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Endocrinology Vol. 139, No. 3 949-954
Copyright © 1998 by The Endocrine Society

ARTICLES

Molecular Cloning and Characterization of Xenopus Insulin-Like Growth Factor-1 Receptor: Its Role in Mediating Insulin-Induced Xenopus Oocyte Maturation and Expression during Embryogenesis1

Li Zhu, Nicholas Ohan, Yehenew Agazie, Cathy Cummings, Sahar Farah and X. Johné Liu2

Ottawa Civic Hospital Loeb Research Institute, Ottawa Civic Hospital; and Department of Biochemistry (S.F., X.J.L.) and Department of Obstetrics & Gynaecology (X.J.L.), University of Ottawa, Ottawa, K1Y 4E9, Canada

Address all correspondence and requests for reprints to: Dr. Johné Liu, Ottawa Civic Hospital Loeb Research Institute, Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa, K1Y 4E9, Canada. E-mail: johne{at}civich.ottawa.on.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have cloned a complementary DNA encoding the putative Xenopus insulin-like growth factor-1 (xIGF-1) receptor. Injection of messenger RNA derived from the cloned complementary DNA into Xenopus oocytes resulted in the expression and correct processing of the receptor’s {alpha}- and ß-subunits. Using antibodies generated against protein expressed against the cloned sequence, we demonstrated that the endogenous xIGF-1 receptor in Xenopus oocytes was activated by nanomolar concentrations of mammalian IGF-1 and by insulin approximately 100-fold higher in concentration. This receptor activation profile correlated with hormone-induced Xenopus oocyte maturation. Furthermore, injection of a neutralizing antiinsulin receptor antibody into Xenopus oocytes inhibited hormone-induced xIGF-1 receptor activation. These results provide molecular and biochemical evidence supporting a role for xIGF-1 receptor in mediating insulin/IGF-1-induced Xenopus oocyte maturation. We also report here that embryonic transcription of xIGF-1 receptor is activated during the formation of the central nervous system in early Xenopus embryos.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FULLY GROWN Xenopus laevis oocytes are physiologically arrested at the prophase of meiosis I. These oocytes must resume meiosis and proceed to the metaphase of meiosis II before fertilization is possible. This process, termed oocyte maturation, is triggered in vivo by a preovulatory gonadotropin surge followed by follicular production of progesterone (1, 2). In addition to progesterone, both insulin and insulin like-growth factor-1 (IGF-1) can induce oocyte maturation (3, 4). Insulin/IGF-1-induced oocyte maturation is mediated by an oocyte receptor that is likely the Xenopus homolog of IGF-1 receptor. This is suggested by the fact that IGF-1 is much more potent than insulin in inducing oocyte maturation (4, 5) and other oocyte responses, including tyrosine phosphorylation (presumably autophosphorylation) of two closely related polypeptides, presumed to be Xenopus IGF-1 (xIGF-1) receptor (6, 7, 8, 9); ligand binding studies also have indicated that oocytes possess high affinity binding sites to IGF-1 (6, 7) but not to insulin (6). Despite the circumstantial evidence, the xIGF-1 receptor protein has not been directly identified in Xenopus oocytes.

Partial complementary DNAs (cDNAs) corresponding to the Xenopus IGF-1 receptor have been identified by RT-PCR (10) and more recently by cDNA library screening (11). Both ligand binding and RT-PCR studies have suggested that the xIGF-1 receptor is expressed in oocytes and throughout early embryogenesis; however, it is not clear whether the IGF-1 binding sites or the xIGF-1 receptor messenger RNA (mRNA) detected throughout early embryonic development was derived from maternal sources or newly synthesized by the developing embryos. Here, we report the molecular cloning of the entire coding sequence for the xIGF-1 receptor and, as well, the biochemical characterization of the xIGF-1 receptor during insulin/IGF-1-induced oocyte maturation. We also demonstrate that the xIGF-1 receptor mRNA present in early embryos was largely derived from embryonic transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of xIGF-1 receptor cDNA and in vitro transcription of the receptor messenger RNA (mRNA)
Standard molecular cloning protocols (12) were followed. A 450-bp PCR insert corresponding to part of the xIGF-1 receptor kinase domain (10) was used as a probe to screen a Xenopus oocyte cDNA library (13). Positive clones were purified and subcloned into pBluescript, and their nucleotide sequence was determined using a semiautomatic sequencer (ABI, Foster City, CA). Any sequence ambiguity was corrected/confirmed by resequencing from the opposite direction.

