and real-time RT-PCR
Total RNA was isolated 24 h posttransfection using the RNeasy Mini Kit (Qiagen) with a simultaneous on-column deoxyribonuclease digestion with the ribonuclease-free deoxyribonuclease set (Qiagen) according to the manufacturer’s instructions. Additionally RNA solution was treated with ribonuclease-free deoxyribonuclease I (Roche). RT-PCR (Promega Corp.), performed with or without reverse transcriptase, showed the presence of hGH transcripts and efficient removal of plasmid DNA. cDNA was synthesized using the Omniscript RT kit (Qiagen). Consecutively, real-time RT-PCR was performed in a Bio-Rad iCycler using the SYBR Green Supermix (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK). The primers were designed to yield products no longer than 150 bp. For each sample, duplicate measurements were performed, and the arithmetic mean was calculated from each duplicate measurement. Threshold cycle values were determined using Bio-Rad iCycler software version 3.0.
hGH and IGFBP-2 measurements
GH values in media and cell lysates and IGFBP-2 values in media were measured with an RIA specific for hGH or human IGFBP-2 as previously described (18, 19).
Western blot analysis
Media or cell extracts (15 μl) were separated on a 15% sodium dodecyl sulfate-polyacrylamide gel, blotted on an Immobilon-P transfer membrane (Millipore Corp., Bedford, MA), and subjected to antibody determination using either a rabbit polyclonal antiserum against recombinant hGH (somatropin, NIBSC code 88/624) or a monoclonal antibody (7B11, gift from C. Strasburger, Berlin, Germany) directed to the N-terminal domain of the hGH molecule. As secondary antibodies, antirabbit immunoglobulin G (New England Biolabs, Frankfurt am Main, Germany) and antimouse immunoglobulin G (New England Biolabs) were used. Chemiluminescence detection was performed using the ECL Plus Western Blotting Detection System (Amersham Biosciences UK Ltd., Little Chalfont, UK). Results were analyzed densitometrically on a Raytest apparatus (Raytest Isotopenmessger?te, Straubenhardt, Germany) using AIDA (advanced image data analyzer) software, version 2.1. In addition, the image was captured on Kodak film (Eastman Kodak Co., Rochester, NY).
Cell counting
In experiments performed in parallel, 4, 24, 48, and 80 h posttransfection, the number of cells transfected with only pwtGH or cotransfected with pwtGH and a del32–71GH-expressing plasmid (pdel32–71GH) was estimated automatically (Cobas Micros, Roche, Montpellier, France; Advia 120, Bayer, Holliston, MA) as well as in a Neubauer-improved counting chamber (Brand, Wertheim, Germany). Vital and nonvital cells were distinguished under the microscope via staining with 0.4% trypan blue solution (Sigma-Aldrich Corp., Irvine, UK).
?-Galactosidase and luciferase activities
?-Galactosidase and luciferase activities were determined in an automated luminometer Wallac 1420 Victor2 (Wallac Oy, Turku, Finland) using the ?-Gal Reporter Gene Assay, chemiluminescent (Roche), and Luciferase Assay System (Promega Corp.), respectively.
?-Galactosidase staining
?-Galactosidase staining was performed directly on culture dish using reagents produced according to the instructions of the In Situ ?-Galactosidase Staining Kit (Stratagene, La Jolla, CA).
Statistical data analysis
Differences between the transfection groups were analyzed by t test.
Results
To investigate the structure-function relationship of hGH mutants with respect to a negative effect on wtGH secretion, a series of hGH constructs that were mutated or deleted in specific aa or stretches of aa was generated. We focused on study of the linker region between -helices 1 and 2 (aa 46 and 71; Fig. 1), because del32–46GH (the 20-kDa GH variant form) exerts no deleterious effect, whereas del32–71GH has a strong adverse effect on wtGH secretion. Therefore, diverse deletion mutants, del32–46, del32–52, del32–53, del32–63, and del32–69 were constructed in which the respective aa stretch was deleted. Of additional interest was the role of the disulfide bridge constituted between Cys53 and Cys165, which is disrupted in del32–71GH. Distinct point mutants were constructed in which the respective cysteine-encoding DNA triplets were mutated to alanine-encoding triplets, resulting in the GH mutants C53A, C165A, and C53A-C165A (Fig. 1).
GH4C1 cells were simultaneously transfected with pwtGH and one of the mutant GH-expressing plasmids. Forty-eight hours posttransfection, GH was measured in incubation media and cell extracts. The GH content in cells cotrans-fected with pwtGH/pwtGH, pwtGH/pdel32–46, pwtGH/pdel32–52, pwtGH/pdel32–53, pwtGH/pdel32–63, pwtGH/pdel32–69, and pwtGH/pdel32–71 was determined using an hGH-specific RIA (Fig. 2). In comparison with the control transfection (pwtGH/pwtGH set at 100%), the amount of hGH secreted into the medium was decreased by 35% when cotransfecting pwtGH/empty vector. It was decreased mildly by 15–30% when coexpressing wtGH and any of the point mutants (C53A, C165A, or C53A-C165A) or mutants deleted in aa 32–46 or 32–52. Deletion of more aa (32–52, 32–63) led to a stronger decrease in secreted hGH by 50–60%. A severe decrease by 75–85% was observed when stretch 32–69 or 32–71 was deleted. The degree of GH reduction was proportional to the increase in size of the deletion. The same relationship between the extent of deletion and the amount of detectable hGH was found in protein extracts of the respectively transfected cells (Fig. 2). The amount of del32–71GH when singly expressed was below the detection limit of our RIA system.
FIG. 2. Relative GH concentrations in media and cell extracts of cotransfected GH4C1 cells measured by RIA. Mean values of five to eight individual experiments ± SD are represented as a percentage of the values obtained for the pwtGH/pwtGH transfection, which was set at 100%. The GH content of each sample was measured twice, using the mean value for calculations. P values are given for the differences between pwtGH/pdel32–52GH and pwtGH/pdel32–53GH on the one side, and between pwtGH/pdel32–63GH and pwtGH/pdel32–69GH on the other side. The extent of deletion played the major role in expression of the dominant negative effect on wtGH.
To determine whether the amount of hGH was underestimated in the RIA due to the intrinsic competition with recombinant hGH, cell extracts and media were analyzed using Western blots (Fig. 3). Densitometric evaluation of protein bands corresponding to wtGH and mutant GH (detected by polyclonal antiserum as well as by monoclonal antibodies directed toward the N terminus of hGH) revealed comparable amounts as determined using RIA. The finding that mutant protein amounts were not underestimated in the RIA was strengthened by the comparability of RIA and Western blot results for cells monotransfected with pwtGH, pdel32–46, pdel32–53, or pdel32–71 (data not shown).
FIG. 3. Western blot analysis of extracts (CE) or media (M) of cotransfected GH4C1 cells using a polyclonal rabbit antihuman GH antiserum (polyclonal rabbit -hGH) or a monoclonal mouse antihuman GH antibody specific for the N-terminal domain (monoclonal mouse -hGH). Protein bands corresponding to the wt and some mutant hGH forms are depicted by arrows and the respective molecular weights.
To investigate whether del32–53GH exerts a dominant negative effect as suggested by the above RIA data, we performed parallel cotransfection experiments in which pdel32–53 or empty vector was cotransfected in varying amounts together with pwtGH (Fig. 4). The total amount of transfected plasmid was kept constant by adapting the amount of pwtGH cotransfected. The concentration of total GH was constantly lower for cells cotransfected with pdel32–53GH and pwtGH than for cells cotransfected with empty vector and pwtGH, indicating a dominant negative effect of del32–53GH on wtGH. This effect became significantly stronger with increasing amounts of pdel32–53GH.
FIG. 4. Relative GH concentrations in media and cell extracts of cotransfected GH4C1 (with pwtGH/pdel32–53GH or pwtGH/vector) were measured by RIA. Mean values of three individual experiments ± SD are represented as a percentage of the values obtained for the pwtGH/vector cotransfection at equimolar amounts, which was set at 100%. The GH content of each sample was measured twice, using the mean value for calculations. The ratio of the respective plasmids used for transfection experiments is depicted on the left. The data reveal a dose-dependent dominant negative effect of del32–53GH on wtGH.
