HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Iliev, D. I Articles by Binder, G. Search for Related Content PubMed PubMed Citation Articles by Iliev, D. I Articles by Binder, G.

Pediatric Endocrinology Section, University Children’s Hospital, Hoppe-Seyler-Street 1, D-72076 Tuebingen, Germany

(Requests for offprints should be addressed to G Binder; Email: gerhard.binder{at}med.uni-tuebingen.de)

N E Wittekindt is now at The Pennsylvania State University, 311 Wartik Building, University Park, Pennsylvania 16802, USA

	Abstract

Top Abstract Introduction Materials and methods Results Discussion References

 
Zinc (Zn²⁺) binding by human GH through amino acid residues His18, His21, and Glu174 has been described as a prerequisite for GH dimerization and storage in secretory granules. Our aim was to investigate in vitro whether disturbed Zn²⁺ binding of mutant GH inhibits wild-type GH (wtGH) secretion and contributes to the pathogenetic mechanisms involved in dominantly transmitted isolated GH deficiency type II. Seven expression vectors harboring mutated human GH cDNAs were constructed in which nucleotide triplets encoding histidine or glutamine at positions 18, 21, and 174 were mutated to triplets encoding alanine: H18A, H21A, G174A, H18A–G174A, H21A–G174A, H18A–H21A, and H18A–H21A–G174A. These vectors were transiently cotransfected with a vector encoding wtGH or were singly transfected into rat pituitary GH₄C₁ cells. Plasmids encoding ß -galactosidase were cotransfected. 48h after transfection, GH in media and cell extracts was measured using a GH-specific RIA, and results were normalized for transfection efficiency by means of ß -galactosidase activity. In comparison with the control transfection (wtGH/wtGH set at 100%), GH secretion remained unaffected when coexpressing wtGH and any of the GH mutants in which Zn²⁺ binding was partially or completely prevented. When these mutants were singly expressed, the amount of GH in both media and cell extracts was decreased by about 50% when compared with cells expressing only wtGH. Our in vitro data do not support the hypothesis of disturbed Zn²⁺ binding as a major pathogenetic mechanism in dominantly transmitted GH deficiency.

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
Zinc (Zn²⁺) plays an important structural role in many proteins including hormones by serving as cross-linking means which bind protein molecules in dimers or big precipitation clusters (Cunningham et al. 1991, Sun et al. 1997). Mutant variants of human insulin which cannot bind Zn²⁺ do not form hexamers and are not properly processed intracellularly, presumably due to the inability of mutant insulin to be concentrated in secretory granules (Carroll et al. 1988, Berg & Shi 1996). Similarly, human prolactin (PRL), which is closely related to human growth hormone (GH), binds Zn²⁺ which is crucial for the intracellular processing and secretion of PRL (Sun et al. 1997, Sankoorikal et al. 2002). Zn²⁺ was shown to be present in human pituitary GH-secretory granules in equimolar amounts with GH, where it forms soluble dimer complexes consisting of two GH molecules and two Zn²⁺ ions (Cunningham et al. 1991). Recombinant human GH is also able to form soluble dimer Zn²⁺ complexes (Yang et al. 2000). Zn²⁺ binding by GH molecules through amino acid residues His18, His21, and Glu174 is a predicted prerequisite for GH dimerization and storage in secretory granules (Cunningham et al. 1991, Yang et al. 2000, Sankoorikal et al. 2002). Thus, it might be hypothesized that loss of affinity of mutant GH to Zn²⁺ could interfere with GH storage and secretion causing disturbed GH secretion as shown for other peptide hormones such as insulin and PRL (Moore & Kelly 1986, Carroll et al. 1988, Berg & Shi 1996, Sun et al. 1997).

