help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boguszewski, C. L.
Right arrow Articles by Carlsson, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Boguszewski, C. L.
Right arrow Articles by Carlsson, B.
The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 8 2878-2885
Copyright © 1998 by The Endocrine Society

Original Studies

Cloning of Two Novel Growth Hormone Transcripts Expressed in Human Placenta1

Cesar L. Boguszewski, Per-Arne Svensson, Thomas Jansson, Ross Clark, Lena M. S. Carlsson and Björn Carlsson

Research Centre for Endocrinology and Metabolism (C.L.B., P-A.S., L.M.S.C., B.C.), Department of Internal Medicine, Department of Physiology and Pharmacology (T.J.), Sahlgrenska University Hospital, Göteborg, Sweden; Research Centre for Developmental Medicine and Biology (R.C.), University of Auckland, New Zealand

Address all correspondence and requests for reprints to: Björn Carlsson, Research Centre for Endocrinology and Metabolism-Sahlgrenska University Hospital, Gröna Stråket 8-S-413 45, Göteborg, Sweden. E-mail: bjorn.carlsson{at}ss.gu.se


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 References
 
Several isoforms of human GH (hGH) are produced by two related genes expressed in the pituitary (hGH-N) and in the placenta (hGH-V). These genes consist of five exons (denoted 1–5) separated by four introns (denoted A-D). In the present report, two new transcripts of the hGH-V gene are described. The coding region of the hGH-V gene was amplified by RT-PCR using placental complementary DNA as template. DNA sequencing of several clones revealed two novel transcripts. One had a 45-bp deletion caused by the use of an alternative splice acceptor site within exon 3, similar to that in the hGH-N gene, predicting a 20-kDa isoform of hGH-V. The other transcript was generated by the use of an alternative splice donor site causing a 4-bp deletion in the end of exon 4, predicting a 24-kDa protein with 219 amino acids, which we refer to as hGH-V3. The carboxy-terminal sequence of hGH-V3 differs from 22-kDa hGH-V and hGH-V2, the two previously reported transcripts of the hGH-V gene, and does not contain a predicted transmembrane domain as described for hGH-V2. Ligase chain reaction was then used to analyze the possible use of the same splicing pattern in transcripts derived from the other genes of the hGH-gene cluster. Alternatively spliced transcripts encoding the 20-kDa hGH isoform were detected from the hGH-N and hGH-V genes, but not from the human chorionic somatomammotropin-A/B genes. The alternative splicing generating hGH-V3 was only demonstrated in transcripts derived from the hGH-V gene. Using competitive RT-PCR, the expression of hGH-V3 was estimated to be 10% of the hGH-V messenger RNA in full-term normal placentas and in placentas from pathological pregnancies. The 20-kDa hGH-V was detected in two of four full-term normal placentas, whereas a weak signal was observed in one of the pathological placentas. We conclude that the hGH-V primary transcript undergoes alternative splicing pathways generating at least four different messenger RNAs, predicting the expression of different hGH isoforms, including two with a complete sequence divergence in the carboxy-terminus.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 References
 
THE human GH (hGH) gene cluster is located on the long arm of chromosome 17 and consists of five highly sequence-conserved genes aligned in the same transcriptional orientation. Among these genes, the hGH-N (N = normal), which is expressed in somatotrophic cells of the anterior pituitary, and the hGH-V (V = variant), which is expressed in syncytiotrophoblastic cells in the placenta, are responsible for the production of different hGH isoforms. In these genes, five exons (designated 1 to 5) are separated by four introns sequences (designated A to D) (1, 2, 3, 4, 5).

In the pituitary, the major product of the hGH-N gene is a single chain polypeptide with 191 amino acids and a molecular mass of approximately 22-kDa (6), which is an important regulator of postnatal growth and metabolism (7, 8). Approximately 10% of the hGH-N transcripts in the pituitary use an alternative splice acceptor site resulting in the 20-kDa hGH isoform of the hGH-N, in which amino acids 32–46 of the 22-kDa hGH-N are deleted (9). The 20-kDa hGH-N shows some differences regarding biological activities, binding affinity, and clearance from blood compared with 22-kDa hGH-N (9, 10, 11, 12, 13). However, both 22-kDa and 20-kDa hGH-N are capable of promoting growth (9, 14). The hGH-V gene encodes a 22-kDa hGH-V isoform, which differs from the 22-kDa hGH-N by 13 amino acids in positions dispersed throughout the mature secreted hormone (15). The 22-kDa hGH-V also undergoes glycosylation to yield a 25-kDa isoform (16, 17). The physiological role of 22-kDa hGH-V is unclear, but it progressively replaces 22-kDa hGH-N in the maternal circulation during pregnancy (18, 19). Placental and pituitary 22-kDa hGH show similar somatogenic activity, but the placental isoform has reduced lactogenic activity (20). In contrast with the hGH-N gene, the use of the alternative splice site generating a 20-kDa isoform has not been documented for the hGH-V gene (21, 22). Instead, a different splicing pathway in which intron D is retained in the processed messenger RNA (mRNA) has been identified, predicting a 230-amino acid protein called hGH-V2. This 26-kDa isoform of the hGH-V gene shows complete sequence divergence in the carboxy-terminus compared with 22-kDa hGH-V, and it is presumably a membrane-bound protein (23).