xIGF-1 receptor subclones R10–1 and R3–6 were ligated together at a PmlI site (at nucleotide 510, as shown in Fig. 1BGo). The ligated clone (with EcoRI sites at both ends) was then cloned into pCS2+ (14), which also had been digested with EcoRI at the polylinker. The plasmid was then linearized with NotI for in vitro synthesis of mRNA using the SP6 Message Machine kit from Ambion (Austin, TX). mRNA was dissolved in water to a final concentration of 1 mg/ml, estimated by comparison in gel electrophoresis to RNA standards of known concentrations (Gibco-BRL, Toronto, Canada). A typical reaction with 1 µg DNA template produced 20 µg mRNA.



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  		Figure 1. Cloning of the xIGF-1 receptor. A, Schematic representation of the various clones used in this study and their comparison with the human IGF-1 receptor (hIGF-1R). Homologies are shown as percent identity in predicted amino acid sequence, except for that between R3–6 and the Xenopus sequence reported by Groigno et al. (11), which is shown as percent identity in nucleotide sequence because it covers the 3' untranslated region (thinner line). TM, transmembrane domain; NPEY, autophosphorylation site responsible for IRS-1 binding; PTK, protein tyrosine kinase domain. Also shown are the proteolytic processing site (RKRR) in the hIGF-1 receptor (to generate the {alpha}- and ß-subunits) and the putative processing site (RRRR) for the xIGF-1 receptor. B, Nucleotide and amino acid (in one-letter code) sequences of the xIGF-1 receptor. The first 350 bases are from clone R10–1, and the remaining ones are from clone R3–6. The EcoRI adaptor sequence derived from the {lambda}gt10 vector is included at both ends. The putative proteolytic processing site is indicated. C, Oocytes injected with water or xIGF-1 receptor cRNA were metabolically labeled with 35S methionine. Immunoprecipitates, using an anti-xIGF-1 receptor antibody, were analyzed by SDS-PAGE and autoradiography. The presumptive {alpha}- and ß-subunits of the xIGF-1 receptor are indicated. The high-molecular-mass band likely represents incompletely processed (uncleaved) receptor (see text).

 
Xenopus oocytes and embryos
All procedures involving live oocytes and embryos were carried out in a room maintained at 18 C, unless otherwise stated. Sexually mature male and female Xenopus laevis were purchased from NASCO (Fort Atkinson, WI) and maintained according to local animal care guidelines. Female frogs were injected with PMS gonadotropin (PMSG, Sigma, Toronto, Canada; 50 IU/frog) 3–10 days before oocyte retrieval. A fragment of ovary was removed surgically under hypothermia. Stage VI oocytes (15) were manually defolliculated according to the procedure described by Smith (16). Ten nanograms (in 10 nl) mRNA was injected per oocyte. Microinjection of oocytes was performed in oocyte incubation medium OR2 (82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na2HPO4, 5 mM HEPES, pH 7.8) lacking CaCl2. Injected oocytes were incubated in OR2 (containing CaCl2) for 6 h to overnight before the addition of hormones. For metabolic labeling, the injected oocytes were immediately placed in OR2 containing 35S-methionine (75 µCi/ml). Labeling was carried out at room temperature for 5 h to overnight. To assay for meiotic maturation, the oocytes were incubated overnight in K+-free OR2 (9, 17) containing the indicated concentrations of insulin (from porcine pancreas, Sigma) or IGF-1 (human recombinant, Boehringer Mannheim, Laval, Canada). Germinal vesicle breakdown (GVBD) was determined by the appearance of a white spot at the center of the animal hemisphere and confirmed, when in doubt, by bisecting the oocytes after fixation in 5% trichloroacetic acid and observing the presence (GVBD negative) or absence (GVBD positive) of a germinal vesicle.