Mutations in the intron 3 donor splice site of one GH-1 allele causing skipping of exon 3 are the most frequent causes of the dominantly transmitted isolated GH deficiency (IGHD type II; Cogan et al. 1994, Binder & Ranke 1995). In addition, three missense mutations have been described in IGHD type II patients, which lead to the exchanges of highly conserved amino acids and also suppress wtGH secretion to varying degrees (P89L, V110F, R183H; Duquesnov et al. 1998, Gertner et al. 1998, Binder et al. 2001, Deladoey et al. 2001). These three amino acids involved are located at protruding sites of the tertiary structure of the GH molecule and are possibly engaged in intramolecular interactions of the four helices or in interactions with other GH molecules (Fig. 1; Ultsch et al. 1994). The cellular mechanisms of the dominant expression of specific GH-1 mutations in humans, which cause IGHD type II, are still incompletely understood and may be manifold (Binder et al. 1996). Reduction of Zn²⁺ binding and GH dimerization by the mutant GH proteins could be one of these mechanisms, although the amino acids closely involved in Zn²⁺ binding are not directly affected by the mutations found in IGHD type II. Misfolding of mutant GH, however, could result in a molecule which does not exhibit the above amino acids at protruding sites yielding reduced affinity to Zn²⁺.

View larger version (37K): [in this window] [in a new window]   Figure 1 Schematic of the wtGH protein tertiary structure. The three Zn2+-binding amino acid residues and the position of Zn2+are depicted.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

 
DNA vectors

Point mutations of human GH cDNA were generated using overlap extension PCR technology (Ho et al. 1989). Human wtGH cDNA inserted in a pcDNA3-vector (pwtGH; kind gift from P Dannies, Yale School of Medicine, New Haven, CT, USA) was used as a template. The oligonucleotides used for site-directed mutagenesis are presented in Table 1.

In a first 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 round of PCR, the primers GH-5' –HindIII and GH-3' –XhoI were used with both amplicons of the first PCR as template. The PCR products were purified using ‘QIAquick Gel Extraction Kit’ (Qiagen GmbH), cut with restriction enzymes specific for HindIII and XhoI sites, and cloned into the transfection vector pcDNA3.1 (Invitrogen GmbH) opened with HindIII and XhoI. Plasmids were transformed into XL-1 blue supercompetent Escherichia coli cells (Stratagene, Cedar Creek, TX, USA) 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 GH cDNA were verified by sequencing (GENterprise GmbH, Mainz, Germany). In addition, we used a previously generated plasmid expressing mutant GH lacking amino acids 32 to 71 which is the mutant GH in most of the cases with IGHD type II (Iliev et al. 2005).

Cell culture

GH₄C₁ cells used for the transfection experiments were purchased from the American Type Culture Collection (ATCC, LGC Promochem, Wesel, Germany). The GH₄C₁ cell line is a radiation-induced rat pituitary adenoma somatotrope cell line, which produces PRL and rat GH, and proteins are secreted both through the constitutive and the regulated secretory pathways (Scammell et al. 1986). Cells were cultured in DMEM/ F-12 (Gibco) supplemented with 15% (v/v) horse serum (Gibco) and incubated at 37 ° C and 5% CO₂.

Transient transfection experiments

The cDNA expression vectors generated, harboring various mutant human GH cDNAs, were in a transient setting, singly transfected or cotransfected with pwtGH and expression vectors for ß -galactosidase (pcDNA3.1.V5/His-lacZ, Invitrogen) using the ‘Effectene Transfection Reagent Kit’ (Qiagen). For transfection, 5 x 10⁵ cells were seeded in 60 mm poly-D-lysine coated transfection dishes (BD Biosciences, Meylan Cedex, France). Transfection was carried out according to the manufacturer’s instructions 24 h after seeding at ~80% confluency. A total amount of 1 µ g DNA was used, the ratio of plasmids encoding wtGH to mutant GH being 1:1 in cotransfection experiments – 0.4 µ g pwtGH, 0.4 µ g plasmid coding for a mutant GH variant, and 0.2 µ g expression vector for ß -galactosidase. 48 h after transfection, medium and cells were harvested and cellular proteins were extracted using reporter lysis buffer (400 µ l/culture dish) according to the manufacturer’s instructions (Promega).

GH measurements

GH values in media and cell lysates were measured using a RIA specific for human GH applying an in-house assay with one polyclonal rabbit anti-hGH antibody against 22 kDa GH as previously described (Hauffa et al. 2004).