In this study, two new transcripts of the hGH-V gene are described. One of the transcripts uses the same alternative splice acceptor site within exon 3, as seen in the hGH-N gene (9), predicting a 20-kDa isoform of the hGH-V gene. The other transcript, which we refer to as hGH-V3, is generated by the use of an alternative splice donor site near the end of the fourth exon causing a 4-bp deletion. The hGH-V3 transcript predicts a 24-kDa protein with 219 amino acids, in which the first 124 amino acids are identical to those of placental 22-kDa hGH, whereas the remaining carboxy-terminal residues shows a complete sequence divergence.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 References
 
Cloning of GH-V complementary DNAs (cDNAs)

hGH-V cDNA was amplified by nested PCR using a human placental cDNA library as template. The outer primers were hGHV-S1 and hGHV-S2, and the inner primers were hGHV-S3 and hGHV-S4, as shown in Table 1Go. Amplification was done in 50 mmol/L KCl, 20 mmol/L Tris-HCl (pH 8.4), 1.5 mmol/L MgCl2, 200 µmol dNTPs, 50 pmol of each primer, and 2 U Taq polymerase in a total volume of 50 µL, using the GeneAmp PCR system 9600 (Perkin-Elmer/Cetus, Norwalk, CT). In the first PCR reaction using primers hGHV-S1 and hGHV-S2, denaturation at 95 C for 5 min was followed by 32 cycles of amplification with denaturation at 94 C for 1 min, annealing at 57 C for 1 min, and extension at 72 C for 1 min, with an extension time increase of 5 sec/cycle. The PCR products were diluted 1:1000, and 1 µL was used as template for amplification with primers hGHV-S3 and hGHV-S4, following the same PCR-profile, with the exception that the annealing temperature was increased to 62 C. The PCR products were separated on 1% agarose gels and purified using the Geneclean II kit (Bio 101, Vista, CA) before restriction analysis or subcloning into pCR-II (Invitrogen, San Diego, CA). Subcloned PCR products were sequenced using the PRISM Sequenase terminator single-stranded DNA sequencing kit (Applied Biosystems, Foster City, CA).


View this table:
[in this window]
[in a new window]
  		Table 1. Primer nomenclature and sequences

 
RNA preparation

Total RNA was isolated from pathological and normal placentas using RNA STAT-60 (Tel-Test B, Friendswood, TX), essentially as described by Chomczynski and Sacchi (24).

Amplification of cDNAs encoding hGH-V, hGH-N, and human chorionic somatomammotropin (hCS)-A/B

First-strand cDNA was generated using 2 µg total placental or pituitary RNA as template, in the presence of RT buffer [50 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 10 mmol/L MgCl2, 10 mmol/L dithiothreitol (DTT), and 0.5 mmol/L spermidine; Promega, Madison, WI], 1 µg random hexamers (Boehringer Mannheim, Mannheim, Germany), 15 U avian myeloblastosis virus reverse transcriptase (Promega), 20 U RNasin (Promega), and 3 µL deoxynucleotides (10 mmol/L of each deoxynucleotide triphosphate; Promega) in a final volume of 20 µL. The reaction was carried out at 42 C for 50 min and terminated at 70 C for 5 min.