Culturing embryos and related methods are according to Sive et al. (18), as described below. Frogs were injected with human CG (hCG, 1000 U per frog) and kept overnight at 18 C to induce ovulation. Eggs were collected in MBS (5 mM HEPES, pH 7.8, 88 mM NaCl, 1 mM KCl, 0.7 mM CaCl2, 1 mM MgSO4, 2.5 mM NaHCO3) plus an additional 20 mM NaCl (high salt-MBS). To fertilize eggs, excess high salt-MBS was removed and a freshly pinched testis was rubbed over the eggs. Immediately after, an excess volume of 0.1x MBS was added to the eggs. After 30 min, fertilized eggs were treated with 2% cysteine (pH 8) for 2–5 min until the jelly coat was removed. Dejellied embryos were rinsed with an excess volume of 0.1x MBS and incubated in 0.1x MBS to various stages (19) before being lysed for RNA isolation using RNAzol (Tel-Test, Inc.), according to the manufacturer’s instructions (Friendswood, TX).

Antibodies against xIGF-1 receptor
The cDNA sequence encoding amino acids 969-1358, which corresponds to the cytoplasmic region of the receptor, was amplified by PCR using the following primers: 5' primer = TAT GGA TCC CTA TGC CTT CTG TGA ACC C; 3' primer = TAT GAA TTC ACT GAT ACA GCG GGG. The amplified cDNA was digested with BamHI and EcoRI, two sites incorporated into the PCR primers (underlined) before being ligated to pGEX-KT (20) that had been similarly digested. The production of the GST fusion protein was induced in bacteria and purified by binding to agarose-immobilized glutathione, as previously described (21). Production of polyclonal antibodies in rabbits was carried out according to standard protocol (22). xIGF-1 receptor-specific IgG was affinity-purified by binding to the GST fusion protein covalently coupled to Sepharose beads.