ß -Galactosidase activity ß

ß-Galactosidase activity was determined in an automated luminometer Wallac 1420 Victor² (Wallac Oy, Turku, Finland) using the ß -Gal Reporter Gene Assay, chemiluminescent (Roche Diagnostics GmbH).

Statistical analysis

Statistical analysis was performed using two-tailed student’s t-test. P < 0.05 was considered to indicate significance.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
The expression vectors that harbored the mutated human GH cDNAs were analyzed by sequencing, which revealed the isolated exchanges of two nucleotide triplets encoding the amino acid histidine at positions 18 and 21, as well as of the triplet encoding the amino acid glutamine at position 174, with a triplet encoding the amino acid alanine. These mutations were predicted to prevent Zn²⁺ binding either partially or completely (Fig. 1).

The mutants were as follows: H18A, H21A, G174A (one Zn²⁺-binding amino acid residue is missing), H18A–G174A, H21A–G174A, H18A–H21A (two Zn²⁺-binding amino acid residues are missing), and H18A–H21A–G174A (all three Zn²⁺-binding amino acid residues are missing). In order to normalize the values obtained by RIA measurements for transfection efficiency, plasmids containing cDNA encoding ß -galactosidase were cotransfected and the activity of the gene product was analyzed in cell extracts.

After 48 h of culture, the secreted and stored GH amounts were found to be not different between cells transfected with one of the seven combinations of pwtGH/pmutantGH and cells transfected with pwtGH alone; this was in contrast to the significant reduction of GH observed when the deletion mutant (del32-71) was cotransfected with wtGH (Fig. 2, top). The mean absolute values of RIA-detected GH in media and cell extracts for the pwt/pwt-transfection were 235 and 137 ng/ml respectively while the amount of GH for untransfected cells was around the low border of the detection limit of our RIA system at 0.23 and 0.51 ng/ml respectively.

View larger version (24K): [in this window] [in a new window]   Figure 2 Relative GH concentrations in cell extracts and media of mono- or cotransfected GH4C1 cells measured by RIA. The mean values of three individual experiments ± S.D. are given as a percentage of the values obtained for the pwtGH/pwtGH transfection, which was set to 100%. All values were normalized for transfection efficiency by measurement of ß -galactosidase expression in cell lysates. The amount of GH in media and in cell extracts decreased by about 50% when GH mutants were singly expressed in comparison with cells expressing only wtGH (P < 0.001).

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
The loss of affinity to Zn²⁺ may be one mechanism of the dominant negative effect exerted by some GH mutants, which are monoallelic expressed in dominantly transmitted IGHD type II. Zn²⁺ binding of GH molecules through amino acid residues His18, His21, and Glu174 is regarded as a prerequisite for GH dimerization and subsequently for GH storage in secretory granules (Cunningham et al. 1991, Yang et al. 2000). Here, we examined GH mutants lacking either one, two, or all three of the above crucial amino acids (due to exchange with alanine) in GH₄C₁ cells, a rat pituitary cell line. When coexpressed with wtGH, the intracellular levels of GH and the amount of GH secreted into the culture medium was not different between cells coexpressing mutant GH and wtGH and those expressing only wtGH. Hence, disturbed Zn²⁺ binding is unlikely to be a relevant mechanism of the dominant negative effect of mutant GH in IGHD type II. However, when mutant GH was singly expressed in GH₄C₁ cells, we observed a decreased overall production and secretion of GH suggesting a role of Zn²⁺ binding for GH intracellular trafficking or storage.

In a similar approach, Sun et al.(1997) stably transfected GH₄C₁ cells with a plasmid encoding mutant human PRL (H27A–PRL) that does not bind Zn²⁺. They found that the secretion of mutant H27A–PRL was decreased to a highly variable degree, while its intracellular stability was constantly reduced. Concomitantly, rat-GH secretion of these stably transfected GH₄C₁ cells was reduced. A decrease of H27A–PRL production was also found in transiently transfected GH₄C₁ cells when transfection efficiency was above 3%. These observations on human PRL are in agreement with our findings on GH in the same GH₄C₁ cell line model. In fact, coexpression studies, including both wt PRL and H27A–PRL, were not performed by Sun et al.(1997). Analysis of the aggregation of wt PRL and H27A–PRL in cell-free solutions suggested that PRL, in contrast to GH, does not require a high-affinity Zn²⁺-binding site to form aggregates (Sankoorikal et al. 2002). This indicates that, although biologically related, GH and PRL show individual distinct behavior.