Using pituitary or placental cDNA as template, PCR reactions were performed in a total volume of 50 µL using a 2-µL aliquot of the RT reactions, 100 mmol/L KCl, 20 mmol/L Tris-HCl (pH 8.4), 3 mmol/L MgCl2, 2 µL deoxynucleotides (2.5 mmol/L of each nucleotide triphosphate), 1.5 U Taq polymerase (Perkin-Elmer/Cetus), and 25 µmol of each primer (Scandinavian Gene Synthesis, Köping, Sweden) (Table 1Go). A schematic representation of the location of the primers used in the reactions is shown in Fig. 1Go. Sequences containing parts of 22-kDa hGH-V (125 bp), 20-kDa hGH-V (118 bp), or the full-length 22-kDa hGH-V (648 bp) were PCR amplified from human placental cDNA using a common forward primer (PO11) and three different reverse primers, the hGH-V-22K, located within exon 3, the hGH-V-20K, which was designed to bridge nucleotides encoding amino acids 31 and 47 of the hGH molecule, and the PO12, located in the 3'-untranslated region of the hGH-V mRNA (Fig. 1Go). hCS-A/B (388 bp) was amplified from human placenta cDNA using primers hCS1 and hCS2. hGH-N was amplified using primers FGH1A and FGH2B with human pituitary cDNA (Clontech, Palo Alto, CA) as template, resulting in a PCR product containing 573 bp. PCR amplifications were carried out using the GeneAmp PCR system 9600 (Perkin-Elmer/Cetus; 30 sec denaturation at 94 C, 15 sec annealing at 57 C, and 40 sec elongation at 72 C; 35 cycles). Aliquots of the amplified samples were separated on a 1% agarose gel, and the PCR products were purified using the the Geneclean II kit (Bio 101).



View larger version (41K):
[in this window]
[in a new window]
  		Figure 1. Nucleotide sequence of hGH-V cDNA. Nucleotides that differ from hGH-V sequence are shown for hGH-N and hCS-A cDNAs. Nucleotides underlined in hCS-A sequence indicate differences from that of hCS-B. Translated region of 22-kDa hGH-V mRNA is shown in triplets, with start and stop codons marked by circles. Bold type denotes codons for first and last amino acids of mature polypeptide. Arrows indicate locations of four introns. Region underlined in exon 3 shows 45-bp deletion observed in 20-kDa hGH. Double underlined sequence GTGG at end of exon 4 indicates bases deleted in hGH-V3 transcript. Note sequence GGGG in hGH-N and hCS-A/B transcripts. Locations of primers used in RT-PCR and competitive PCR (Table 1Go) are shown by boxes. Note that sequence of primer hGHV-20K starts at end of exon 2 and continues within exon 3, after 45-bp region deleted in 20-kDa hGH-V.

 
Ligase chain reaction (LCR)

LCR (25, 26) was used to characterize the transcripts of the hGH-V, hGH-N, and hCS-A/B genes. LCR was performed in a reaction mixture containing 20 mmol/L Tris-HCl (pH 7.5), 20 mmol/L KCl, 10 mmol/L MgCl2, 0.1% NP-40, 0.1 mmol/L ATP, 1 mmol/L DTT, 0.4 µg/µL sonicated salmon sperm DNA, 150 nmol/L of all four LCR primers (Genset, Paris, France; Table 1Go), and using 10–150 ng hGH-V, hGH-N, or hCS-A/B cDNAs as template. Previously, 30 pmol of primers hGH-V-20K-LCR B and hGH-V3-LCR B were end-labeled in a 20-µL reaction at 37 C for 1 h, consisting of 1.5 µM [{gamma}32P]ATP (Du Pont, Dreieich, Germany), 16 U of T4 polynucleotide kinase (Promega), 70 mM Tris-HCl (pH 7.6), 10 mM MgCl2, and 5 mM DTT. In the LCR, 4 U of Pyrococcus furiosus (Pfu) ligase (Stratagene, La Jolla, CA) was added to the reaction after an initial denaturation step for 4 min at 94 C. The reactions were then performed with 35 cycles for analysis of 20-kDa hGH-V (20 sec at 94 C; 50 sec at 48 C) and 25 cycles for analysis of hGH-V3 (20 sec at 94 C; 20 sec at 60 C) in the GeneAmp PCR system 9600 (Perkin-Elmer/Cetus). The reactions were stopped by adding loading dye containing formamide. The LCR products were separated on 6% polyacrylamide gels and analyzed by the Phosphor Imager (Molecular Dynamics; Sunnyvale, CA). The size of the LCR products was compared with radiolabeled size markers (Promega).

Competitive PCR assay

To estimate the relative proportions of the new transcripts in relation to the total hGH-V mRNA, competitive PCRs for 20-kDa hGH-V and hGH-V3 were performed using primers PO11/PO14 and GHV3A/GHV3B, respectively (Table 1Go and Fig. 1Go). The forward primers were end-labeled in a reaction similar to that described for the LCR primers. Competitive PCR was performed with placental cDNA as template (20 sec at 94 C, 15 sec at 57 C (20-kDa hGH-V) or 60 C (hGH-V3), and 30 sec at 72 C; 35 cycles). The PCR products were electrophoresed on 6% polyacrylamide gels, and the bands were visualized and quantified by the Phosphor Imager (Molecular Dynamics).