The total cell numbers and viability of cells expressing only wtGH or coexpressing wtGH and del32–71GH stayed equivalent in individual experiments over periods of up to 80 h, thus making an acute toxic effect of del32–71GH on the cells unlikely (data not shown).
The transfection efficiency was determined in individual experiments by counting the ?-galactosidase-stained cells directly on the culture dish and was between 10–15%. No intraexperimental variation in transfection efficiency was found between cells transfected only with pwtGH and those cotransfected with both pwtGH and pdel32–46, pdel32–53, or pdel32–71GH. Interestingly, the intensity of the staining decreased with the increasing length of the deleted stretch (data not shown).
To normalize the values obtained by RIA measurements for transfection efficiency, plasmids containing cDNA encoding ?-galactosidase, firefly luciferase, or IGFBP-2 were cotransfected, and the activity or expression of the respective gene products was analyzed in cell extracts (?-galactosidase and luciferase) or media (IGFBP-2). Unexpectedly, the ?-galactosidase activity measured in extracts from cells cotransfected with pwtGH/pdel32–71GH was approximately 5-fold lower than that of cells transfected only with pwtGH or cotransfected with pwtGH and an empty vector (Fig. 5). In addition, the dominant negative effect of del32–71GH on both wtGH and ?-galactosidase appeared to be dose dependent. When constant amounts of pwtGH and expression plasmid for ?-galactosidase were cotransfected with increasing amounts of pdel32–71GH, a reverse correlation was found between the quantity of pdel32–71GH transfected, on the one hand, and the ?-galactosidase activity and GH concentrations measured, on the other hand (Fig. 6). Similar results were obtained when firefly luciferase was coexpressed. The activity of this enzyme found in extracts from cells cotransfected with pwtGH and either of the expression plasmids for the deletion mutant del32–69 or del32–71 was approximately 5-fold lower than that of the pwtGH/pwtGH control transfection, and the degree of inhibition was also dose dependent (data not shown). To investigate whether a molecular interaction between wtGH and mutant GH proteins is a prerequisite for the observed effects on heterologous proteins, luciferase activity was measured in cells cotransfected only with pdel32–71GH and pGL2LUC. In this case, luciferase activity was barely detectable and was comparable to levels detected in extracts of untransfected cells, whereas those cotransfected with pwtGH and pGL2LUC yielded considerable luciferase activity (data not shown). Therefore, the effect of del32–71GH on heterologously coexpressed proteins was independent of the presence of wtGH.
FIG. 5. ?-Galactosidase activities in cell lysates. Cells were always cotransfected with 0.4 μg of each of the plasmids denoted and 0.2 μg plasmid pcDNA3.1.V5/His-lacZ. The mean ± SD of triplicate transfections are presented. ?-Galactosidase activity was severely diminished in the presence of del32–71GH.
FIG. 6. ?-Galactosidase activities in cell lysates and GH concentrations determined by RIA in media and cell extracts. For ?-galactosidase analysis, cells were cotransfected with 0.2 μg pcDNA3.1.V5/His-lacZ, 0.4 μg pwtGH, and 0.4, 0.2, or 0.1 μg pdel32–71GH. For GH analysis, cells were cotransfected with 0.4 μg pwtGH and 0.4, 0.2, or 0.1 μg pdel32–71GH. A total of 1 μg transfected DNA was achieved by cotransfection of respective amounts of an empty vector. Values represent the mean of duplicate determinations of one experiment and indicate that the dominant negative effect of del32–71GH on wtGH as well as on the heterologously coexpressed ?-galactosidase was dose dependent.
Analogous results were obtained when IGFBP-2 levels were determined in culture media of cells that were cotransfected with pwtGH, either of the expression plasmids for a GH mutant, and pIGFBP-2. The concentrations of IGFBP-2 detected in medium were inversely correlated to the extent of the deletion greater than 32–52 in the transfected mutant constructs. Cotransfection of either of the point mutants showed no significant effect on the amount of IGFBP-2 (Fig. 7).
FIG. 7. IGFBP-2 concentrations measured using RIA in media of cells cotransfected with pIGFBP-2, pwtGH, and plasmids encoding GH deletion or point mutants. The mean values of three individual experiments ± SD are represented as a percentage of the values obtained for the pwtGH/pwtGH transfection, which was set at 100%. The IGFBP-2 concentration in each sample was measured twice, using the mean value for calculations. P values are given for the indicated comparisons.
Real-time RT-PCR results revealed comparable mRNA expression levels for wtGH as well as total hGH and for the marker protein IGFBP-2 (Fig. 8), indicating that the observations at the protein level were not based on a decrease in transcription or RNA instability.
FIG. 8. Relative expression levels of wtGH, total GH, and IGFBP-2 mRNA for cells cotransfected with pwtGH, plasmids encoding GH deletion mutants, and pIGFBP-2. Data are represented as a percentage of the values obtained for the pwtGH/pwtGH transfection, which was set at 100%. For each sample, duplicate measurements were performed, using the mean value for calculations.
Discussion
The presence of specific GH protein mutants in the pituitary somatotrophs results in a dominant restriction of wtGH secretion, whereas others are tolerated at the heterozygous state. Therefore, it was of great interest to analyze the relationship between GH-1 mutations and effects in further detail. The focus was set on the role of the linker region between helices 1 and 2 of GH, the deletion of which is frequently the cause of IGHD II. This region contains a cysteine at position 53 that is necessary for the formation of an intramolecular disulfide bridge with Cys165. Mutation of one cysteine residue at position 53 or 165 eliminated formation of the intramolecular disulfide bridge Cys53-Cys165 and made the establishment of irregular intramolecular, with cysteine residues at positions 182 or 189, or intermolecular disulfide bridges possible. The last mechanism would potentially facilitate the formation of GH molecule dimers covalently bound to each other. Mutations of both cysteine residues at positions 53 and 165 totally eliminated the formation of disulfide bridges. Our results suggest that disruption of the disulfide bridge alone is not sufficient for the dominant negative effect observed, because the GH concentration in cells cotransfected with pwtGH and an expression plasmid for either of the point mutants was only slightly reduced. In addition, the point mutants with one free cysteine (C53A or C165A) displayed similar behavior compared with the mutant harboring two aa exchanges (C53A-C165A). Our results are in line with those of a previous study suggesting that the presence of the free cysteine residue 165 has only a minor influence on exhibition of the dominant negative effect (12). These findings are contradictory to the assumption that del32–71GH affects wtGH secretion only by forming dimers due to free cysteine residues (10).
Our construct del32–52GH was related to the naturally occurring 20-kDa GH variant (del32–46) lacking additional six aa but preserving the cysteine at position 53 and, therefore, the intramolecular disulfide bridge. The concentrations of GH detected in both media and cell extracts of cells cotransfected with pwtGH and either pdel32–46GH or pdel32–52GH were similar. Deletion of one additional aa in the construct del32–53, thereby hampering formation of the disulfide bridge between aa residues 53 and 165, led to a statistically significant decrease (P = 0.006) in the concentration of secreted GH compared with the effect of del32–52GH. Moreover, the inhibitory effect of del32–53GH on wtGH secretion was shown to be dose dependent. This observation indicates that the disruption of the disulfide bridge is a step-change event. This disruption evidently affects the tertiary structure of the GH molecule critically when residue 53 is removed in the context of the whole deleted aa stretch 32–53; thus, the relationship between structure and function is not simply linearly correlated to the extent of the peptide deletion. Deletion of 10 additional aa up to the beginning of the second minihelix in construct del32–63GH induced no further reduction of the hGH detected, whereas the additional deletion of the second minihelix encompassing six supplementary aa in del32–69GH resulted in the exhibition of a negative effect comparable to that of del32–71GH. One hypothesis for the exhibition of the dominant negative effect exerted by GH mutants explains the decreased secretion with a disorder of the aggregation of GH molecules. It has been suggested that the binding of Zn2+ by GH molecules is a prerequisite for GH dimerization and subsequently for GH storage in secretory granules (20). Because deletion of the linker region between -helices 1 and 2 presumably results in a strongly changed tertiary structure of the GH molecule, this might also affect Zn2+ binding and, as a consequence, influence aggregation or sorting of GH proteins or maturation of the secretory granules. An increase in the extent of the deletion and, in particular, deletion of pronounced structures such as the second minihelix are likely to result in major structural changes and, consequently, strong disturbances of aggregation.