The diminished human GH content in cell extracts and medium when GH₄C₁ cells were singly transfected with one of the different GH mutants cannot be fully explained by decreased immunoreactivity to our polyvalent antibodies of the GH RIA. In a previous study of hGH mutants, we analyzed results of western blot analysis using the polyvalent RIA antibody as well as monoclonal antibodies with RIA measurements and found a good correlation (Iliev et al. 2005). Although these GH mutants were different (i.e., del32-71, C53A, C165A), we suppose that the immunoreactivity measured by the polyclonal RIA truly reflects the total amounts of GH. Therefore, the decrease in GH measured points to a role of Zn²⁺ binding in GH stability and secretion. This conclusion was also supported by the trend to lower GH levels in the presence of three-point mutations compared with the presence of only one, which may still allow for high-affinity binding to Zn²⁺. Nevertheless, as coexpression with wtGH did not yield diminished GH levels, there is no indication that loss of high-affinity Zn²⁺binding is a major component of the dominant negative effect of mutant GH in IGHD II.

McGuinness et al.(2003) has established the only mouse model for IGHD II where, in addition to the endogenous mouse GH, the human del32-71 GH was expressed. They found severe damage to somatropic cells of the pituitary gland and panhypopituitarism. Severe cellular damage was not a prominent finding in in vitro studies with pituitary cell lines transfected with del32-71 GH (Lee et al. 2000, Iliev et al. 2005). In addition, the panhypopituitarism of the mice was not in line with the main clinical phenotype observed in children with IGHD II, who present with no other hormone deficiency but GH (Binder et al. 2001). However, our recent long-term study on IGHD II indicated that a minority of affected individuals develop additional pituitary hormone deficiencies over time (Mullis et al. 2005). These partly contradictory data suggest limitations of in vitro and in vivo experiments in the field of the research on IGHD II.

In conclusion, the mutated GH peptides, which poorly bind Zn²⁺, did not have a major effect on the secretion of the wtGH isoform; thus, our in vitro data do not support the hypothesis that disturbed Zn²⁺binding of mutant GH is involved in the mechanism of dominantly transmitted GHD.

	Acknowledgements

 
We thank Claudia Striepe for technical assistance and C Philipp Schwarze for language editing.

	Funding

This work has been supported by an ESPE Research fellowship sponsored by Novo Nordisk Comp. and by an ESPE Visiting Scholarship sponsored by Pfizer Comp. to D I I and by an Eli Lilly Foundation grant to G B. There is no conflict of interest that would prejudice its impartiality.

	References

Top Abstract Introduction Materials and methods Results Discussion References

 
Berg JM & Shi Y 1996 The galvanization of biology: a growing appreciation for the roles of zinc. Science 271 1081–1085.[Abstract]

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. Journal of Clinical Endocrinology and Metabolism 80 1247–1252.[Abstract]

Binder G, Brown M & Parks JS 1996 Mechanisms responsible for dominant expression of human growth hormone gene mutations. Journal of Clinical Endocrinology and Metabolism 81 4047–4050.[Abstract/Free Full Text]

Binder G, Keller E, Mix M, Massa GG, Stokvis-Brantsma WH, Wit JM & Ranke MB 2001 Isolated GH deficiency with dominant inheritance: new mutations, new insights. Journal of Clinical Endocrinology and Metabolism 86 3877–3881.[Abstract/Free Full Text]

Carroll RJ, Hammer RE, Chan SJ, Swift HH, Rubenstein AH & Steiner DF 1988 A mutant human proinsulin is secreted from islets of Langerhans in increased amounts via an unregulated pathway. PNAS 85 8943–8947.[Abstract/Free Full Text]

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. Journal of Clinical Endocrinology and Metabolism 79 1261–1265.[Abstract]

Cunningham BC, Mulkerrin MG & Wells JA 1991 Dimerization of human growth hormone by zinc. Science 253 545–548.[Abstract/Free Full Text]

Deladoey J, Stocker P & Mullis PE 2001 Autosomal dominant GH deficiency due to an Arg183His GH-1 gene mutation: clinical and molecular evidence of impaired regulated GH secretion. Journal of Clinical Endocrinology and Metabolism 86 3941–3947.[Abstract/Free Full Text]

Duquesnov P, Simon D, Netchine I, Dastot F, Sobrier ML, Goossens M, Czernichow P & Amselem S 1998 Familial isolated growth hormone deficiency with slight height reduction due to a heterozygote mutation in GH gene. Proc. 80th Meeting of The Endocrine Society P2–202 (Abstract).

Gertner JM, Wajnrajch MP & Leibel RL 1998 Genetic defects in the control of growth hormone secretion. Hormone Research 49 9–14.[CrossRef][Web of Science][Medline]

Hauffa BP, Lehmann N, Bettendorf M, Mehls O, Dorr HG, Partsch CJ, Schwarz HP, Stahnke N, Steinkamp H, Said E et al. 2004 Central reassessment of GH concentrations measured at local treatment centers in children with impaired growth: consequences for patient management. European Journal of Endocrinology 150 291–297.[Abstract]

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.[CrossRef][Web of Science][Medline]

Iliev DI, Wittekindt NE, Ranke MB & Binder G 2005 Structural analysis of human growth hormone with respect to the dominant expression of GH mutations in isolated growth hormone deficiency type II. Endocrinology 146 1411–1417.[Abstract/Free Full Text]

Lee MS, Wajnrajch MP, Kim SS, Plotnick LP, Wang J, Gertner JM, Leibel RL & Dannies P 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.[Abstract/Free Full Text]

McGuinness L, Magoulas C, Sesay AK, Mathers K, Carmignac D, Manneville JB, Christian H, Philips JA, III & Robinson IC 2003 Autosomal dominant growth hormone deficiency disrupts secretory vesicles in vitro and in vivo in transgenic mice. Endocrinology 144 720–731.[Abstract/Free Full Text]

Moore HH & Kelly RB 1986 Re-routing of a secretory protein by fusion with human growth hormone sequences. Nature 321 443–446.[CrossRef][Medline]

Mullis PE, Robinson IC, Salemi S, Eble A, Besson A, Vuissoz JM, Deladoey J, Simon D, Czernichow P & Binder G 2005 Isolated autosomal dominant growth hormone deficiency: an evolving pituitary deficit? A multicenter follow-up study Journal of Clinical Endocrinology and Metabolism 90 2089–2096.[Abstract/Free Full Text]

Sankoorikal BJ, Zhu YL, Hodsdon ME, Lolis E & Dannies PS 2002 Aggregation of human will-type and H27A prolactin in cells and solution: role of Zn²⁺, Cu²⁺, and pH. Endocrinology 143 1302–1309.[Abstract/Free Full Text]

Scammell JG, Burrage TG & Dannies PS 1986 Hormonal induction of secretory granules in a pituitary tumor cell line. Endocrinology 119 1543–1548.[Abstract/Free Full Text]

Sun Z, Lee MS, Rhee HK, Arrandale JM & Dannies PS 1997 Inefficient secretion of human H27A-PRL, a mutant that does not bind Zn²⁺. Molecular Endocrinology 11 1544–1551.[Abstract/Free Full Text]

Ultsch MH, Somers W, Kossiakoff AA & de Vos AM 1994 The crystal structure of affinity-matured human growth hormone at 2 A resolution. Journal of Molecular Biology 236 286–299.[CrossRef][Web of Science][Medline]

Yang TH, Cleland JL, Lam X, Meyer JD, Jones LS, Randolph TW, Manning MC & Carpenter JF 2000 Effect of zinc binding and precipitation on structures of recombinant human growth hormone and nerve growth factor. Journal of Pharmaceutical Sciences 89 1480–1485.[CrossRef][Web of Science][Medline]

Received in final form 3 May 2007
Accepted 12 June 2007
Made available online as an Accepted Preprint 19 June 2007

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Iliev, D. I Articles by Binder, G. Search for Related Content PubMed PubMed Citation Articles by Iliev, D. I Articles by Binder, G.