Hydrophilicity plots

The hydrophilicity plots (scale Kyte-Doolittle) of 22-kDa hGH-V, hGH-V2, and hGH-V3 were obtained using the software MacVector version 4.5 (Eastman Kodak, Rochester, NY).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 References
 
Identification of novel hGH splice variants in human placenta

A scheme of the splicing mechanisms of hGH-V gene is shown in the Fig. 2AGo. Sequencing of several clones obtained by PCR of the coding region of the hGH-V gene using human placental cDNA as template revealed two novel transcripts. One of the transcripts had a 45-bp deletion within exon 3, similar to that described in the hGH-N gene, predicting a 20-kDa isoform of hGH-V (GeneBank accession number: AF006060). Using RT-PCR and primers PO11 and hGHV-20K, which were designed for specific amplification of placental 20-kDa hGH-V, a DNA fragment of 118 bp was identified (data not shown). The other new transcript, designated hGH-V3, resulted from the use of an alternative splice donor site near the end of exon 4, causing a deletion of 4 bp (GTGG) upstream of the regular splice site (GeneBank accession number: AF006061). The hGH-V3 transcript is predicted to encode a 24-kDa protein with 219 amino acids (245 with the signal peptide) with a frameshift after residue 124 compared with placental 22-kDa hGH. This alternative splice donor site is not present in either hGH-N or hCS-A/B because of a nucleotide difference in the sequence of these genes (GGGG instead of GTGG) (Fig. 2BGo).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 2. Schematic representation of hGH-V gene and its alternative spliced products. A, hGH-V gene with its five exons (1 2 3 4 5 ) and four introns (A-D) is shown in upper part of diagram. Most common splicing mechanism of hGH-V primary transcript removes all intron sequences resulting in 22-kDa hGH-V mRNA. Twenty-kilodalton hGH-V mRNA is generated by use of an alternative splice site within exon 3, which eliminates codons for amino acids 32–46, similar to that described for hGH-N. In hGH-V2 mRNA, intron D is retained in processed mRNA, resulting in frameshift. hGH-V3 mRNA is produced by use of an alternative splice site near end of exon 4, predicting a protein with a unique carboxy-terminus. Right arrows in hGH-V2 and hGH-V3 transcripts denote sequence divergence carboxy-terminal to splice site. *, In hGH-V3 indicates sequence after exon 5 that is translated in this isoform. B, Characterization of hGH-V3 (left) and 20-kDa hGH (right) mRNA splicing of hGH-V gene. Consensus sequences for splice donor and acceptor sites are shown. Bold type indicates highly conserved bases. Predominant splicing site is indicated by solid lines, and alternative splicing sites is indicated by dotted lines. In hGH-V3 mRNA, alternative site is located only 4 bp upstream of normal splice donor site (GTGG, in italics). Note T to G transition present in hGH-N and hCS-A/B genes.

 
LCR was then used to analyze the use of the same splicing pattern in transcripts derived from the other genes of the hGH-gene cluster. The LCR products of 50 bp corresponding to transcripts encoding the 20-kDa isoforms of hGH-N and hGH-V were detected in hGH-N cDNA and in hGH-V cDNA, respectively, but not in hCS-A/B cDNA (Fig. 3Go). In addition, the 50-bp band corresponding to hGH-V3 transcript was only observed in placental cDNA from the hGH-V gene, but not in cDNAs derived from the other genes of the cluster (Fig. 3Go).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 3. LCR analysis of use of novel splice donor and acceptor sites mRNAs derived from hGH-gene cluster. Arrows indicate 50-bp LCR products and 25-bp unligated primers. A, Use of alternative splice acceptor site for generation of 20-kDa hGH transcripts: lane 1, template was 150 ng hCS-A/B cDNA; lane 2, 150 ng hGH-V cDNA; lane 3, 150 ng hGH-N cDNA; lane 4, control with no template; lane 5, control with all four primers; lane 6, control with labeled LCR-primer. B, Use of alternative splice donor site for generation of hGH-V3 transcript: lanes 1 and 2, templates were 10 and 50 ng hCS-A/B cDNA; lanes 3 and 4, 10 and 50 ng hGH-V cDNA; lanes 5 and 6, 10 and 50 ng hGH-N cDNA; lanes 7–9, control samples as shown in left panel.