Immunoprecipitation and Western blotting
Oocytes were lysed by forcing them through pipette tips in PBS lysis buffer (10 mM sodium phosphate, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 µg/ml each of leupeptin and aprotinin, 1 mM phenylmethylsulfonate, and 1 mM sodium orthovanadate; 10 µl lysis buffer/oocyte). The homogenate was centrifuged in an Eppendorf centrifuge for 15 min at 4 C. Under these conditions, the yolk protein (vitellogenin) was not solubilized and was discarded as a pellet. For immunoprecipitation, the homogenate (containing 2 mg total oocyte protein, as determined by protein assays using a Bio-Rad protein assay kit, usually corresponding to the amount derived from 20 oocytes), was incubated with 5 µg affinity-purified anti-xIGF-1 receptor antibodies and protein A Sepharose at 4 C for 2 h. The beads were washed three times with lysis buffer before analysis by SDS-PAGE, followed by Western blotting. The primary antibodies (pY20 from Transduction Laboratory, Lexington, KY, or anti-xIGF-1 receptor) were used at a concentration of 1 µg/ml, and the blots were developed with ECL reagents (Amersham, Toronto, Canada). If reprobing with a different antibody was required, the blots were stripped of the primary and secondary antibodies according to the protocol provided with the ECL kit.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning and expression of xIGF-1 receptors
An RT-PCR fragment corresponding to part of the kinase domain of the xIGF-1 receptor (10) was used as a probe to clone the xIGF-1 receptor cDNA. Initial screening of an oocyte cDNA library identified multiple clones, the longest of which (R3–6) was subcloned and sequenced. A comparison of the cloned sequence to that of the human IGF-1 receptor (23) revealed that R3–6 contained the entire ß-subunit of the receptor and part of the {alpha}-subunit but lacked the amino terminal 80 or so amino acids including the signal peptide. The nucleotide sequence of R3–6 was more than 99% identical to the partial xIGF-1 receptor cDNA sequence determined by Groigno et al. (11), over the entire reported sequence (>3 kb), including the 3' untranslated region. Therefore, the differences likely represented polymorphic variation and/or sequencing errors. Further screening of the same library with a probe corresponding to the 5' 500 bp of R3–6 resulted in the isolation of several clones of similar sizes (about 2 kB). Sequence analyses of one of these clones (R10–1) revealed that it contained the nucleotide sequence encoding the missing amino-terminal region of the xIGF-1 receptor. A comparison of the overlapping sequences of R10–1 and R3–6 revealed a 5% variation between the two clones (Fig. 1AGo). This sequence variation was confirmed by sequencing both R10–1 and the corresponding region of R3–6 from both directions. Sequence analyses of two other clones indicated that they were identical to clone R10–1. Thus, the assembled xIGF-1 receptor cDNA sequence presented here was largely derived from clone R3–6, with the first 350 nucleotides being added from clone R10–1. The predicted amino acid sequence of the xIGF-1 receptor (Fig. 1BGo) was 76% and 57% identical to that of the human IGF-1 receptor and the human insulin receptor, respectively. The highest homology was in the tyrosine kinase domain (90% and 81%, respectively). Its putative ß-subunit contained all the functionally important elements identified in the mammalian insulin receptor and IGF-1 receptor, including the putative IRS-1-binding site (NPEY976) and all three catalytically important autophosphorylation sites within the kinase domain Y1157ETDY1161Y1162. One notable change was the presence of RRRR at the putative cleavage site (to generate {alpha}- and ß-subunits) instead of the RKRR sequence found in both the human IGF-1 receptor and human insulin receptor. Interestingly, the corresponding region of the Drosophila insulin receptor contains an RRRR sequence (24). Not surprisingly, its putative {alpha}-subunit is highly similar to that of the human IGF-1 receptor (75% identical), given that mammalian IGF-I binds and activates xIGF-1 receptor at very low concentrations.

The entire xIGF-1 receptor coding region was subcloned into pCS2+ and transcribed in vitro using SP6 polymerase. The in vitro synthesized mRNA was injected into Xenopus oocytes, which were then metabolically labeled with 35S-methionine. An immunoprecipitation experiment, using xIGF-1 receptor-specific polyclonal antibodies (Materials and Methods), precipitated two polypeptides of approximately 95 kDa and approximately 130 kDa, presumably representing the {alpha}- and ß-subunits, respectively, of the xIGF-1 receptor. In addition, a high-molecular-mass (>200 kDa) protein was also precipitated. In fact, it was usually the most prominent species of the three. This larger protein likely represented the unprocessed precursor protein, although it seemed to have been glycosylated, because, like the processed receptor ({alpha}- and ß-subunits), it was precipitable with wheat germ agglutinin (not shown).

Characterization of xIGF-1 receptor in Xenopus oocytes
To test whether tyrosine (auto)phosphorylation of the endogenous xIGF-1 receptor correlates with hormone-induced GVBD, we incubated oocytes with various concentrations of insulin or IGF-1. Oocytes were either stimulated for 10 min, followed by analysis of the autophosphorylation of endogenous xIGF-1 receptor and tyrosine phosphorylation of other cellular proteins, or incubated with the hormones overnight followed by GVBD scoring. Maximum tyrosine phosphorylation of the previously identified 95-kDa protein doublet (6, 9, 17) was achieved in the presence of 1 µM insulin or at a much lower concentration (10 nM) of IGF-1 (Fig. 2Go). The maximum hormone-induced tyrosine phosphorylation of the endogenous xIGF-1 receptor ß-subunit correlated well with the maximum hormone-induced oocyte maturation (Fig. 2BGo). No other tyrosine phosphorylated protein [perhaps most strikingly, no phosphorylated protein resembling xIRS-1 (21)] could be detected (see upper panel of Fig. 2AGo).



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  		Figure 2. Insulin/IGF-1-induced oocyte maturation correlates with activation of endogenous xIGF-1 receptor. A, Groups of 20 or more oocytes were incubated for 10 min with K+-free OR2 containing the indicated concentrations of insulin or IGF-1. Total lysates (100 µg of proteins, upper panel) or anti-xIGF-1 receptor antibody immunoprecipitates (lower panel) were analyzed by SDS-PAGE, followed by Western blotting using antibodies against phosphotyrosine (PY20), followed by ECL detection. The phosphorylated doublets (more obvious in the upper panel) are indicated. B, Groups of at least 30 or more oocytes were incubated overnight with the indicated concentrations of insulin or IGF-1 before GVBD scoring. Data from both A and B were from the same experiment and were representative of several similar experiments.

 
Previous studies have shown that a neutralizing antihuman insulin receptor antibody (17A3) inhibits insulin/IGF-1-induced Xenopus oocyte maturation, presumably by blocking the function of the endogenous IGF-1 receptor (6, 9, 25). To test this directly, we injected 17A3 into oocytes and stimulated the injected oocytes with insulin. Fig. 3Go shows that injection of 17A3, but not of control mouse IgG, significantly reduced insulin-induced tyrosine phosphorylation of the endogenous xIGF-1 receptor. It should be pointed out that the anti-xIGF-1 receptor antibodies generated against the cytoplasmic portion of the receptor (Materials and Methods) did not inhibit insulin-induced GVBD, nor did they inhibit insulin-induced autophosphorylation of the endogenous xIGF-1 receptors (not shown).



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  		Figure 3. A neutralizing antiinsulin receptor antibody (17A3) inhibits autophosphorylation of the xIGF-1 receptor. Groups of 20 or more oocytes were left uninjected (control) or injected with nonspecific mouse IgG (10 ng/oocyte) or with 17A3 (10 ng/oocyte). Injected oocytes were incubated in OR2 for 2 h before the addition of insulin (5 µM). Oocytes were lysed 10 min after the addition of insulin. Immunoprecipitation with antibodies against the xIGF-1 receptor and Western blotting with pY20 were as described in the legend to Fig. 2AGo. The blot was stripped and reprobed with antibodies against the xIGF-1 receptor (B).

 
Expression of xIGF-1 receptor during early embryogenesis
Previous RT-PCR studies have suggested that xIGF-1 receptor mRNA is present in the eggs and throughout early embryogenesis (10, 11). It is not clear whether the mRNA detected after the midblastula transition (MBT, the point at which embryonic transcription is activated in Xenopus laevis) was from a maternal source or derived from embryonic transcription. To address this, we employed the more quantitative technique of Northern blot analysis. Figure 4Go shows that although xIGF-1 receptor mRNA (larger than 9 kB) was readily detectable in fertilized eggs and throughout MBT, it was almost undetectable at midgastrula. The mRNA was again readily detectable at early neurula, and the level remain unchanged throughout early embryogenesis. This biphasic pattern of xIGF-1 receptor mRNA accumulation suggests the turnover of maternal mRNA, followed by the subsequent activation of the zygotic genome and embryonic transcription of xIGF-1 receptor mRNA. A duplicate gel was stained with ethidium bromide to reveal the presence of the 18S and 28S ribosomal RNA. Densitometric analysis of the stained gel confirmed that the amounts of 18S or 28S RNA were within 80% of each other (not shown).



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  		Figure 4. Expression of embryonic xIGF-1 receptor. Total RNA (10 µg each lane) from eggs and embryos at various stages were analyzed by Northern blotting using a probe corresponding to nucleotides 350-2355 of the xIGF-1 receptor (upper panel). A duplicate gel was stained with ethidium bromide (bottom panel).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have isolated the nucleotide sequence of the coding region of Xenopus IGF-1 receptor. The sequence variation between clone R10–1 and clone R3–6 (5% over more than 500 amino acids) suggests that they represent two nonallelic xIGF-1 receptors rather than polymorphic differences of the same locus. Xenopus laevis has a pseudotetraploid genetic makeup (26), and many genes are present in nonallelic duplicates, including, for example, the proinsulin genes (27) and the insulin receptor genes (10). However, our Northern blot analyses, using R3–6 (Fig. 4Go) and R10–1 (not shown), revealed only one (and the same) prominent message band in eggs and in early embryos. Therefore, either the two genes encode xIGF-1 receptors of indistinguishable sizes or one of them is expressed at a much lower (and therefore, undetectable) level. An attempt to clarify the issue of gene duplication by Southern blotting did not produce conclusive results because of the lack of knowledge about the intron/exon structure of the xIGF-1 receptor genes (not shown). Although R10–1 and R3–6 likely represent two nonallelic genes, reconstitution of the entire coding region of the xIGF-1 receptor seems to have generated a functional xIGF-1 receptor in oocytes injected with xIGF-1 receptor mRNA, as suggested by the production of two polypeptides of sizes consistent with being the receptor’s {alpha}- and ß-subunits.

Many previous studies have suggested that the Xenopus IGF-1 receptor is the mediator of insulin and IGF-1 responses in Xenopus oocytes (4, 6, 9). We have now provided molecular cloning and biochemical evidence supporting this notion. First, antibodies generated against a recombinant xIGF-1 receptor protein (produced as a GST fusion protein) detected a receptor doublet previously identified by antibodies against phosphotyrosine, or by autophosphorylation assays. Second, tyrosine phosphorylation of the doublet was induced by low nanomolar concentrations of IGF-1 and by insulin at about 100-fold higher concentrations. Importantly, the hormone concentrations required for xIGF-1 receptor phosphorylation correspond precisely with those required for activation of oocyte maturation. Third, we provide direct biochemical evidence that insulin/IGF-1-induced tyrosine phosphorylation of the xIGF-1 receptor doublet was significantly reduced by injection of the 17A3 antibody, which has been previously shown to inhibit insulin/IGF-1-induced oocyte maturation (9, 25, 28).

However, the identity of the receptor doublet remains unresolved, because there was no evidence that the cloned cDNA produced a similar protein doublet (Fig. 1CGo and data not shown). One intriguing possibility is that the receptor doublet corresponds to the two nonallelic xIGF-1 receptor genes (R10–1 and R3–6), although the confirmation of this must await the cloning of full-length cDNAs for both genes. We consider it unlikely that one band in the receptor doublet represents the ß-subunit of Xenopus insulin receptor, because tyrosine phosphorylation of both bands followed the same kinetics in response to varying concentrations of insulin and IGF-1.

Finally, the biphasic expression of xIGF-1 receptor suggests that, like in mammals (29, 30), the xIGF-1 receptor may play an important role in early embryonic development in Xenopus laevis. The activation of embryonic transcription of the xIGF-1 receptor during the early neurula stages further suggests that the xIGF-1 receptor may be involved in the development of the central nervous system, a notion that is under investigation with the availability of the cloned xIGF-1 receptor cDNA.


   Acknowledgments
 
We thank A. R. Shuldiner for the RT-PCR fragment of the xIGF-1 receptor, R. A. Roth for the 17A3 antiinsulin receptor antibody, and D. L. Turner for the pCS2+ vector.


   Footnotes
 
1 This study was supported by grants from the National Cancer Institute of Canada and Cancer Research Society (to X.J.L). Back

2 A scholar of the Medical Research Council of Canada. Back

Received August 27, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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