GH immunoreactivity assayed using a polyclonal RIA was the main parameter studied in our in vitro experiments. The polyclonal antiserum of this RIA has a presumed wide spectrum of epitope specificities (9). It has been well characterized for the determination of 22-kDa GH (18), but its cross-reactivity to mutant GH proteins, especially del32–71GH (17.5-kDa GH), is not known. Therefore, there is no doubt that in those experiments in which wtGH was expressed using the same amounts of pwtGH a substantial decrease in RIA values did represent a decrease in 22-kDa GH. In experiments with a minor decrease in RIA values, the change could be caused by a decrease in the amount of GH, by decreased cross-reactivity, or by a combination of the two from a theoretical standpoint. However, our data from Western blot analysis, which avoided the direct competition between wt and mutant GH, correlated well with RIA values. In addition, Western blot analysis using a monoclonal antibody raised against an epitope not affected by the GH mutations confirmed this finding. These experimental data strongly argue that immunoreactivity measured using the polyclonal RIA reflects total GH amounts rather than lack of GH cross-reactivity. Therefore, these results underline the usefulness of the applied GH RIA for the detection of the various GH mutants.
GH RIA results are presented without correction, because the transfection efficiency, determined by counting ?-galactosidase-stained cells, was equal. The discrepancy compared with the luminometric data could be due to different expressions at the protein level despite the equivalent efficiency of transfection as the intensity of staining observed under the microscope decreased in the presence of del32–71GH. In addition, the absence of significant differences in expression at the mRNA level of total GH and wtGH was indicative of comparable transfection efficiencies.
Toxic effects in cells cotransfected with pwtGH and pdel32–71GH were not found in a cell viability analysis. There was no decrease in cell numbers 80 h posttransfection, which presumably would have been the case if del32–71GH exerted cell toxicity, despite the relatively low percentage of the cell population being transfected. In addition, mRNA expression of the marker protein IGFBP-2 was unaffected by coexpression of del32–71GH. These results are in agreement with an earlier report in which a lack of toxicity over a 24-h period was suggested by the observation that the secretory pathway in GH4C1 cells expressing del32–71GH functioned properly (12). It has to be emphasized that our data were obtained over a relatively short period of time. Therefore, we cannot exclude effects on cell viability due to inhibition of non-GH protein production over a longer term.
The observed severe negative effect of del32–69GH and del32–71GH not only on wtGH, but also on IGFBP-2 secretion as well as on ?-galactosidase and firefly luciferase activities suggests a general disturbance of cells expressing mutant GH beyond the molecular interaction of wtGH and mutant GH. Also, ?-galactosidase and luciferase are not expected to be transported through the regulated secretory pathway. Our findings are not in line with the results of experiments applying the neuroendocrine cell lines MtT/S and AtT-20, in which del32–71GH had no clear decreasing effect on ?-galactosidase activity (11). However, in the experimental setting cited, the effect of del32–71GH on wtGH secretion was much less pronounced, requiring a high excess of mutant GH for an observable effect, thereby making the missing effect on ?-galactosidase less significant. These divergent data may be explained by different transfection conditions. In addition, some effects of del32–69GH and del32–71GH may be cell type specific; GH4C1, a rat prolactin- and GH-producing cell line, is a somatotroph-derived cell line, whereas AtT-20 is adrenocorticotroph derived.
Recently, McGuinness et al. (15) reported that rat pituitary GC cells, which were stably monotransfected with a plasmid expressing human del32–71GH, showed a decreased proliferation rate, reduced attachment, and disturbed morphology, followed by early cell apoptosis, thus indicating toxic-like effects of del32–71GH on this cell line. This discrepancy from our data may be explained by cell specificities, because GH4C1 cells stably cotransfected with pwtGH and pdel32–71GH exhibit viability and proliferation rates similar to those of cells monotransfected with pwtGH (data not shown). Therefore, every cell system may potentially react in a specific manner to the mutant GH expression, possibly different from that of human somatotrophs. However, we believe that our cell system is a good model for studying specific features of mutant human GH because GH4C1 cells are neuroendocrine somatotroph cells.
The phenomena of suppression of wtGH secretion and the negative effect exerted on the other proteins discussed presumably share a common genesis. The presence of the deletion mutant possibly disturbs various biological functions of cells without affecting their viability and proliferation rate. The expression of misfolded proteins is a source of cell stress, which results in degradation of proteins due to activation of unfolded protein responses (21). Presumably, the level of heterologous proteins is affected to a higher degree than that of endogenous proteins, because the former are produced in higher quantities and are thus more prone to be affected by degradation systems induced by unfolded protein responses.
Our data suggest that besides a minor role for the integrity of the disulfide bridge Cys53-Cys165, the extent of deletion plays the major role in expression of the dominant negative effect. The inhibitory effect of GH mutants on heterologously expressed non-GH proteins suggests that the dominant negative effect is not limited to GH or even to proteins of the regulated secretory pathway, but may depend on expression levels.
Acknowledgments
We thank Christian Strasburger for providing monoclonal antibody 7B11, Burkhardt Schuett for fruitful discussions as well as for provision of the vector pIGFBP-2, and Klaus Frommer for support in performing the real-time PCR. We acknowledge the technical assistance of Evelina Goetz and Karin Weber as well as the assistance of Priscilla Herrmann in the language editing of this manuscript, and the support of Peter-Michael Weber in the design of some figures.