 
Expression of new transcripts in human placenta

The expression of the new transcripts in relation to the total hGH-V mRNA was estimated by competitive PCR in four full-term normal placentas and three placentas from pregnancies with complications. A diagram showing the location of the primers used in the competitive PCRs is shown in Fig. 4AGo. The expression of hGH-V3 did not vary greatly in normal and pathological placentas, corresponding to approximately 10% of the hGH-V transcripts. The 20-kDa hGH-V mRNA was only detected in two of the four normal placentas, and a weak signal was observed in one of the abnormal placentas (Fig. 4BGo).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 4. Competitive RT-PCR for 20-kDa hGH-V and hGH-V3. A, Schematic illustration of hGH-V gene organization showing location of primers used in reactions for placental 20-kDa hGH (left) and hGH-V3 (right). Exons are shown as boxes and introns as horizontal lines. Vertical lines indicate alternative splice sites. Radiolabeled forward primers are shown by *. B, Lanes 1–4, normal full-term placentas; lane 5, gestational diabetes, full-term; lane 6, intrauterine growth retardation (IUGR), 35 weeks of gestation; lane 7, pregestational and insulin-treated diabetes + IUGR, full-term. In upper panel, 74- and 70-bp bands denote transcripts encoding 22-kDa hGH-V and hGH-V3, respectively, whereas in lower panel, 179- and 134-bp bands denote those for 22-kDa hGH-V and 20-kDa hGH-V, respectively.

 
Hydrophilicity of hGH-V3

The predicted proteins encoded by the hGH-V2 and hGH-V3 transcripts have distinct carboxy-termini compared with other hGH isoforms (Fig. 5Go). The hGH-V3 is a 24-kDa protein with 219 amino acids, in which the first 124 amino acids are identical to those of 22-kDa hGH-V, with a frameshift occurring in the remaining carboxy-terminal sequence. In this part of hGH-V3, there is no transmembrane domain, as predicted for the hGH-V2 (23) (Fig. 6Go).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 5. Predicted amino acid sequences of 22-kDa hGH-V, hGH-V2, and hGH-V3. *, Denote identical residues in hGH-V2 and hGH-V3 proteins compared with 22-kDa hGH-V. Underlined amino acids are those deleted in 20-kDa hGH-V.

 


View larger version (40K):
[in this window]
[in a new window]
  		Figure 6. Hydrophilicity plots of 22-kDa hGH-V, hGH-V2, and hGH-V3. Sequences include signal peptide (26 amino acids). Circle indicates predicted transmembrane domain of hGH-V2 that is not present in either placental 22-kDa hGH and hGH-V3.

 

   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
References
 
Sequence analysis of several hGH-V cDNA clones revealed the presence of two novel transcripts of this gene, in addition to those two previously described encoding 22-kDa hGH-V (27, 28) and hGH-V2 (23). One of the novel transcripts predicts a 20-kDa hGH-V isoform, generated through the use of an alternative splice site similar to that described for the hGH-N gene (9, 29, 30). The other novel transcript, hGH-V3, uses an alternative splice donor site near the end of exon 4 of the hGH-V gene, only 4 bp upstream of the regular splicing site. hGH-V3 mRNA is predicted to encode a 24-kDa protein with 219 amino acids and an unique carboxy-terminus. In our study, LCR products corresponding to 20-kDa hGH-V and hGH-V3 were detected by oligonucleotides that were correctly base-paired at the junction in both transcripts, using placental cDNAs as template. As expected, the 20-kDa hGH-transcript was also detected in cDNA derived from the hGH-N gene, but not in cDNA derived from the hCS-A/B genes. In contrast, the alternative splice pattern generating hGH-V3 mRNA was only observed in transcripts derived from the hGH-V gene.

Although the hGH-V gene is highly similar in structure to the hGH-N gene, processing of its primary transcript has not been shown to use the alternative acceptor site that generates pituitary 20-kDa hGH. Instead, transfection studies using genes encoding hGH-N, hGH-V, or chimeric genes, have identified striking differences between hGH-N and hGH-V genes regarding the use of this acceptor site (21, 31). Sequences in the immediate proximity of the alternative splice acceptor sites, as well as more remote sequences, seem to contribute to the splice site selection (22). Our results using placental cDNA as template show that primary transcripts derived from the hGH-V gene can be alternatively spliced within exon 3 in a manner similar to that observed for the hGH-N gene. The expression of the placental 20-kDa hGH transcript seems to vary among different full-term normal placentas and placentas with abnormalities. In fact, the transcript encoding 20-kDa hGH-V was not detected in all placentas. This variation in expression of the 20-kDa hGH-V transcript might partly explain the previous unsuccessfully attempts in detecting this transcript (21). Interestingly, altered hGH/hCS gene expression has been described in human trophoblastic neoplasms in comparison with normal placentas (32), and further studies are needed to address whether the expression of various transcripts differs under physiological or pathological conditions.

In contrast to the transcript encoding 20-kDa hGH-V, the hGH-V3 transcript seems to be expressed in similar amounts in different placentas, comprising approximately 10% of the total hGH-V transcripts. However, it is unclear whether the abundance of these transcripts reflects the production of the predicted proteins. The alternative splice site generating hGH-V3 is not present in the other genes of the cluster. This is probably because of a T to G transition observed in the splice donor site of the other genes that presumably prevents this splicing in the hGH-N and hCS-A/B genes. Interestingly, the predicted hGH-V3 protein exhibits a complete frameshift carboxy-terminal to the splice site, similar to what is observed in hGH-V2 (23) and bovine GH (33). However, in contrast to hGH-V2, there was no putative transmembrane domain in the carboxy-terminus of the predicted hGH-V3 protein as analyzed by hydrophilicity plots. The predicted hGH-V3 protein is a member of a group of GH-related proteins with distinct carboxy-terminal, which includes hGH-V2 and bovine GH (23, 33).

The interaction between hGH and its receptor is a sequential event, in which hGH initially binds to one hGH receptor (hGHR) molecule through a large contact surface area (site 1), involving amino acids of the carboxy-terminal part of the fourth helix bundle, to form an inactive 1:1 complex. This complex then associates with a second hGHR molecule via binding to site 2, which represents a smaller surface area on the N-terminal part of the hGH molecule (34, 35). The distinct carboxy-terminus in the predicted hGH-V2 and hGH-V3 proteins completely changes the amino acids in binding site 1. Thus, it remains to be elucidated if these new hGH isoforms are able to bind and activate the hGHR (36). Alternatively, it is possible that hGH-V3 and related molecules act through specific receptors, as suggested for other hGH and human PRL fragments (37, 38) and more recently, for 22-kDa hGH-V (39).

The role of placental hGH-V is unclear, but there are indications that it may participate in the regulation of maternal metabolism (40). The 22-kDa hGH-V levels are reduced in most cases of intrauterine growth retardation in comparison with normal pregnancies (41). Moreover, hGH-V2 is probably a membrane-bound protein, and its expression is controlled independently from the expression of 22-kDa hGH-V during gestation, supporting the idea that hGH-V and related peptides play a role in placental growth or function (42, 43). The results of the present study showing that the hGH-V primary transcript undergoes alternative splicing generating at least four different mRNAs, two of them predicting proteins with completely distinct carboxy-termini, add new complexity to the physiology of hGH during pregnancy.


   Footnotes
 
1 This study was supported by grants from the Swedish Medical Research Council (11576, 11331, 11285, and 11502). Back

Received March 10, 1998.

Revised April 29, 1998.

Accepted May 5, 1998.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 References
 