References
Phillips JA, Cogan JD 1994 Genetic basis of endocrine disease. VI. Molecular basis of familial human growth hormone deficiency. J Clin Endocrinol Metab 78:11–16
Phillips III JA, Hjelle BL, Seeburg PH, Zachmann M 1981 Molecular basis for familial isolated growth hormone deficiency. Proc Natl Acad Sci USA 78:6372–6375
Akinci A, Kanaka C, Eble A, Akar N, Vidinlisan S, Mullis PE 1992 Isolated growth hormone (GH) deficiency type IA associated with a 45-kilobase gene deletion within the human GH gene cluster. J Clin Endocrinol Metab 75:437–441
Procter AM, Phillips JA, Cooper DN 1998 The molecular genetics of growth hormone deficiency. Hum Genet 103:255–272
Baumann G, Maheshwari H 1997 The dwarfs of Sindh: severe growth hormone (GH) deficiency caused by a mutation in the GH-releasing hormone receptor gene. Acta Paediatr 423(Suppl):33–38
Binder G, Ranke MB 1995 Screening for growth hormone (GH) gene splice-site mutations in sporadic cases with severe isolated GH deficiency using ectopic transcript analysis. J Clin Endocrinol Metab 80:1247–1252
Cogan JD, Phillips JA, Schenkman SS, Milner RD, Sakati N 1994 Familial growth hormone deficiency: a model of dominant and recessive mutations affecting a monomeric protein. J Clin Endocrinol Metab 79:1261–1265
Ultsch MH, Somers W, Kossiakoff AA, de Vos AM 1994 The crystal structure of affinity-matured human growth hormone at 2 A resolution. J Mol Biol 236:286–299
Baumann G 1991 Growth hormone heterogeneity: genes, isohormones, variants, and binding proteins. Endocr Rev 12:424–449
Binder G, Brown M, Parks JS 1996 Mechanisms responsible for dominant expression of human growth hormone gene mutations. J Clin Endocrinol Metab 81:4047–4050
Hayashi Y, Yamamoto M, Ohmori S, Kamijo T, Ogawa M, Seo H 1999 Inhibition of growth hormone (GH) secretion by a mutant GH-I gene product in neuroendocrine cells containing secretory granules: an implication for isolated GH deficiency inherited in an autosomal dominant manner. J Clin Endocrinol Metab 84:2134–2139
Lee MS, Wajnrajch MP, Kim SS, Plotnick LP, Wang J, Gertner JM, Leibel RL, Dannies PS 2000 Autosomal dominant growth hormone (GH) deficiency type II: the del32–71-GH deletion mutant suppresses secretion of wild-type GH. Endocrinology 141:883–890
Dannies PS 2000 Protein folding and deficiencies caused by dominant-negative mutants of hormones. Vitam Horm 58:1–26
Graves TK, Patel S, Dannies PS, Hinkle PM 2001 Misfolded growth hormone causes fragmentation of the Golgi apparatus and disrupts endoplasmic reticulum-to-Golgi traffic. J Cell Sci 114:3685–3694
McGuinness L, Magoulas C, Sesay AK, Mathers K, Carmignac D, Manneville JB, Christian H, Phillips III JA, Robinson IC 2003 Autosomal dominant growth hormone deficiency disrupts secretory vesicles in vitro and in vivo in transgenic mice. Endocrinology 144:720–731
Binder G, Nagel BH, Ranke MB, Mullis PE 2002 Isolated GH deficiency (IGHD) type II: imaging of the pituitary gland by magnetic resonance reveals characteristic differences in comparison with severe IGHD of unknown origin. Eur J Endocrinol 147:755–760
Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR 1989 Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59
Hauffa BP, Lehmann N, Bettendorf M, Mehls O, Dorr HG, Partsch CJ, Schwarz HP, Stahnke N, Steinkamp H, Said E, Sander S, Ranke MB 2004 Central reassessment of GH concentrations measured at local treatment centers in children with impaired growth: consequences for patient management. Eur J Endocrinol 150:291–297
Elmlinger MW, Wimmer K, Biemer E, Blum WF, Ranke MB Dannecker GE 1996 Insulin-like growth factor binding protein 2 is differentially expressed in leukaemic B- and T-cell lines. Growth Regul 6:152–157
Cunningham BC, Mulkerrin MG, Wells JA 1991 Dimerization of human growth hormone by zinc. Science 253:545–548
Kauffman KJ, Pridgen EM, Doyle III FJ, Dhurjati PS, Robinson AS 2002 Decreased protein expression and intermittent recoveries in BiP levels result from cellular stress during heterologous protein expression in Saccharomyces cerevisiae. Biotechnol Prog 18:942–950(Daniel I. Iliev, Nicola E)
Address all correspondence and requests for reprints to: Dr. Gerhard Binder, University Children’s Hospital, Pediatric Endocrinology Section, Hoppe-Seyler-Strasse 1, D-72076 Tuebingen Germany. E-mail: gerhard.binder@med.uni-tuebingen.de.
Abstract
Human GH protein consists of four -helices and contains two disulfide bridges. Isolated GH deficiency type II (IGHD II) is mainly caused by heterozygous splice site mutations of GH-1 leading to the disruption of one disulfide bridge (Cys53-Cys165) and to the loss of amino acids (aa) 32–71, which comprise the complete loop between -helices 1 and 2. The mutant GH protein exerts a dominant negative effect on wild-type (wt) GH secretion by unclear mechanisms. For study of the structure-function relationship of GH mutants concerning the dominant negative effect, expression vectors harboring mutated GH cDNAs were transiently cotransfected with a vector encoding wtGH (pwtGH) into GH4C1 cells. Plasmids encoding ?-galactosidase, luciferase, or IGF-binding protein-2 were cotransfected with pwtGH and either of the GH mutants. Compared with the control transfection with pwtGH, GH secretion was mildly decreased by coexpressing wtGH and different GH point mutants with isolated disruption of the disulfide bridge Cys53-Cys165. Similar results were observed with GH mutants deleted in aa 32–46 or 32–52. Deletion of more aa (32–53, 32–63, 32–69, 32–71) ascendingly decreased GH secretion and content in parallel with the increasing length of the deleted stretch. An inhibitory dose-dependent effect of del32–69GH and del32–71GH on the activity/amount of coexpressed ?-galactosidase, luciferase, and IGF-binding protein-2 was found, whereas mRNA levels were unaffected. Hence, the extent of deletion played the major role in expression of the dominant negative effect. The inhibitory effect of GH mutants on heterologously expressed, non-GH proteins suggests that the dominant negative effect is not limited to GH or to proteins of the regulated secretory pathway, but may depend on expression levels.
Introduction
MONOGENETIC GH DEFICIENCY is subclassified into three groups: autosomal recessive [isolated GH deficiency type I (IGHD I)], autosomal dominant [isolated GH deficiency type II (IGHD II)], and X-chromosomal [isolated GH deficiency type III (IGHD III)] (1). The group with IGHD I is further subdivided into patients with total absence of GH (IGHD IA) and those with severe GH deprivation (IGHD IB). Southern blot analysis demonstrated that most of the children with IGHD IA have a homozygous GH-1 gene deletion and, as a consequence, no genetic information for GH (2). The healthy parents of these children are hemizygous for GH-1, showing that the presence of a single GH-1 allele is sufficient for GH production and secretion, resulting in normal GH levels in serum (3). The genetic basis for the most common type of inheritance IGHD IB is unclear in most cases. In a few patients, splice site mutations in intron IV of GH-1 were described, resulting in skipping the nucleotides coding for amino acids (aa) 103–126 and subsequently in a frame shift in exon 5 (4). Inactivating mutations of the gene encoding the GHRH receptor were found as an alternative genetic basis of IGHD IB (5).
IGHD II has mainly been described in patients harboring mutations in the intron III donor splice site of one GH-1 allele, which cause skipping of exon 3 (6, 7). The resulting gene product lacks aa 32–71 and, therefore, the entire loop connecting the first -helix of the GH molecule to the second one (8) (Fig. 1). The mutant GH protein is thus subject to serious structural changes. In patients harboring this kind of mutation, GH concentrations are extremely low even though one intact GH-1 allele is present. Therefore, the presence of del32–71GH in the somatotrophs causes a blockade of wild-type (wt) GH secretion by an as yet unknown mechanism. In contrast, the splicing variant del32–46GH (the 20-kDa GH variant), which also lacks part of the loop connecting -helices 1 and 2 (8), accounts for approximately 10% of the GH found in serum of normal individuals and evidently does not have any adverse effect on the secretion of wtGH (9).
FIG. 1. Schematic representation of the wtGH protein tertiary structure. The two intramolecular disulfide bridges and distinct aa positions corresponding to end points of deleted aa stretches are outlined.
When human wtGH and del32–71GH were coexpressed in different cell types, the dominant negative effect was observed only in neuroendocrine cells, whereas no impairment of wtGH secretion was found in other cell types (e.g. COS, Chinese hamster ovary, and Epstein-Barr virus-transformed lymphocytes) (10, 11, 12). Neuroendocrine cells differ from these cells by having specific modes of protein transport, sorting, storage, and release (13). In transient transfection studies involving rat GH4C1 neuroendocrine cells, it was observed that del32–71GH suppresses both intracellular accumulation and secretion of wtGH without itself being accumulated or secreted, whereas wtGH and del32–71GH mRNAs were shown to be present in approximately equal quantities (12). Studies with COS cells revealed an uneven distribution of wtGH and del32–71GH, the former being localized in the Golgi apparatus and the latter retained in the endoplasmic reticulum. It was suggested that the presence of the misfolded protein causes Golgi apparatus fragmentation, thus disrupting transport from the endoplasmic reticulum to the Golgi apparatus also involving other membrane and secretory proteins (14). These findings in nonneuroendocrine cells were not observed in GH4C1 neuroendocrine cells, showing no deleterious effect of human del32–71GH on the production and secretion of heterologously expressed human prolactin, suggesting that the effects of mutant GH are cell type specific (12). In transgenic mice expressing human del32–71GH, a marked decrease in GH levels was found in pituitary extracts, and the affected animals developed short stature (15). Moreover, multiple anterior pituitary deficiencies, pituitary hypoplasia, morphological abnormalities of somatotrophs with few secretory vesicles, and macrophage hypophyseal invasion were detected. In contrast, the pituitary abnormalities observed in patients with IGHD II are not as severe as those evident in the in vivo mouse model, because a small to normal size pituitary gland was confirmed by magnetic resonance tomographic analysis (16).
Several hypotheses have been discussed in the literature to explain the basic mechanisms of the interference of some mutant GH forms with wtGH. These include 1) accumulation of toxic aggregates of mutant proteins, 2) decrease in intracellular stability of wtGH due to cellular responses induced by unfolded proteins (overload response, unfolded protein response, and induction of cell type-specific degradation systems), 3) specific blockade of GH aggregation and/or sorting into secretory granules, and 4) impaired maturation of secretory granules.
The aim of this in vitro study using GH4C1 cells was to elucidate the importance of specific aa or stretches of aa in the context of the GH tertiary structure for exhibition of the dominant negative effect.
Materials and Methods
DNA vectors
Deletion and point mutations of GH cDNA were performed using overlap extension PCR technology (17). The wtGH cDNA inserted in a pcDNA3-vector (pwtGH; gift from P. Dannies, Yale School of Medicine, New Haven, CT) was used as a template. The oligonucleotides used for site-directed mutation were: C53A forward, 5'-GAC CTC CCT CGC ATT CTC AG-3'; C53A reverse, 5'-CTG AGA ATG CGA GGG AGG TC-3' (for C53A-GH and C53A-C165A-GH); C165A forward, 5'-GCT CTA CGC ATT CAG GAA GGA-3'; C165A reverse, 5'-TCC TTC CTG AAT GCG TAG AGC-3' (for C165A-GH and C53A-C165A-GH); del32–46 forward, 5'-CAG GAG TTT AAC CCC CAG ACC TCC CTC-3'; del32–46 reverse, 5'-CTG GGG GTT AAA CTC CTG GTA GGT GTC-3' (for del32–46-GH); del32–52 forward, 5'-CAG GAG TTT TGT TTC TCA GAG T-3'; del32–52 reverse, 5'-ACT CTG AGA AAC AAA ACT CCT G-3' (for del32–52-GH); del32–53 forward, 5'-CTA CCA GGA GTT TTT CTC AGA G-3'; del32–53 reverse, 5'-GAC TCT GAG AAA AAC TCC TGG T-3' (for del32–53-GH); del32–63 forward, 5'-TAC CAG GAG TTT AGG GAG GAA-3'; del32–63 reverse, 5'-TTC CTC CCT AAA CTC CTG GTA-3' (for del32–63-GH); del32–69 forward, 5'-CCA GGA GTT TAA ATC CAA CCT-3'; del32–69 reverse, 5'-GGT TGG ATT TAA ACT CCT GGT-3' (for del32–69-GH); GH-5'-HindIII, 5'-GTT AAG CTT CCT GTG GAC AGC TCA C-3'; and GH-3'-XhoI, 5'-AGA CTC GAG TAT TAG GAC AAG GCT GGT-3' (homologous to the 5' and 3' noncoding regions of the hGH cDNA, including, in addition, HindIII and XhoI restriction sites, respectively).
In a first round of PCR, amplicons were generated using either GH-5'-HindIII and one of the mutant reverse primers or GH-3'-XhoI primer and one of the mutant forward primers. In a second PCR round, the primers GH-5'-HindIII and GH-3'-XhoI were used with both amplicons of the first PCR round as template. The PCR products were purified using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), cut with restriction enzymes specific for HindIII and XhoI sites, and cloned into the transfection vector pcDNA3.1 (Invitrogen Life Technologies, Inc., Karlsruhe, Germany) opened with HindIII and XhoI. Plasmids were transformed into XL-1 Blue supercompetent Escherichia coli cells (Stratagene, Cedar Creek, CA) and screened for efficient cloning by use of restriction enzyme analysis (HindIII and XhoI). Plasmid DNA was isolated from a suitable colony using EndoFree Plasmid Maxi Kit (Qiagen). The presence of the point or deletion mutation and the integrity of the human GH (hGH) cDNA were verified by sequencing (GENterpise, Mainz, Germany).
Cell culture
GH4C1 cells used for the transfection experiments were purchased from American Type Culture Collection (LGC Promochem, Wesel, Germany). GH4C1 cells were cultured in DMEM/Ham’s F-12 (Invitrogen Life Technologies, Inc., Paisley, UK) supplemented with 15% horse serum (Invitrogen Life Technologies, Inc.) and incubated at 37 C in 5% CO2.
Transfection experiments
The cDNA expression vectors generated, harboring various mutant GH cDNAs, were cotransfected with pwtGH using the Effectene Transfection Reagent Kit (Qiagen). Cotransfection experiments with expression vectors for ?-galactosidase (pcDNA3.1.V5/His-lacZ, Invitrogen Life Technologies, Inc.), firefly luciferase (pGL2LUC, Promega Corp., Mannheim, Germany), or human IGF-binding protein 2 (pcDNA3.1-IGFBP-2) were performed. For transfection, 5 x 105 cells were seeded in 60-mm poly-D-lysine-coated transfection dishes (BD Biosciences, Meylan, France). Transfection was carried out according to the manufacturer’s instructions 24 h after seeding at approximately 80% confluence. A total of 1 μg DNA and a DNA ratio of wtGH to mutant GH of 1:1 were used, if not otherwise stated. If the amounts of mutant constructs were varied, the total amount of the transfected plasmid DNA was adjusted to 1 μg DNA using an empty pcDNA3.1 vector. Forty-eight hours posttransfection, medium and cells were harvested, and cellular proteins were extracted using reporter lysis buffer (400 μl/culture dish) according to the manufacturer’s instructions (Promega Corp., Madison, WI). Shorter or longer incubation periods (18, 24, and 72 h, respectively) were demonstrated to be inappropriate for the RIA used, because the GH values measured were beyond the detection capacity of the system.
RNA extraction and real-time RT-PCR
Total RNA was isolated 24 h posttransfection using the RNeasy Mini Kit (Qiagen) with a simultaneous on-column deoxyribonuclease digestion with the ribonuclease-free deoxyribonuclease set (Qiagen) according to the manufacturer’s instructions. Additionally RNA solution was treated with ribonuclease-free deoxyribonuclease I (Roche). RT-PCR (Promega Corp.), performed with or without reverse transcriptase, showed the presence of hGH transcripts and efficient removal of plasmid DNA. cDNA was synthesized using the Omniscript RT kit (Qiagen). Consecutively, real-time RT-PCR was performed in a Bio-Rad iCycler using the SYBR Green Supermix (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK). The primers were designed to yield products no longer than 150 bp. For each sample, duplicate measurements were performed, and the arithmetic mean was calculated from each duplicate measurement. Threshold cycle values were determined using Bio-Rad iCycler software version 3.0.
hGH and IGFBP-2 measurements
GH values in media and cell lysates and IGFBP-2 values in media were measured with an RIA specific for hGH or human IGFBP-2 as previously described (18, 19).
Western blot analysis
Media or cell extracts (15 μl) were separated on a 15% sodium dodecyl sulfate-polyacrylamide gel, blotted on an Immobilon-P transfer membrane (Millipore Corp., Bedford, MA), and subjected to antibody determination using either a rabbit polyclonal antiserum against recombinant hGH (somatropin, NIBSC code 88/624) or a monoclonal antibody (7B11, gift from C. Strasburger, Berlin, Germany) directed to the N-terminal domain of the hGH molecule. As secondary antibodies, antirabbit immunoglobulin G (New England Biolabs, Frankfurt am Main, Germany) and antimouse immunoglobulin G (New England Biolabs) were used. Chemiluminescence detection was performed using the ECL Plus Western Blotting Detection System (Amersham Biosciences UK Ltd., Little Chalfont, UK). Results were analyzed densitometrically on a Raytest apparatus (Raytest Isotopenmessger?te, Straubenhardt, Germany) using AIDA (advanced image data analyzer) software, version 2.1. In addition, the image was captured on Kodak film (Eastman Kodak Co., Rochester, NY).