  1. Barsh GS, Seeburg PH, Gelinas RE. 1983 The human growth hormone gene family: structure and evolution of the chromosomal locus. Nucleic Acids Res. 11:3939–3958.[Abstract/Free Full Text]
  2. Chen EY, Liao Y-C, Smith DH, Barrera-Saldaña HA, Gelinas RE, Seeburg PH. 1989 The human growth hormone locus: nucleotide sequence, biology, and evolution. Genomics. 4:479–497.[CrossRef][Medline]
  3. Parks JS. 1989 Molecular biology of growth hormone. Acta Paediatr Scand Suppl. 349:127–135.[Medline]
  4. Lewis UJ. 1992 Growth hormone: what is it and what does it do? Trends Endocrinol Metab. 3:117–121.[CrossRef][Medline]
  5. Baumann G. 1991 Growth hormone heterogeneity: genes, isohormones, variants, and binding proteins. Endocr Rev. 12:424–449.[Abstract/Free Full Text]
  6. Li CH, Dixon JS. 1971 Human pituitary growth hormone. The primary structure of the hormone: revision. Arch Biochem Biophys. 146:233–236.[CrossRef][Medline]
  7. Isaksson OGP, Lindahl A, Nilson AL, Isgaard J. 1987 Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr Rev. 8:426–438.[Abstract/Free Full Text]
  8. Berneis K, Keller U. 1996 Metabolic actions of growth hormone: direct and indirect. Baillieres Clin Endocrinol Metab. 10:337–352.[CrossRef][Medline]
  9. Lewis UJ, Markoff E, Culler FL, Hayek A, Vanderlaan WP. 1987 Biologic properties of the 20k-dalton variant of human growth hormone: a review. Endocrinol Jpn. 34[Suppl 1]:73–85.
  10. McCarter J, Shaw MA, Winer LA, Baumann G. 1990 The 20,000 dalton variant of human growth hormone does not bind to growth hormone receptors in human liver. Mol Cell Endocrinol. 73:11–14.[CrossRef][Medline]
  11. Baumann G, Shaw MA. 1990 Plasma transport of the 20,000-dalton variant of human growth hormone (20K): evidence for a 20K-specific binding site. J Clin Endocrinol Metab. 71:1339–1343.[Abstract/Free Full Text]
  12. Baumann G, Stolar MW, Buchanan TA. 1985 Slow metabolic clearance rate of the 20,000-dalton variant of human growth hormone: implications for biological activity. Endocrinology. 117:1309–1313.[Abstract/Free Full Text]
  13. Wada M, Uchida H, Ikeda M, Naito N, Honjo M. 1997 Unique properties of authentic 20K human growth hormone in receptor dimerization and receptor-mediated cell proliferation. Proc. 79th Meeting of The Endocrine Society, 1997, Minneapolis, MN. p. 337.
  14. Stewart TA, Clift S, Pitts-Meek S, et al. 1992 An evaluation of the functions of the 22-kilodalton (kDa), the 20-kDa, and the n-terminal polypeptide forms of human growth hormone using transgenic mice. Endocrinology. 130:405–414.[Abstract/Free Full Text]
  15. Seeburg PH. 1982 The human growth hormone gene family: nucleotide sequences show recent divergence and predict a new polypeptide hormone. DNA. 1:239–249.[Medline]
  16. Ray J, Jones BK, Liebhaber SA, Cooke NE. 1989 Glycosylated human growth hormone variant. Endocrinology. 125:566–568.[Abstract/Free Full Text]
  17. Frankenne F, Scippo M, Van Beeumen J, Igout A, Hennen G. 1990 Identification of placental human growth hormone as the growth hormone-V gene expression product. J Clin Endocrinol Metab. 71:15–18.[Abstract/Free Full Text]
  18. Hennen G, Frankenne F, Closset J, Gomez F, Pirens G, El Khayat N. 1985 A human placental GH: increasing levels during second half of pregnancy with pituitary GH suppression as revealed by monoclonal antibody radioimmunoassays. Int J Fertil. 30:27–33.[Medline]
  19. Evain-Brion D, Alsat E, Igout A, Frankenne F, Hennen G. 1994 Placental growth hormone variant: assay and clinical aspects. Acta Paediatr Suppl. 399:49–51.[Medline]
  20. Igout A, Frankenne F, L’Hermite-Balériaux M, Martin A, Hennen G. 1995 Somatogenic and lactogenic activity of the recombinant 22 kDa isoform of human placental growth hormone. Growth Regul. 5:60–65.[Medline]
  21. Cooke NE, Ray J, Watson MA, Estes PA, Kuo BA, Liebhaber SA. 1988 Human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns. J Clin Invest. 82:270–275.
  22. Estes PA, Urbanek M, Ray J, Liebhaber SA, Cooke NE. 1994 Alternative spliced site selection in the human growth hormone gene transcript and synthesis of the 20 kDa isoform: role of higher order transcript structure. Acta Paediatr Suppl. 399:42–47.[Medline]
  23. Cooke NE, Ray J, Emery JG, Liebhaber SA. 1988 Two distinct species of human growth hormone-variant mRNA in the human placenta predict the expression of novel growth hormone proteins. J Biol Chem. 263:9001–9006.[Abstract/Free Full Text]
  24. Chomczynski P, Sacchi N. 1987 Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 162:156–159.[Medline]
  25. Landegren U, Kaiser R, Sanders J, Hood L. 1988 A ligase-mediated gene detection technique. Science. 241:1077–1080.[Abstract/Free Full Text]
  26. Barany F. 1991 The ligase chain reaction in a PCR world. PCR Methods Appl. 1:5–16.[Medline]