Cell counting
In experiments performed in parallel, 4, 24, 48, and 80 h posttransfection, the number of cells transfected with only pwtGH or cotransfected with pwtGH and a del32–71GH-expressing plasmid (pdel32–71GH) was estimated automatically (Cobas Micros, Roche, Montpellier, France; Advia 120, Bayer, Holliston, MA) as well as in a Neubauer-improved counting chamber (Brand, Wertheim, Germany). Vital and nonvital cells were distinguished under the microscope via staining with 0.4% trypan blue solution (Sigma-Aldrich Corp., Irvine, UK).
?-Galactosidase and luciferase activities
?-Galactosidase and luciferase activities were determined in an automated luminometer Wallac 1420 Victor2 (Wallac Oy, Turku, Finland) using the ?-Gal Reporter Gene Assay, chemiluminescent (Roche), and Luciferase Assay System (Promega Corp.), respectively.
?-Galactosidase staining
?-Galactosidase staining was performed directly on culture dish using reagents produced according to the instructions of the In Situ ?-Galactosidase Staining Kit (Stratagene, La Jolla, CA).
Statistical data analysis
Differences between the transfection groups were analyzed by t test.
Results
To investigate the structure-function relationship of hGH mutants with respect to a negative effect on wtGH secretion, a series of hGH constructs that were mutated or deleted in specific aa or stretches of aa was generated. We focused on study of the linker region between -helices 1 and 2 (aa 46 and 71; Fig. 1), because del32–46GH (the 20-kDa GH variant form) exerts no deleterious effect, whereas del32–71GH has a strong adverse effect on wtGH secretion. Therefore, diverse deletion mutants, del32–46, del32–52, del32–53, del32–63, and del32–69 were constructed in which the respective aa stretch was deleted. Of additional interest was the role of the disulfide bridge constituted between Cys53 and Cys165, which is disrupted in del32–71GH. Distinct point mutants were constructed in which the respective cysteine-encoding DNA triplets were mutated to alanine-encoding triplets, resulting in the GH mutants C53A, C165A, and C53A-C165A (Fig. 1).
GH4C1 cells were simultaneously transfected with pwtGH and one of the mutant GH-expressing plasmids. Forty-eight hours posttransfection, GH was measured in incubation media and cell extracts. The GH content in cells cotrans-fected with pwtGH/pwtGH, pwtGH/pdel32–46, pwtGH/pdel32–52, pwtGH/pdel32–53, pwtGH/pdel32–63, pwtGH/pdel32–69, and pwtGH/pdel32–71 was determined using an hGH-specific RIA (Fig. 2). In comparison with the control transfection (pwtGH/pwtGH set at 100%), the amount of hGH secreted into the medium was decreased by 35% when cotransfecting pwtGH/empty vector. It was decreased mildly by 15–30% when coexpressing wtGH and any of the point mutants (C53A, C165A, or C53A-C165A) or mutants deleted in aa 32–46 or 32–52. Deletion of more aa (32–52, 32–63) led to a stronger decrease in secreted hGH by 50–60%. A severe decrease by 75–85% was observed when stretch 32–69 or 32–71 was deleted. The degree of GH reduction was proportional to the increase in size of the deletion. The same relationship between the extent of deletion and the amount of detectable hGH was found in protein extracts of the respectively transfected cells (Fig. 2). The amount of del32–71GH when singly expressed was below the detection limit of our RIA system.
FIG. 2. Relative GH concentrations in media and cell extracts of cotransfected GH4C1 cells measured by RIA. Mean values of five to eight individual experiments ± SD are represented as a percentage of the values obtained for the pwtGH/pwtGH transfection, which was set at 100%. The GH content of each sample was measured twice, using the mean value for calculations. P values are given for the differences between pwtGH/pdel32–52GH and pwtGH/pdel32–53GH on the one side, and between pwtGH/pdel32–63GH and pwtGH/pdel32–69GH on the other side. The extent of deletion played the major role in expression of the dominant negative effect on wtGH.
To determine whether the amount of hGH was underestimated in the RIA due to the intrinsic competition with recombinant hGH, cell extracts and media were analyzed using Western blots (Fig. 3). Densitometric evaluation of protein bands corresponding to wtGH and mutant GH (detected by polyclonal antiserum as well as by monoclonal antibodies directed toward the N terminus of hGH) revealed comparable amounts as determined using RIA. The finding that mutant protein amounts were not underestimated in the RIA was strengthened by the comparability of RIA and Western blot results for cells monotransfected with pwtGH, pdel32–46, pdel32–53, or pdel32–71 (data not shown).
FIG. 3. Western blot analysis of extracts (CE) or media (M) of cotransfected GH4C1 cells using a polyclonal rabbit antihuman GH antiserum (polyclonal rabbit -hGH) or a monoclonal mouse antihuman GH antibody specific for the N-terminal domain (monoclonal mouse -hGH). Protein bands corresponding to the wt and some mutant hGH forms are depicted by arrows and the respective molecular weights.
To investigate whether del32–53GH exerts a dominant negative effect as suggested by the above RIA data, we performed parallel cotransfection experiments in which pdel32–53 or empty vector was cotransfected in varying amounts together with pwtGH (Fig. 4). The total amount of transfected plasmid was kept constant by adapting the amount of pwtGH cotransfected. The concentration of total GH was constantly lower for cells cotransfected with pdel32–53GH and pwtGH than for cells cotransfected with empty vector and pwtGH, indicating a dominant negative effect of del32–53GH on wtGH. This effect became significantly stronger with increasing amounts of pdel32–53GH.
FIG. 4. Relative GH concentrations in media and cell extracts of cotransfected GH4C1 (with pwtGH/pdel32–53GH or pwtGH/vector) were measured by RIA. Mean values of three individual experiments ± SD are represented as a percentage of the values obtained for the pwtGH/vector cotransfection at equimolar amounts, which was set at 100%. The GH content of each sample was measured twice, using the mean value for calculations. The ratio of the respective plasmids used for transfection experiments is depicted on the left. The data reveal a dose-dependent dominant negative effect of del32–53GH on wtGH.
The total cell numbers and viability of cells expressing only wtGH or coexpressing wtGH and del32–71GH stayed equivalent in individual experiments over periods of up to 80 h, thus making an acute toxic effect of del32–71GH on the cells unlikely (data not shown).
The transfection efficiency was determined in individual experiments by counting the ?-galactosidase-stained cells directly on the culture dish and was between 10–15%. No intraexperimental variation in transfection efficiency was found between cells transfected only with pwtGH and those cotransfected with both pwtGH and pdel32–46, pdel32–53, or pdel32–71GH. Interestingly, the intensity of the staining decreased with the increasing length of the deleted stretch (data not shown).
To normalize the values obtained by RIA measurements for transfection efficiency, plasmids containing cDNA encoding ?-galactosidase, firefly luciferase, or IGFBP-2 were cotransfected, and the activity or expression of the respective gene products was analyzed in cell extracts (?-galactosidase and luciferase) or media (IGFBP-2). Unexpectedly, the ?-galactosidase activity measured in extracts from cells cotransfected with pwtGH/pdel32–71GH was approximately 5-fold lower than that of cells transfected only with pwtGH or cotransfected with pwtGH and an empty vector (Fig. 5). In addition, the dominant negative effect of del32–71GH on both wtGH and ?-galactosidase appeared to be dose dependent. When constant amounts of pwtGH and expression plasmid for ?-galactosidase were cotransfected with increasing amounts of pdel32–71GH, a reverse correlation was found between the quantity of pdel32–71GH transfected, on the one hand, and the ?-galactosidase activity and GH concentrations measured, on the other hand (Fig. 6). Similar results were obtained when firefly luciferase was coexpressed. The activity of this enzyme found in extracts from cells cotransfected with pwtGH and either of the expression plasmids for the deletion mutant del32–69 or del32–71 was approximately 5-fold lower than that of the pwtGH/pwtGH control transfection, and the degree of inhibition was also dose dependent (data not shown). To investigate whether a molecular interaction between wtGH and mutant GH proteins is a prerequisite for the observed effects on heterologous proteins, luciferase activity was measured in cells cotransfected only with pdel32–71GH and pGL2LUC. In this case, luciferase activity was barely detectable and was comparable to levels detected in extracts of untransfected cells, whereas those cotransfected with pwtGH and pGL2LUC yielded considerable luciferase activity (data not shown). Therefore, the effect of del32–71GH on heterologously coexpressed proteins was independent of the presence of wtGH.
FIG. 5. ?-Galactosidase activities in cell lysates. Cells were always cotransfected with 0.4 μg of each of the plasmids denoted and 0.2 μg plasmid pcDNA3.1.V5/His-lacZ. The mean ± SD of triplicate transfections are presented. ?-Galactosidase activity was severely diminished in the presence of del32–71GH.
FIG. 6. ?-Galactosidase activities in cell lysates and GH concentrations determined by RIA in media and cell extracts. For ?-galactosidase analysis, cells were cotransfected with 0.2 μg pcDNA3.1.V5/His-lacZ, 0.4 μg pwtGH, and 0.4, 0.2, or 0.1 μg pdel32–71GH. For GH analysis, cells were cotransfected with 0.4 μg pwtGH and 0.4, 0.2, or 0.1 μg pdel32–71GH. A total of 1 μg transfected DNA was achieved by cotransfection of respective amounts of an empty vector. Values represent the mean of duplicate determinations of one experiment and indicate that the dominant negative effect of del32–71GH on wtGH as well as on the heterologously coexpressed ?-galactosidase was dose dependent.
Analogous results were obtained when IGFBP-2 levels were determined in culture media of cells that were cotransfected with pwtGH, either of the expression plasmids for a GH mutant, and pIGFBP-2. The concentrations of IGFBP-2 detected in medium were inversely correlated to the extent of the deletion greater than 32–52 in the transfected mutant constructs. Cotransfection of either of the point mutants showed no significant effect on the amount of IGFBP-2 (Fig. 7).
FIG. 7. IGFBP-2 concentrations measured using RIA in media of cells cotransfected with pIGFBP-2, pwtGH, and plasmids encoding GH deletion or point mutants. The mean values of three individual experiments ± SD are represented as a percentage of the values obtained for the pwtGH/pwtGH transfection, which was set at 100%. The IGFBP-2 concentration in each sample was measured twice, using the mean value for calculations. P values are given for the indicated comparisons.
Real-time RT-PCR results revealed comparable mRNA expression levels for wtGH as well as total hGH and for the marker protein IGFBP-2 (Fig. 8), indicating that the observations at the protein level were not based on a decrease in transcription or RNA instability.
FIG. 8. Relative expression levels of wtGH, total GH, and IGFBP-2 mRNA for cells cotransfected with pwtGH, plasmids encoding GH deletion mutants, and pIGFBP-2. Data are represented as a percentage of the values obtained for the pwtGH/pwtGH transfection, which was set at 100%. For each sample, duplicate measurements were performed, using the mean value for calculations.
Discussion
The presence of specific GH protein mutants in the pituitary somatotrophs results in a dominant restriction of wtGH secretion, whereas others are tolerated at the heterozygous state. Therefore, it was of great interest to analyze the relationship between GH-1 mutations and effects in further detail. The focus was set on the role of the linker region between helices 1 and 2 of GH, the deletion of which is frequently the cause of IGHD II. This region contains a cysteine at position 53 that is necessary for the formation of an intramolecular disulfide bridge with Cys165. Mutation of one cysteine residue at position 53 or 165 eliminated formation of the intramolecular disulfide bridge Cys53-Cys165 and made the establishment of irregular intramolecular, with cysteine residues at positions 182 or 189, or intermolecular disulfide bridges possible. The last mechanism would potentially facilitate the formation of GH molecule dimers covalently bound to each other. Mutations of both cysteine residues at positions 53 and 165 totally eliminated the formation of disulfide bridges. Our results suggest that disruption of the disulfide bridge alone is not sufficient for the dominant negative effect observed, because the GH concentration in cells cotransfected with pwtGH and an expression plasmid for either of the point mutants was only slightly reduced. In addition, the point mutants with one free cysteine (C53A or C165A) displayed similar behavior compared with the mutant harboring two aa exchanges (C53A-C165A). Our results are in line with those of a previous study suggesting that the presence of the free cysteine residue 165 has only a minor influence on exhibition of the dominant negative effect (12). These findings are contradictory to the assumption that del32–71GH affects wtGH secretion only by forming dimers due to free cysteine residues (10).
Our construct del32–52GH was related to the naturally occurring 20-kDa GH variant (del32–46) lacking additional six aa but preserving the cysteine at position 53 and, therefore, the intramolecular disulfide bridge. The concentrations of GH detected in both media and cell extracts of cells cotransfected with pwtGH and either pdel32–46GH or pdel32–52GH were similar. Deletion of one additional aa in the construct del32–53, thereby hampering formation of the disulfide bridge between aa residues 53 and 165, led to a statistically significant decrease (P = 0.006) in the concentration of secreted GH compared with the effect of del32–52GH. Moreover, the inhibitory effect of del32–53GH on wtGH secretion was shown to be dose dependent. This observation indicates that the disruption of the disulfide bridge is a step-change event. This disruption evidently affects the tertiary structure of the GH molecule critically when residue 53 is removed in the context of the whole deleted aa stretch 32–53; thus, the relationship between structure and function is not simply linearly correlated to the extent of the peptide deletion. Deletion of 10 additional aa up to the beginning of the second minihelix in construct del32–63GH induced no further reduction of the hGH detected, whereas the additional deletion of the second minihelix encompassing six supplementary aa in del32–69GH resulted in the exhibition of a negative effect comparable to that of del32–71GH. One hypothesis for the exhibition of the dominant negative effect exerted by GH mutants explains the decreased secretion with a disorder of the aggregation of GH molecules. It has been suggested that the binding of Zn2+ by GH molecules is a prerequisite for GH dimerization and subsequently for GH storage in secretory granules (20). Because deletion of the linker region between -helices 1 and 2 presumably results in a strongly changed tertiary structure of the GH molecule, this might also affect Zn2+ binding and, as a consequence, influence aggregation or sorting of GH proteins or maturation of the secretory granules. An increase in the extent of the deletion and, in particular, deletion of pronounced structures such as the second minihelix are likely to result in major structural changes and, consequently, strong disturbances of aggregation.
GH immunoreactivity assayed using a polyclonal RIA was the main parameter studied in our in vitro experiments. The polyclonal antiserum of this RIA has a presumed wide spectrum of epitope specificities (9). It has been well characterized for the determination of 22-kDa GH (18), but its cross-reactivity to mutant GH proteins, especially del32–71GH (17.5-kDa GH), is not known. Therefore, there is no doubt that in those experiments in which wtGH was expressed using the same amounts of pwtGH a substantial decrease in RIA values did represent a decrease in 22-kDa GH. In experiments with a minor decrease in RIA values, the change could be caused by a decrease in the amount of GH, by decreased cross-reactivity, or by a combination of the two from a theoretical standpoint. However, our data from Western blot analysis, which avoided the direct competition between wt and mutant GH, correlated well with RIA values. In addition, Western blot analysis using a monoclonal antibody raised against an epitope not affected by the GH mutations confirmed this finding. These experimental data strongly argue that immunoreactivity measured using the polyclonal RIA reflects total GH amounts rather than lack of GH cross-reactivity. Therefore, these results underline the usefulness of the applied GH RIA for the detection of the various GH mutants.
GH RIA results are presented without correction, because the transfection efficiency, determined by counting ?-galactosidase-stained cells, was equal. The discrepancy compared with the luminometric data could be due to different expressions at the protein level despite the equivalent efficiency of transfection as the intensity of staining observed under the microscope decreased in the presence of del32–71GH. In addition, the absence of significant differences in expression at the mRNA level of total GH and wtGH was indicative of comparable transfection efficiencies.
Toxic effects in cells cotransfected with pwtGH and pdel32–71GH were not found in a cell viability analysis. There was no decrease in cell numbers 80 h posttransfection, which presumably would have been the case if del32–71GH exerted cell toxicity, despite the relatively low percentage of the cell population being transfected. In addition, mRNA expression of the marker protein IGFBP-2 was unaffected by coexpression of del32–71GH. These results are in agreement with an earlier report in which a lack of toxicity over a 24-h period was suggested by the observation that the secretory pathway in GH4C1 cells expressing del32–71GH functioned properly (12). It has to be emphasized that our data were obtained over a relatively short period of time. Therefore, we cannot exclude effects on cell viability due to inhibition of non-GH protein production over a longer term.
The observed severe negative effect of del32–69GH and del32–71GH not only on wtGH, but also on IGFBP-2 secretion as well as on ?-galactosidase and firefly luciferase activities suggests a general disturbance of cells expressing mutant GH beyond the molecular interaction of wtGH and mutant GH. Also, ?-galactosidase and luciferase are not expected to be transported through the regulated secretory pathway. Our findings are not in line with the results of experiments applying the neuroendocrine cell lines MtT/S and AtT-20, in which del32–71GH had no clear decreasing effect on ?-galactosidase activity (11). However, in the experimental setting cited, the effect of del32–71GH on wtGH secretion was much less pronounced, requiring a high excess of mutant GH for an observable effect, thereby making the missing effect on ?-galactosidase less significant. These divergent data may be explained by different transfection conditions. In addition, some effects of del32–69GH and del32–71GH may be cell type specific; GH4C1, a rat prolactin- and GH-producing cell line, is a somatotroph-derived cell line, whereas AtT-20 is adrenocorticotroph derived.
Recently, McGuinness et al. (15) reported that rat pituitary GC cells, which were stably monotransfected with a plasmid expressing human del32–71GH, showed a decreased proliferation rate, reduced attachment, and disturbed morphology, followed by early cell apoptosis, thus indicating toxic-like effects of del32–71GH on this cell line. This discrepancy from our data may be explained by cell specificities, because GH4C1 cells stably cotransfected with pwtGH and pdel32–71GH exhibit viability and proliferation rates similar to those of cells monotransfected with pwtGH (data not shown). Therefore, every cell system may potentially react in a specific manner to the mutant GH expression, possibly different from that of human somatotrophs. However, we believe that our cell system is a good model for studying specific features of mutant human GH because GH4C1 cells are neuroendocrine somatotroph cells.
The phenomena of suppression of wtGH secretion and the negative effect exerted on the other proteins discussed presumably share a common genesis. The presence of the deletion mutant possibly disturbs various biological functions of cells without affecting their viability and proliferation rate. The expression of misfolded proteins is a source of cell stress, which results in degradation of proteins due to activation of unfolded protein responses (21). Presumably, the level of heterologous proteins is affected to a higher degree than that of endogenous proteins, because the former are produced in higher quantities and are thus more prone to be affected by degradation systems induced by unfolded protein responses.
Our data suggest that besides a minor role for the integrity of the disulfide bridge Cys53-Cys165, the extent of deletion plays the major role in expression of the dominant negative effect. The inhibitory effect of GH mutants on heterologously expressed non-GH proteins suggests that the dominant negative effect is not limited to GH or even to proteins of the regulated secretory pathway, but may depend on expression levels.
Acknowledgments
We thank Christian Strasburger for providing monoclonal antibody 7B11, Burkhardt Schuett for fruitful discussions as well as for provision of the vector pIGFBP-2, and Klaus Frommer for support in performing the real-time PCR. We acknowledge the technical assistance of Evelina Goetz and Karin Weber as well as the assistance of Priscilla Herrmann in the language editing of this manuscript, and the support of Peter-Michael Weber in the design of some figures.
References
Phillips JA, Cogan JD 1994 Genetic basis of endocrine disease. VI. Molecular basis of familial human growth hormone deficiency. J Clin Endocrinol Metab 78:11–16
Phillips III JA, Hjelle BL, Seeburg PH, Zachmann M 1981 Molecular basis for familial isolated growth hormone deficiency. Proc Natl Acad Sci USA 78:6372–6375
Akinci A, Kanaka C, Eble A, Akar N, Vidinlisan S, Mullis PE 1992 Isolated growth hormone (GH) deficiency type IA associated with a 45-kilobase gene deletion within the human GH gene cluster. J Clin Endocrinol Metab 75:437–441
Procter AM, Phillips JA, Cooper DN 1998 The molecular genetics of growth hormone deficiency. Hum Genet 103:255–272
Baumann G, Maheshwari H 1997 The dwarfs of Sindh: severe growth hormone (GH) deficiency caused by a mutation in the GH-releasing hormone receptor gene. Acta Paediatr 423(Suppl):33–38
Binder G, Ranke MB 1995 Screening for growth hormone (GH) gene splice-site mutations in sporadic cases with severe isolated GH deficiency using ectopic transcript analysis. J Clin Endocrinol Metab 80:1247–1252
Cogan JD, Phillips JA, Schenkman SS, Milner RD, Sakati N 1994 Familial growth hormone deficiency: a model of dominant and recessive mutations affecting a monomeric protein. J Clin Endocrinol Metab 79:1261–1265
Ultsch MH, Somers W, Kossiakoff AA, de Vos AM 1994 The crystal structure of affinity-matured human growth hormone at 2 A resolution. J Mol Biol 236:286–299
Baumann G 1991 Growth hormone heterogeneity: genes, isohormones, variants, and binding proteins. Endocr Rev 12:424–449
Binder G, Brown M, Parks JS 1996 Mechanisms responsible for dominant expression of human growth hormone gene mutations. J Clin Endocrinol Metab 81:4047–4050
Hayashi Y, Yamamoto M, Ohmori S, Kamijo T, Ogawa M, Seo H 1999 Inhibition of growth hormone (GH) secretion by a mutant GH-I gene product in neuroendocrine cells containing secretory granules: an implication for isolated GH deficiency inherited in an autosomal dominant manner. J Clin Endocrinol Metab 84:2134–2139
Lee MS, Wajnrajch MP, Kim SS, Plotnick LP, Wang J, Gertner JM, Leibel RL, Dannies PS 2000 Autosomal dominant growth hormone (GH) deficiency type II: the del32–71-GH deletion mutant suppresses secretion of wild-type GH. Endocrinology 141:883–890
Dannies PS 2000 Protein folding and deficiencies caused by dominant-negative mutants of hormones. Vitam Horm 58:1–26
Graves TK, Patel S, Dannies PS, Hinkle PM 2001 Misfolded growth hormone causes fragmentation of the Golgi apparatus and disrupts endoplasmic reticulum-to-Golgi traffic. J Cell Sci 114:3685–3694
McGuinness L, Magoulas C, Sesay AK, Mathers K, Carmignac D, Manneville JB, Christian H, Phillips III JA, Robinson IC 2003 Autosomal dominant growth hormone deficiency disrupts secretory vesicles in vitro and in vivo in transgenic mice. Endocrinology 144:720–731
Binder G, Nagel BH, Ranke MB, Mullis PE 2002 Isolated GH deficiency (IGHD) type II: imaging of the pituitary gland by magnetic resonance reveals characteristic differences in comparison with severe IGHD of unknown origin. Eur J Endocrinol 147:755–760
Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR 1989 Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59
Hauffa BP, Lehmann N, Bettendorf M, Mehls O, Dorr HG, Partsch CJ, Schwarz HP, Stahnke N, Steinkamp H, Said E, Sander S, Ranke MB 2004 Central reassessment of GH concentrations measured at local treatment centers in children with impaired growth: consequences for patient management. Eur J Endocrinol 150:291–297
Elmlinger MW, Wimmer K, Biemer E, Blum WF, Ranke MB Dannecker GE 1996 Insulin-like growth factor binding protein 2 is differentially expressed in leukaemic B- and T-cell lines. Growth Regul 6:152–157
Cunningham BC, Mulkerrin MG, Wells JA 1991 Dimerization of human growth hormone by zinc. Science 253:545–548
Kauffman KJ, Pridgen EM, Doyle III FJ, Dhurjati PS, Robinson AS 2002 Decreased protein expression and intermittent recoveries in BiP levels result from cellular stress during heterologous protein expression in Saccharomyces cerevisiae. Biotechnol Prog 18:942–950(Daniel I. Iliev, Nicola E)