  27. Frankenne F, Closset J, Gomez F, Scippo ML, Smal J, Hennen G. 1988 The physiology of growth hormones in pregnant women and partial characterization of the placental GH variant. J Clin Endocrinol Metab. 66:1171–1180.[Abstract/Free Full Text]
  28. Igout A, Van Beeumen J, Frankenne F, Scippo ML, Devreese B, Hennen G. 1993 Purification and biochemical characterization of recombinant human placental growth hormone produced in Escherichia coli. Biochem J. 295:719–724.
  29. DeNoto FM, Moore DD, Goodman HM. 1981 Human growth hormone DNA sequence and mRNA structure: possible alternative splicing. Nucleic Acids Res. 9:3719–3730.[Abstract/Free Full Text]
  30. Lewis UJ, Bonewald LF, Lewis LJ. 1980 The 20,000 dalton variant of human growth hormone: location of the amino acid deletions. Biochem Biophys Res Commun. 92:511–516.[CrossRef][Medline]
  31. Estes PA, Cooke NE, Liebhaber SA. 1992 A native RNA secondary structure controls alternative splice-site selection and generates two human growth hormone isoforms. J Biol Chem. 267:14902–14908.[Abstract/Free Full Text]
  32. Lytras A, Bock ME, Dodd JG, Cattini PA. 1994 Detection of placental growth hormone variant and chorionic somatomammotropin ribonucleic acid expression in human trophoblastic neoplasms by reverse transcriptase-polymerase chain reaction. Endocrinology. 134:2461–2467.[Abstract/Free Full Text]
  33. Hampson RK, Rottman FM. 1987 Alternative processing of bovine growth hormone mRNA: nonsplicing of the final intron predicts a high molecular weight variant of bovine growth hormone. Proc Natl Acad Sci USA. 84:2673–2677.[Abstract/Free Full Text]
  34. de Vos A, Ultsch M, Kossiakoff AA. 1992 Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science. 255:306–312.[Abstract/Free Full Text]
  35. Clackson T, Wells JA. 1995 A hot spot of binding energy in a hormone-receptor interface. Science. 267:383–386.[Abstract/Free Full Text]
  36. Goffin V, Shiverick KT, Kelly PA, Martial JA. 1996 Sequence-function relationships within the expanding family of prolactin, growth hormone, placental lactogen, and related proteins in mammals. Endocr Rev. 17:385–410.[Abstract/Free Full Text]
  37. Clapp C, Weiner RI. 1992 A specific, high affinity, saturable binding site for the 16-kilodalton fragment of prolactin on capillary endothelial cells. Endocrinology. 130:1380–1386.[Abstract/Free Full Text]
  38. Rowlinson SW, Waters M, Lewis UJ, Barnard R. 1996 Human growth hormone fragments 1–43 and 44–191: in vitro somatogenic activity and receptor binding characteristics in human and nonprimate systems. Endocrinology. 137:90–95.[Abstract]
  39. Mélot F, Igout A, Hennen G. 1997 Demonstration of a specific binding site for placental growth hormone in the human placenta. Prog 79th Meeting of The Endocrine Society, 1997, Minneapolis, MN. p. 338.
  40. Anthony RV, Pratt SL, Liang R, Holland MD. 1995 Placental-fetal hormonal interactions: impact on fetal growth. J Anim Sci. 73:1861–1871.[Abstract]
  41. Evain-Brion D, Alsat E, Igout A, Frankenne F, Hennen G. 1994 Placental growth hormone variant: assay and clinical aspects. Acta Paediatr. 399:49–51.
  42. Hill DJ. 1992 What is the role of growth hormone and related peptides in implantation and the development of the embryo and fetus. Horm Res. 38[Suppl 1]:28–34.
  43. MacLeod JN, Lee AK, Liebhaber SA, Cooke NE. 1992 Developmental control and alternative splicing of the placentally expressed transcripts from the human growth hormone gene cluster. J Biol Chem. 267:14219–14226.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. H. Vickers, S. Gilmour, A. Gertler, B. H. Breier, K. Tunny, M. J. Waters, and P. D. Gluckman
20-kDa placental hGH-V has diminished diabetogenic and lactogenic activities compared with 22-kDa hGH-N while retaining antilipogenic activity
Am J Physiol Endocrinol Metab, September 1, 2009; 297(3): E629 - E637.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M.-L. Baudet, B. Martin, Z. Hassanali, E. Parker, E. J. Sanders, and S. Harvey
Expression, Translation, and Localization of a Novel, Small Growth Hormone Variant
Endocrinology, January 1, 2007; 148(1): 103 - 115.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
K.-C. Leung, G. Johannsson, G. M. Leong, and K. K. Y. Ho
Estrogen Regulation of Growth Hormone Action
Endocr. Rev., October 1, 2004; 25(5): 693 - 721.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
D. Le Roith, C. Bondy, S. Yakar, J.-L. Liu, and A. Butler
The Somatomedin Hypothesis: 2001
Endocr. Rev., February 1, 2001; 22(1): 53 - 74.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boguszewski, C. L.
Right arrow Articles by Carlsson, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Boguszewski, C. L.
Right arrow Articles by Carlsson, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals