help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
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 García-de Lacoba, M.
Right arrow Articles by de Pablo, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by García-de Lacoba, M.
Right arrow Articles by de Pablo, F.
Endocrinology Vol. 140, No. 1 233-243
Copyright © 1999 by The Endocrine Society

ARTICLES

Insulin/Insulin-Like Growth Factor-I Hybrid Receptors with High Affinity for Insulin Are Developmentally Regulated during Neurogenesis1

Mario García-de Lacoba, Cristina Alarcón, Enrique J. de la Rosa and Flora de Pablo

Department of Cell and Developmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, E-28006 Madrid, Spain

Address all correspondence and requests for reprints to: Flora de Pablo, Department of Cell and Developmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, E-Madrid, Spain. E-mail: cibfp1f{at}fresno.csic.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The extensive colocalization of insulin receptor (IR) and insulin-like growth factor-I receptor (IGFR) messenger RNAs during central nervous system development, together with the effects of insulin and IGF-I in neurogenesis, raises the question of how stage- and factor-specific signaling occurs. Thus, it is necessary to characterize the receptor proteins present in vivo to start addressing this issue. Here we have studied the chick embryonic neuroretina at day 6 (E6), when it is predominantly proliferative, and at E12, when neuronal differentiation is advanced. Developmentally regulated high-affinity binding sites for both insulin and IGF-I were detected at E6 and E12. In proliferative neuroretina, typical IGFR with the highest affinity for IGF-I coexisted with separate atypical insulin binding sites, which had similar high affinity for insulin and IGF-I. Immunoprecipitation of ligand-cross-linked receptors with specific antibodies for the IR {alpha}-subunit, the IR ß-subunit, or the IGFR ß-subunit demonstrated the presence of IR/IGFR hybrids. They were more abundant in E6 than in E12 retina. These hybrid receptors bound most of radiolabeled insulin, but little radiolabeled IGF-I, at tracer concentrations. At E12, the specificity of the insulin binding sites changed, and it was closer to that found with IR in liver, where hybrids were undetectable. The basal autophosphorylation level of these atypical hybrid receptors was high, although insulin and, even more so, IGF-I modestly increased the phosphorylation of two IR ß-subunits of 95 and 105 kDa. The high-affinity/low-discriminative IR/IGFR hybrids predominantly found in a proliferative stage of neurogenesis can mediate the effects of proinsulin and insulin, previously demonstrated in organoculture at this stage. More importantly, this hybrid receptor may be physiologically relevant for the action of the locally produced proinsulin found in early neurogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN receptor (IR) and insulin-like growth factor receptor (IGFR) messenger RNAs (mRNAs), as well as their corresponding proteins, are widely expressed throughout the developing central nervous system (CNS) (reviewed in Ref. 1). The characteristics of both receptors have been studied mostly in cell culture, which may poorly reflect the in vivo regulation of receptor proteins (2, 3, 4). Here we study the chick embryo neuroretina, a well characterized model of vertebrate CNS development, at two representative stages, embryonic day 6 (E6) and embryonic day 12 (E12). Since the developing neuroretina expresses both proinsulin mRNA and IGF-I mRNA (5), it represents an autocrine/paracrine system suitable for the study of the coordinated actions of (pro)insulin and IGF-I in CNS development (1). At E6, the neuroretina consists of more than 90% proliferating neuroepithelial cells that express abundant IR and IGFR mRNAs, demonstrated by RT-PCR (5) and in situ hybridization (6). In a whole E6 retina organoculture system, IGF-I, insulin, and proinsulin stimulated cell proliferation and differentiation at similar low concentrations (5). Further, these three peptides protected the neuroepithelial cells from apoptosis with nearly equal potency (7). At E12, most retinal neural precursors have withdrawn from the cell cycle and have differentiated as either one of the five types of neurons in the chick retina or as Müller glial cells (8). Similar to earlier stages, there are widespread IR and IGFR mRNAs in the E12 retina (6, 9). This wide and relatively unchanged distribution of receptor mRNAs expression, observed with semiquantitative RT- PCR and in situ hybridization, suggests that part of the developmental regulation of central nervous system (CNS) ligand-binding sites found previously (10, 11) could be explained by posttranscriptional control.

The canonical IR and IGFR are disulfide-linked homodimeric glycoproteins composed of disulfide-linked {alpha}-ß monomers that form the ß{alpha}{alpha}ß quaternary structure in the mature complex. The extracellular {alpha}-subunit contains the ligand binding domain, whereas the ß-subunit bears the ligand-sensitive tyrosine kinase activity in the cytosolic domain (12, 13). Before the main proreceptor posttranslational modifications take place, i.e. N-glycosylation and proteolytic processing, the IR acquires ligand binding capacity (14). The fully processed typical receptors assemble in homodimers that have the highest affinity for the homologous ligand. The {alpha}-ß monomers can also assemble in heterodimers, IR/IGFR hybrids, as initially reported in human placenta, and in fibroblast and hepatocyte cell lines (15, 16). Hyperexpression studies in transfected cells allowed further characterization of the formation of hybrid receptors depending on their relative mRNA concentration (17). In these systems, the general conclusion was that IR/IGFR hybrids behaved mostly as typical IGFR in terms of relative affinities for the ligands: the binding of IGF-I was similar to that found for IGFR, whereas the affinity for insulin was low. Whether this is the behavior of hybrid receptors in embryonic tissues is not known. The biochemical characterization has been hampered by the very low number of receptors present in nontransfected cells and the small amount of embryo tissues. Hybrid receptors may be a significant fraction of total insulin/IGF-I binding sites, even in adult tissues, as reported in rabbit where more than 50% of the IGF-I binding was to hybrid receptors in brain, kidney, adipose tissue, muscle, and heart (18). Similar results were obtained more recently in human tissues (19).

In addition to the typical and hybrid receptors, another subpopulation of atypical receptors coexists in human placenta and lymphocytes (18). These binding sites are immunologically similar to IR but have unusual high affinity for IGF-I (20). Finally, further heterogeneity of this family of receptors affects the ß-subunit of the IGFR, with several tissue-specific variants so far described. In fetal rat muscle, two ß-subunit forms of 105 kDa (fetal form) and 95 kDa (adult form) have been documented (21, 22). In rat CNS, a variant ß-subunit has been found in growth cones that comigrates in bidimensional gels with the ß-subunit of the IGF-I receptor but is immunologically neither an insulin nor an IGF-I typical receptor (23). Clearly, more studies in primary tissues well characterized in their cellular and developmental aspects are needed to eventually understand the role of the IR and IGFR multiple forms. Here we show, in proliferative stages of neurogenesis, the presence of hybrid receptors with the unexpected characteristic of binding insulin with high affinity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of tissue and crude cell membranes
Fertilized white Leghorn eggs (Avícola Rodríguez-Serrano, Sala-manca, Spain) were incubated at 38.2 C and 60–80% relative humidity. Neural retina (free of connective tissue, pigmented epithelium, and the other surrounding eye structures) and liver were carefully microdissected on ice from chicken embryos at selected days, pooled, immediately frozen on dry-ice, and stored at -70 C. Crude cell membranes were obtained by differential centrifugation, as previously described (10, 24). Briefly, pooled tissues were homogenized with a hand-held ground glass homogenizer in ice-cold 1 mM NaHCO3 buffer, pH 7.8, containing 2 mM phenylmethylsulfonyl fluoride and 10 µg/ml leupeptin. The homogenate was centrifuged at 600 x g for 15 min at 4 C, and the pellet was discarded. The supernatant was centrifuged at 20,000 x g for 30 min at 4 C. The resulting pellet was resuspended in fresh buffer and recentrifuged at 20,000 x g. Finally, the high-speed supernatant was discarded and the pellet was resuspended in HEPES buffer (50 mM HEPES, pH 7.8, 120 mM NaCl, 15 mM sodium acetate, 10 mM glucose, 2.5 mM KCl, 1.2 mM MgSO4, and 1 mM EDTA) and stored at -70 C until assay utilization. The protein content of the membrane preparations was measured by the method of Bradford (25) using a commercial assay (Bio-Rad Laboratories, Inc., Hercules, CA) and BSA as a standard. The yield of protein was 5.5 ± 0.5 µg per E6 retina and 42 ± 2 µg per E12 retina. Pools typically consisted of 30–60 retinas, and a total of about 3,000 were needed for the study.

Antisera
Antiserum B-10 is a human polyclonal serum that recognizes the {alpha}-subunit of the IR (26), and it was used at 1:50–1:100 dilution for immunoprecipitation. In preliminary experiments using chick (c) embryo brain membranes, B-10 immunoprecipitated [125I]cIns cross-linked receptors but not [125I]cIGF-I cross-linked receptors. Antiserum P5 (a generous gift of Dr. Robert Garofalo, Pfizer Inc., Groton, CT) is a rabbit polyclonal peptide-specific antiserum generated against the COOH terminus of the IR ß-subunit (27). It was used at 1:150–1:200 dilution. Antiphosphotyrosine antiserum was a polyclonal rabbit serum generated as previously described (28), and it was used at 1:2000 dilution for immunoblot. M2 and M3 are mouse polyclonal antibodies generated in our laboratory against the sequence GRKNERALPLPQSSAC, corresponding to amino acids 1318–1333 in the ß-subunit of the cIGF-I receptor (29), plus a N-terminal cysteine to allow coupling to KLH (Calbiochem, San Diego, CA) using standard procedures. This sequence is 50% different from the amino acids 1328–1343 of the human IR, which is the peptide sequence recognized by the antiserum P5. The S/W mice were immunized with 75–100 µg of the immunogen in adjuvant MPL+TDM (Sigma Chemical Co., St. Louis, MO) according to the procedure of Ou et al. (30), using T-180 sarcoma cells (kindly provided by Dr. Paul H. Patterson, California Institute of Technology, Pasadena, CA). Titration and specificity of the antibodies (equivalent) were carried out by dot blot (not shown), and the dilution chosen for immunoprecipitations was 1:50–1:100. When these antibodies were used for immunoprecipitation, a secondary rabbit antimouse IgG was used before the incubation step with Protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) to allow better recognition.

Peptides and binding assays
cIns (Litron Laboratory, Rochester, NY), recombinant cIGF-I (GroPep Pty. Ltd., Adelaide, Australia), and recombinant human proinsulin (a kind gift from Eli Lilly & Co., Indianapolis, IN) were used. Tracers were radiolabeled with Na125I (Amersham Pharmacia Biotech, Aylesbury, UK) by the lactoperoxidase method (31). The specific activities of [125I]cIns and [125I]cIGF-I yielded 1000–1800 Ci/mmol. Ligand binding of [125I]cIns and [125I]cIGF-I to chick embryo tissues was determined by incubation of crude membranes (30–60 µg total protein in 50 µl) with the tracer (~30,000 cpm) and a range of concentrations of unlabeled peptides, as indicated. Binding buffer (50 mM HEPES, pH 7.8, 120 mM NaCl, 15 mM sodium acetate, 10 mM glucose, 2.5 mM KCl, 1.2 mM MgSO4, 0.1% insulin-free BSA, and 1.4 mg/ml bacitracin) was added to a final volume of 150 µl, and incubation at 4 C was continued overnight. Then, the membrane-bound ligand was recovered by centrifugation at approximately 20,000 x g for 10 min at 4 C. The supernatant was aspirated off, and the pellet was washed once with ice-cold buffer. The 125I-radioactivity in the pellet was measured in a {gamma}-counter (LKB Instruments, Inc., Rockville, MD). Nonspecific binding, determined as the amount of [125I]cIns or [125I]cIGF-I bound in the presence of 0.1 µM unlabeled cIns or cIGF-I, was subtracted from the total binding to yield the specific binding. In previous studies we had demonstrated that, under these conditions, the degradation of the tracer bound to embryo membranes was less than 2% of the initial activity (10). Competitive binding data were analyzed by the nonlinear least squares computer program LIGAND (32; Ligand Program 2.0, Elsevier-Biosoft, Cambridge, UK) and then transformed according to the method of Scatchard (33).

Cross-linking of 125I-labeled peptides to membrane receptors
Binding of [125I]cIns or [125I]cIGF-I to membranes was performed as described above except that 50–100 µg of total protein and 100,000–150,000 cpm (to adjust to 1 nM) of tracer was used. The binding was terminated by centrifugation, and the membrane pellets were washed once with binding buffer without BSA and resuspended in 150 µl of cold HEPES buffer (as described above but without BSA). The cross-linking of the 125I-labeled peptide to the receptor was performed with 0.1 mM disuccinimidyl suberate for 15 min at 4 C (34). The chemical reaction was quenched by adding 150 µl of 10 mM Tris, pH 7.8, 1 mM EDTA. The cross-linked membranes were washed once and either processed for immunoprecipitation with antisera P5 or M2/M3, as described below for B-10, or directly resuspended in PAGE sample buffer (31.25 mM Tris, pH 7.4, 1% SDS, 5% glycerol, 0.05% bromophenol blue, and with or without 2.5% ß-mercaptoethanol). Samples were then electrophoresed on 5% or 7.5% gels. Dried gels were exposed to films (X-Omat, Eastman Kodak Co., Rochester, NY) for the indicated times.

Autophosphorylation, immunoprecipitation, and immunoblotting of receptors
Crude membranes (25 µg of total protein) were preincubated for 60 min at 4 C in the absence or presence of the indicated concentrations of cIns or cIGF-I in 50 µl of 50 mM HEPES, pH 7.8, and 2.5 mM MnCl2. The receptor autophosphorylation reaction was initiated by the addition of 100 µM ATP and 100 µM sodium orthovanadate and stopped after a 15-min incubation at room temperature by adding 200 µl of solubilization buffer (50 mM HEPES, pH 7.8, 150 mM NaCl, 1% Triton X-100, 2 mM sodium orthovanadate, 0.1 M NaF, 4 mM sodium pyrophosphate, 2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin). After 2 h at 37 C, the solubilized membrane receptors were immunoprecipitated with B-10 antiserum for 16 h at 4 C. The receptor-antibody complex was precipitated with 30 µl of 150 µg/ml Protein A-Sepharose per 200 µl of membranes. Immunocomplexes were centrifuged, washed three times, resuspended in PAGE sample buffer, denatured by boiling for 10 min, and subjected to SDS-PAGE as described for affinity cross-linking. Proteins were then transferred into nitrocellulose (0.2 µm, Bio-Rad Laboratories, Inc., Hercules, CA) in Towbin buffer (25 mM Tris, pH 8.3, 192 mM glycine, and 20% methanol) using a semidry transfer unit (Bio-Rad Laboratories, Inc.). The blots were incubated with the indicated antibody for 1 h at room temperature and then with a horseradish peroxidase-linked IgG for 45 min. Phosphoproteins were revealed by the enhanced chemiluminescent procedure (Amersham Pharmacia Biotech). All incubations and washes were carried out at room temperature with PBS-0.1% Tween 20 buffer, containing 2 mM orthovanadate, 0.1 M NaF, and 4 mM sodium pyrophosphate as phosphatase inhibitors.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Typical IGF-I receptors coexist with atypical IRs in proliferative neuroretina
As a first approach to characterize the ligand binding specificity of [125I]cIGF-I and [125I]cIns to the developing neuroretina, displacement curves were generated (Fig. 1Go). Typical high-affinity binding sites for IGF-I were found in E6 and E12 retina. The lowest half-maximal displacement (ED50) of [125I]cIGF-I binding was in both ages obtained with cIGF-I (Table 1Go), whereas proinsulin was a very poor competitor of this binding (ED50 > 10-7 M). Scatchard analysis of the displacement curves adjusted to a linear plot in both E6 (r = -0.67) and E12 (r = -0.87) neuroretina (data not shown). The affinity of [125I]cIGF-I binding decreased slightly from the proliferative to the differentiative stage (Table 2Go), while it was in the subnanomolar range in both ages. The IGF-I binding sites increased slightly with development (per unit of membrane protein) (Table 2Go), although there was a minimal decrease of the average number of IGF-I receptor sites per cell from 60 sites per cell at E6 to 50 sites per cell at E12 (calculations based on data from Table 2Go and counting cell number in a E6 retina, 5 x 106 cells, and a E12 retina, 40 x 106 cells). These receptors, thus, are very low in abundance in the developing neuroretina but display typical specificity and very high affinity for IGF-I.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 1. Binding-competition curves of [125I]cIns and [125I]cIGF-I in embryonic retina and liver. Aliquots of crude membranes (30–50 µg protein per tube) were incubated with the indicated tracer in the absence or presence of unlabeled cIns, cIGF-I, and human proinsulin (hPro) as competitors. The nonspecific binding (obtained in the presence of 0.1 µM homologous unlabeled ligand) has been subtracted. Each point is expressed as the percent of the maximum binding and represents the average of triplicate samples in two to four separate assays.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Half maximal displacement (ED50) of ligand tracer by unlabeled insulin (Ins), IGF-I, and proinsulin (ProIns)

 

View this table:
[in this window]
[in a new window]
  		Table 2. Receptor affinities (Kd) and receptor number (Ro) derived from Scatchard analysis

 
As opposed to the maintained specificity of the [125I]cIGF-I binding sites during neurogenesis, there was a shift in the specificity of the [125I]cIns binding sites (Fig. 1Go). In the E6 retina, this binding was nearly equally well displaced by cIGF-I as by cIns, with an ED50 in the subnanomolar range (Table 1Go). Human proinsulin, despite being heterologous in species, was also a good competitor of [125I]cIns binding sites, with an ED50 very similar in the E6 retina and in the liver. Therefore, the atypical insulin binding sites are not the more abundant IGFR, since the later bind proinsulin very poorly. The atypical insulin binding sites, however, not only differed from the typical liver IR in that they did not discriminate well between insulin and IGF-I (Fig. 1Go and Table 1Go) as liver sites do, but they did not fit to a curvilinear Scatchard; in addition, they indeed fit poorly to a linear Scatchard (r = -0.43), suggesting heterogeneity in the E6 receptor population (results not shown). In contrast, the liver [125I]cIns binding presented a curvilinear Scatchard plot that could be analyzed as high- and low-affinity binding sites, both more abundant than the retina sites (Table 2Go). Remarkably, the E6 retina [125I]cIns atypical binding sites had higher affinity (6.4 x 10-10 M) than that of typical [125I]cIns liver binding sites (1.8 x 10-9 M). As development proceeds in the differentiative E12 neuroretina, the specificity of these atypical binding sites changed. [125I]cIns binding was best displaced by cIns > cIGF-I > proinsulin (Fig. 1Go and Table 1Go). The preferential insulin binding sites were, however, still distinct from the liver insulin binding sites in two features. First, they displayed good fitting to a linear Scatchard plot (r = -0.87) as opposed to the curvilinear Scatchard found in liver (results not shown); and second, they presented a hook effect, i.e. an increase in tracer binding in the presence of low-intermediate concentrations of unlabeled IGF-I and proinsulin that was not found with liver IRs (Fig. 1Go). The affinity of the insulin binding sites from E12 neuroretina was, nevertheless, in the nanomolar range, similar to the high-affinity binding sites in the liver (Table 2Go). In contrast, the number of insulin binding sites was much higher in the liver than in the neuroretina (Table 2Go). Calculated on a per cell basis for neuroretina, there was no change in the very low number of insulin binding sites, which remained about half of the number of IGF-I binding sites between E6 and E12.

The unusual [125I]cIns binding characteristics found in the proliferative neuroretina were confirmed by a ligand cross-linking analysis that identified the {alpha}-subunit of the receptors. [125I]cIGF-I (Fig. 2Go) and [125I]cIns (Fig. 3Go) bound to {alpha}-subunits of 125 kDa in both E6 and E12 neuroretina, slightly smaller than the corresponding liver {alpha}-subunit (135 kDa), as expected for a neural tissue (34). The homologous and heterologous competition of the cross-linked tracer confirmed that IGFR bound with highest affinity [125I]cIGF-I at the two developmental stages, with cIGF-I best competing for this binding and very poor competition by proinsulin (Fig. 2Go). These experiments also confirmed the atypical behavior of insulin binding sites at E6. At this age, [125I]cIns cross-linked to receptors that recognized cIGF-I and cIns equally well. In E12 neuroretina, we observed more typical [125I]cIns binding sites, with slightly better competition of the cross-linking by cIns but efficient competition as well with proinsulin and IGF-I (Fig. 3Go).



View larger version (91K):
[in this window]
[in a new window]
  		Figure 2. Cross-linking of receptors with [125I]cIGF-I and specificity in developing retina. Tracer concentrations of [125I]cIGF-I were cross-linked to E6 and E12 retina and E8 liver (Li) crude membranes, either in the absence or in the presence of the indicated concentrations of unlabeled peptides, cIns, cIGF-I, and human proinsulin (hPro). After SDS-PAGE under reducing conditions, the {alpha}-subunit of the retina receptor migrated as a 125-kDa band (arrow), and the corresponding liver subunit (used as control) migrated as a 135-kDa band (arrowhead). The autoradiograms were exposed for 20–25 days. The positions of molecular mass standards are indicated. Note that in both E6 and E12 retina the best competitor of [125I]cIGF-I binding is cIGF-I.

 


View larger version (90K):
[in this window]
[in a new window]
  		Figure 3. Cross-linking of receptors with [125I]cIns and specificity in developing retina. Tracer concentrations of [125I]cIns were cross-linked to E6 and E12 neuroretina and E8 liver (Li) crude membranes, either in the absence or in the presence of the indicated concentrations of unlabeled peptides, cIns, cIGF-I, and human proinsulin (hPro). After SDS-PAGE under reducing conditions, the {alpha}-subunit of the retina receptor migrated as a 125-kDa band (arrow), and the corresponding liver subunit (used as control) migrated as a 135-kDa band (arrowhead). The autoradiograms were exposed for 20–25 days. Note that in E6 retina the competition of [125I]cIns binding by cIns and cIGF-I is similar, whereas in E12 the best competitor is cIns.

 
Insulin binds to hybrid IR/IGFR and monomeric IR, but IGF-I binds predominantly to dimeric IGFR in neuroretina
The unusual binding affinities for insulin and IGF-I found in proliferative neuroretina could be a consequence of the presence of IR/IGFR hybrids. Thus, this possibility was further investigated. After ligand cross-linking, the solubilized receptors were immunoprecipitated with either P5, a specific IR antibody, or M2/M3, specific antisera generated against chicken IGFR. We used M2/M3 since none of the available antisera against the mammalian IGFR recognized it specifically in the chick. A receptor that contained at least a monomer of the IR was immunoprecipitated with P5 from both E6 and E12 retinas, as well as from liver, after [125I]cIns cross-linking (Fig. 4Go). In contrast, virtually all the [125I]cIGF-I was cross-linked to a receptor that did not immunoprecipitate with the IR antibody. Since the immunoprecipitation was performed before reduction of the dimeric receptor, these results excluded that at 1 nM concentration IGF-I bound in vivo to a putative IR/IGFR hybrid in retina. The complementary experiment was compatible with the presence of [125I]cIns binding to hybrid receptors. Immunoprecipitation of IGFR cross-linked with [125I]cIGF-I by M2 antibody showed the expected {alpha}-subunit to be present in reducing gels and very faintly in liver, as corresponds to the low binding in this tissue (Fig. 5AGo). As expected, [125I]cIns was cross-linked in liver to a much more abundant {alpha}-subunit, the IR demonstrated in the previous experiment (Fig. 4Go), which was not immunoprecipitated by M3 (Fig. 5AGo). In the retina, however, there was a [125I]cIns cross-linked {alpha}-subunit, relatively more abundant in E6 than E12, which most likely was part of a IR/IGFR hybrid immunoprecipitable by M3 antibody. Counting the radioactivity associated with each type of receptor in several immunoprecipitation experiments similar to those described, we concluded that less than 10% of the [125I]cIGF-I was bound to hybrid receptors, whereas at least 40% at E6 and 30% at E12 of total [125I]cIns associated with immunoprecipitable receptors was bound to IR/IGFR hybrids. In the liver, most of the [125I]cIns was bound to the typical, abundant IRs (Figs. 4Go and 5AGo). To assess further the molecular characteristics of the atypical receptors, the ligand cross-linked and immunoprecipitated receptors were electrophoresed under nonreducing conditions. Consistent with the typical binding characteristics described, [125I]cIGF-I was exclusively bound to a receptor with the apparent molecular mass of an {alpha}2ß2 IGFR dimer (>=350 kDa). This molecular form was only recognized by the anti-IGFR antiserum (M2 and M3 had similar specificity in a series of experiments) (Fig. 5BGo). In marked contrast, [125I]cIns was bound to two molecular receptor forms, one an IR/IGFR hybrid dimer, immunoprecipitated by M3, and another receptor form with the apparent molecular mass of an {alpha}ß-monomer (~200 kDa), corresponding to an IR, since it was only recognized by the antiserum P5 (Fig. 5BGo). Although at relatively lower concentration, the monomeric form of the IR was also present in liver (Fig. 5BGo).



View larger version (64K):
[in this window]
[in a new window]
  		Figure 4. Immunoprecipitation of [125I]cIns and [125I]cIGF-I cross-linked receptors by an IR antibody. [125I]cIns or [125I]cIGF-I (1 nM) was cross-linked to membranes from E6 and E12 neuroretinas (Ret) and from E8 liver (Li). After solubilization, immunoprecipitation (IP) was performed with antiserum P5 or an irrelevant control Ig (IgY). The precipitate (P) and an aliquot of the supernatant (S) were subjected to SDS-PAGE under reducing conditions. The autoradiogram was exposed for 40 days. Note that the [125I]cIns is bound to a receptor immunoprecipitated in all three tissues by the anti-IR antiserum while [125I]cIGF-I is bound to a receptor not recognized by P5.

 


View larger version (56K):
[in this window]
[in a new window]
  		Figure 5. Immunoprecipitation of [125I]cIGF-I and [125I]cIns cross-linked receptors by IGFR- or IR-specific antibodies. [125I]cIGF-I or [125I]cIns (1 nM) was cross-linked (c-l) to membranes from E6 and E12 neuroretinas and from liver (Li). After solubilization, immunoprecipitation (IP) was performed with antisera against the IGFR (M2/M3) or against the IR (P5). A, The precipitate (P) of all samples was subjected to SDS-PAGE under reducing conditions. An aliquot of the supernatant (S) from the control liver was also electrophoresed. The autoradiogram was exposed for 40 days. Note that the [125I]cIns bound in liver is to a receptor not recognized by the M3 antiserum, whereas in both E6 and E12 retina there are immunoprecipitated receptors bound to [125I]cIns and [125I]cIGF-I. B, The precipitate of all samples in another similar experiment was subjected to SDS-PAGE under nonreducing conditions. The autoradiogram corresponding to [125I]cIGF-I was exposed for 25 days and the [125I]cIns was exposed for 90 days. The dimeric ({alpha}2ß2) and monomeric ({alpha}ß) receptors are indicated. The experiment was replicated three times.

 
Total IGF-I and insulin binding are both higher in proliferative than differentiative neuroretina
The very heterogeneous insulin binding sites from neuroretina were, nevertheless, capable of significant total binding when compared with liver, a typical target tissue for insulin. The highest specific binding for both ligands was found in the proliferative E6 retina, with predominant binding of [125I]cIGF-I (24.8%/100 µg protein) over [125I]cIns binding (4.0%/100 µg protein). At E12, there was approximately an 8-fold decrease in [125I]cIGF-I binding and a 2-fold decrease in [125I]cIns binding. In the embryonic liver, the binding of [125I]cIns was lower (2.5%/100 µg protein) than that in the E6 retina, and the [125I]cIGF-I binding, consistent with the previous experiments, was very low (1.6%/100 µg protein) (Fig. 6Go).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 6. [125I]cIns and [125I]cIGF-I total specific binding in embryonic retina and liver. Crude membranes from E6 (Ret E6) and E12 neuroretina (Ret E12) and from E8 liver (Li E8) were prepared, and specific binding of the indicated ligand tracer was determined in triplicate tubes. The mean percentage of specific counts per min bound per 100 µg of membrane protein obtained in up to three independent experiments is shown. Error bars correspond to the SEM. Note that the scale for [125I]cIns binding is 3-fold expanded with respect to the scale for [125I]cIGF-I.

 
The IR from neuroretina at proliferative and differentiative stages autophosphorylate in vitro
To analyze the basic activity of the retina receptors containing at least one IR monomer, we performed initial autophosphorylation experiments with the whole neuroretina in organoculture (data not shown) and with isolated membranes. Autophosphorylation of retina and liver receptors in tyrosines was observed after immunoprecipitation with the IR antibody B-10. In both E6 and E12 neuroretina, there were two phosphorylated bands, one corresponding to the expected apparent molecular mass of the ß-subunit of the IR, 95-kDa, and a larger, 105-kDa band (Fig. 7AGo). The latter was also present in the liver, although in some experiments it was not phosphorylated as consistently as in the retina. The 105-kDa band could be either a form of the IGFR ß-subunit part of the hybrid receptors demonstrated in retina, but this was unlikely in the case of liver, or it could be alternatively a tyrosine-phosphorylated substrate of the receptor that coimmunoprecipitated with it even in basal conditions. Direct immunoblots of liver and E12 retina extracts probed with P5 antibody, however, recognized strongly both the 95-kDa and the 105-kDa bands in retina, while the 95-kDa band was predominant in liver (Fig. 7BGo). This result suggests that the 105-kDa phosphorylated band likely corresponds to an alternative ß-subunit of the IR, more abundant in neuroretina. A high basal tyrosine phosphorylation level of the receptors was noticed. Accordingly, modest ligand-dependent autophosphorylation was elicited in both retina and liver. Induction of receptor autophosphorylation by insulin and IGF-I was more significant in the E12 retina than in the E6 retina and slightly higher with IGF-I. In liver, in agreement with the low IGF-I binding, and the specificity of the immunoprecipitated IR observed previously, no stimulation of receptor autophosphorylation occurred with IGF-I. When total cell phosphorylation was measured in E6 retina in toto, IGF-I was clearly a several fold better stimulus than insulin (7).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 7. Ligand-stimulated autophosphorylation of IR/IGFR hybrids and IR in retina membranes (A) and recognition in immunoblot of the IR ß-subunit (B). In panel A, crude membranes from E6 and E12 neuroretinas (Ret) and E8 liver (Li) were preincubated for 60 min at 4 C with the indicated concentrations of cIns or cIGF-I. The in vitro phosphorylation reaction was then carried out as described in Materials and Methods. The membranes were solubilized and the receptors immunoprecipitated (IP) with antiserum B-10, specific for the {alpha}-subunit of the IR, electrophoresed, and immunoblotted (ID) with antiphosphotyrosine antiserum ({alpha}PTyr). Similar results were obtained in a parallel experiment using antiserum P5 for immunoprecipitation. B, Total cell extracts from E12 retina and E8 liver were resolved in SDS-PAGE and then immunoblotted with P5 antiserum and developed by the enhanced chemiluminescent system. Two bands of 95 kDa (arrow) and 105 kDa (arrowhead) were identified.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The balance of apoptosis, proliferation, and differentiation, cell processes essential in neurogenesis, is affected by insulin and IGFs signaling (1). The main objective of this study was to characterize, as close to the in vivo situation as possible, the receptors that mediate the described biological effects of insulin, proinsulin, and IGF-I in retinal neurogenesis (5, 6, 7) in relation to the state of cell differentiation. We found IR/IGFR hybrids with high affinity for insulin predominantly in the E6 chick embryo neuroretina, a prototypic proliferative neuroepithelium, and less abundant in the E12 neuroretina, at advanced differentiation stage (8). To maintain the physiological approach, we avoided the enrichment of the receptor proteins present in crude membranes and did not use lectin purification, since it is known that not all receptor species bind lectins equally well (35). Moreover, the affinity of the receptors semipurified on lectins can change (36), and this change is not equal for different populations of receptors in a primary tissue (37, 38). The major drawback of using crude membranes is the very low concentration of receptors, <100 per retinal cell, that limited the feasibility of the biochemical analysis. Important also for comparison with other studies is the fact that we have used homologous species tracers, which is essential in the case of insulin binding studies, since the affinity of cIns for the IR is at least 10-fold higher than the affinity of mammalian insulins (39). The demonstration of receptor-specific and stage-specific changes in retinal IR and IGFR, including hybrid and monomeric forms, when their respective mRNAs show little change (6), suggests that posttranscriptional regulation of these receptors is important and may be a major mechanism with which to introduce versatility in their downstream action during neurogenesis.

The level of specific [125I]cIns and [125I]cIGF-I binding to the proliferative neuroretina is remarkable, several fold higher for both ligands than the level found in whole brain or liver at this developmental stage (10, 34, 40). This finding confirms the retina as a major target organ for insulin and IGF-I action (Refs. 5, 6, 41, 42) and strongly supports the proposed coordinated role of insulin and IGF-I in CNS development (1, 6, 43). The high-affinity insulin binding sites found in proliferative neuroretina, in addition to having similar affinity for [125I]cIns and [125I]cIGF-I, also recognized human proinsulin, even better than the typical IRs from liver, a feature that distinguishes them further from the [125I]cIGF-I typical binding sites. We suspect that the proinsulin binding would be closer to that of insulin and IGF-I if chicken proinsulin could have been used. The poorly discriminative specificity of the [125I]cIns binding sites was transient and at E12, despite a decrease of the binding level by half, the sites had the specificity of an IR: 10-fold higher affinity for insulin than for IGF-I. The decrease in [125I]cIns binding from E6 to E12 must be due to a decrease in affinity since the receptor number (Ro) increased slightly between the two ages. This developmental regulation is retina specific, different, for example, from that of whole brain and heart in which [125I]cIns binding increased in the same period (40). The decrease in [125I]cIGF-I binding from E6 to E12, approximately 6-fold, is more difficult to justify by the small decrease in affinity measured in steady state, in the presence of a slight increase in Ro, and suggests differences in the IGFR population, perhaps in relation to the presence of IR/IGFR hybrids. In addition, IGF binding proteins may remain in higher proportion in the crude membrane preparation of E6 than E12 neuroretina, causing an increase in [125I]cIGF-I binding. IGFBP 2 is found in vitreous humor, in close contact with neuroretina, in higher abundance in E6 than E12 (44).

We demonstrated that hybrid receptors were present in neuroretina by using ligand cross-linking followed by specific receptor antibody immunoprecipitation. Unexpectedly, however, [125I]cIGF-I did not bind significantly to the hybrid receptor at tracer concentration, as shown by immunoprecipitation with the IR-specific antibody P5 and recovery of most of the [125I]cIGF-I in the supernatant (Fig. 4Go). We interpret this result as a preference of [125I]cIGF-I for its own homodimeric high- affinity receptor, which is relatively more abundant, within the very low concentration of receptors found in this primary tissue. The immunoprecipitation of receptors cross-linked to both tracers with the specific antibody for the chicken IGFR (M2/M3) showed, in turn, that hybrid receptors bound a significant proportion of tracer insulin. In liver, in contrast, [125I]cIns binding was to a typical IR, and no receptor hybrids were found. Although this high affinity of the hybrid receptors for insulin is in disagreement with reported studies in transfected cells, we need to recall that these cells express approximately 105 receptors per cell (17). We speculate that the relatively large spatial separation of the very few (an average of <100 sites per cell, in this study and in Ref. 45) receptors present in the embryonic retina cells may cause different behavior. With the limitation of comparing a quite heterogeneous, at least in E12, cell population, we found that the average number of [125I]cIns binding sites per cell was half than that of [125I]cIGF-I binding sites. The less abundant insulin proreceptor may assemble in the majority of dimers in a hybrid form instead of in homodimers. While this correlation was found in overexpression studies (17), it has not been confirmed in adult mammalian tissues, where it has been proposed that the receptor dimerization may occur at random between IR and IGFR monomers (19).

How much of the atypical [125I]cIns binding characteristics in E6 retina is due to IR/IGFR hybrids is difficult to confirm. The contribution of the monomeric IR found, also unexpected, may be important for the high affinity [125I]cIns binding. We think that it is the heterogeneous mix of hybrids, IR monomers, and more abundant IGFR dimers that confers the overall unusual steady-state binding specificity to the proliferative neuroretina. The proportion of receptor hybrids was, in the semiquantitative estimation of immunoprecipitation experiments, lower in differentiative neuroretina, in parallel to a change in the characteristics of [125I]cIns binding approaching a typical IR. This decrease in hybrids formation may be related to a slightly higher abundance of insulin proreceptor molecules, relative to total protein (Ro) between the two ages (Table 2Go), or to unknown regulatory mechanisms of receptor assembly that may operate in neural development. Changes in the proportion of hybrid receptors in relation to the proliferation/differentiation state of cells have been found also in carcinoma cell lines (46) and may be cell type specific and receptor concentration dependent. In muscle of patients with insulinoma it has been recently reported that IR/IGFR hybrids are more abundant than in control subjects, suggesting that insulin may play a role in the regulation of these receptors (47). Further work will be necessary to understand proreceptor assembly in vivo and particularly in early embryogenesis.

Further differences between neuroretina and liver IR were revealed by the initial autophosphorylation experiments performed. Immunoprecipitation with IR antibodies of unstimulated and ligand-stimulated receptors confirmed that the atypical IR (monomeric and/or hybrid receptors) of E6 neuroretina had an active tyrosine kinase. In neuroretina membranes in vitro, the 95-kDa protein band appeared to be slightly more responsive to ligand-induced phosphorylation than the 105-kDa form. This 105-kDa species may be the ß-subunit preferentially used in the hybrid receptor form, as shown in the case of brain and muscle fetal rat hybrid receptors (21). Alexandrides et al. (22) have postulated that this alternative ß-subunit is part of an IGFR different in primary structure from the typical IGFR. Interestingly, this 105-kDa ß-subunit was preferentially phosphorylated in the basal state in fetal muscle and less responsive to ligand stimulation than the 95-kDa ß-subunit (21). In the present study, the direct recognition of the 105-kDa protein, together with the 95-kDa protein by the P5 antibody in immunoblot, suggests that the 105-kDa is an alternative IR ß-subunit. The IR gene has not yet been fully characterized in chicken, and it remains possible, as has been recently reported in fish cartilage (48), that more than one IR and IGFR gene may exist in chicken.

The signaling capacity and biological relevance of IR/IGFR hybrids and other atypical receptors have been a matter of debate (18, 19, 49). It should be emphasized that a low-discriminative receptor, such as the hybrid receptor found in E6 neuroretina, would be cost effective for signaling in embryogenesis. This strategy, in the context of receptor function diversification (50), may allow any ligand of the insulin family involved in autocrine/paracrine loops in neuroretina, including proinsulin and perhaps IGF-II, to bind an active receptor. In fact, both proinsulin mRNA and IGF-I mRNA expression are markedly regulated in proliferative and differentiative chick neuroretina (Table 3Go). In E5-E6, proinsulin mRNA expression predominates while IGF-I mRNA is low. The reverse is true at differentiative stages. Thus, in proliferative neuroretina the atypical hybrid receptor appears suitable for binding unprocessed proinsulin, the ligand predominantly available in vivo at that age (Ref. 5 and Table 3Go). Proinsulin is, in our hands, as potent an antiapoptotic factor as insulin and IGF-I for the E5-E6 neuroretina (7). This survival function may be very similar to the function in the chick embryo during neurulation, where the endogenous proinsulin and the IR are involved in prevention of apoptosis (51).


View this table:
[in this window]
[in a new window]
  		Table 3. Summary of ligands and receptors expression in developing retina

 
In conclusion, the stage- and receptor-specific regulation of the IR, IGFR, and IR/IGFR hybrids in developing neuroretina supports the physiological, complementary but not identical, implication of these receptor forms in the cellular processes leading to a fully differentiated neuroretina.


   Acknowledgments
 
We thank E. Martínez and V. Quesada for technical assistance, Dr. R. Garofalo for the P5 antiserum, and Dr. C. Bernabeu for critical reading of the manuscript.


   Footnotes
 
1 This study was funded by Grants PB94–0052 (to F.d.P.) and PM96–0003 (to E.J.d.l.R.) from the Dirección General de Investigación Científica y Técnica. A contract (to C.A.) was awarded by the Ministerio de Educación y Cultura (Spain). Back

Received June 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. De Pablo F, De la Rosa EJ 1995 The developing CNS: a scenario for the action of proinsulin, insulin and insulin-like growth factors. Trends Neurosci 18:143–150[CrossRef][Medline]
  2. Recio-Pinto E, Rechler MM, Ishii DN 1986 Effects of insulin, insulin-like growth factor-II, and nerve growth factor on neurite formation and survival in cultured sympathetic and sensory neurons. J Neurosci 6:1211–1219[Abstract]
  3. Shemer J, Raizada MK, Masters BA, Ota A, LeRoith D 1987 Insulin-like growth factor I receptors in neuronal and glial cells. Characterization and biological effects in primary culture. J Biol Chem 262:7693–7699[Abstract/Free Full Text]
  4. Nissley P, Lopaczynski W 1991 Insulin-like growth factor receptors. Growth Factors 5:29–43[Medline]
  5. Hernández-Sánchez C, López-Carranza A, Alarcón C, De la Rosa EJ, De Pablo F 1995 Autocrine/paracrine role of insulin-related growth factors in neurogenesis: local expression and effects on cell proliferation and differentiation in retina. Proc Natl Acad Sci USA 92:9834–9838[Abstract/Free Full Text]
  6. De la Rosa EJ, Bondy CA, Hernández-Sánchez C, Wu X, Zhou J, López-Carranza A, Scavo LM, De Pablo F 1994 Insulin and insulin-like growth factor system components gene expression in the chicken retina from early neurogenesis until late development and their effects on neuroepithelial cells. Eur J Neurosci 6:1801–1810[CrossRef][Medline]
  7. De Pablo F, Díaz B, García de Lacoba M, Vega E, De la Rosa EJ 1998 The early embryonic neuroretina: a CNS site of production and action of (pro)insulin and IGF-I. In: Takano K, Hizuka N, Takahashi S-I (eds) Molecular Mechanisms to Regulate the Activities of Insulin-Like Growth Factors. Elsevier Science, Amsterdam, The Netherlands, pp 155–161
  8. Prada C, Puga J, Pérez-Méndez L, López R, Ramírez G 1991 Spatial and temporal patterns of neurogenesis in the chick retina. Eur J Neurosci 3:559–569[CrossRef][Medline]
  9. De Pablo F, Alarcón C, Díaz B, García de Lacoba M, López-Carranza A, Morales, AV, Pimentel B, Serna J, De la Rosa EJ 1996 Complementary roles of the insulin family of factors and receptors in early development and neurogenesis. Int J Dev Biol [Suppl 1]:109S–110S
  10. Bassas L, De Pablo F, Lesniak MA, Roth J 1985 Ontogeny of receptors for insulin-like peptides in chick embryo tissues: early dominance of insulin-like growth factor over insulin receptors in brain. Endocrinology 117:2321–2329[Abstract/Free Full Text]
  11. Bassas L, Girbau M, Lesniak MA, Roth J, De Pablo F 1989 Development of receptors for insulin and insulin-like growth factor-I in head and brain of chick embryos: autoradiographic localization. Endocrinology 125:2320–2327[Abstract/Free Full Text]
  12. Massagué J, Czech MP 1982 The subunit structures of two distinct receptors for insulin-like growth factors I and II and their relationship to the insulin receptor. J Biol Chem 257:5038–5045[Free Full Text]
  13. Kasuga M, Zick Y, Blith DL, Karlsson FA, Haring HU, Kahn CR 1982 Insulin stimulation of phosphorylation of the beta subunit of the insulin receptor. Formation of both phosphoserine and phosphotyrosine. J Biol Chem 257:9891–9894[Abstract/Free Full Text]
  14. Olson TS, Bamberger MJ, Lane M.D 1988 Posttranslational changes in tertiary and quaternary structure of the insulin proreceptor. Correlation with acquisition of function. J Biol Chem 263:7342–7351[Abstract/Free Full Text]
  15. Soos MA, Siddle K 1989 Immunological relationships between receptors for insulin and insulin-like growth factor I. Evidence for structural heterogeneity of insulin-like growth factor I receptors involving hybrids with insulin receptors. Biochem J 263:553–563[Medline]
  16. Moxham CP, Duronio V, Jacobs S 1989 Insulin-like growth factor I receptor beta-subunit heterogeneity. Evidence for hybrid tetramers composed of insulin-like growth factor I and insulin receptor heterodimers. J Biol Chem 264:13238–13244[Abstract/Free Full Text]
  17. Frattali AL, Pessin JE 1993 Relationship between {alpha} subunit ligand occupancy and ß subunit autophosphorylation in insulin/insulin-like growth factor-I hybrid receptors. J Biol Chem 268:7393–7400[Abstract/Free Full Text]
  18. Soos MA, Nave BT, Siddle K 1993 Immunological studies of type I IGF receptors and insulin receptors: characterisation of hybrid and atypical receptor subtypes. Adv Exp Med Biol 343:145–157[Medline]
  19. Bailyes EM, Nave BT, Soos MA, Orr SR, Hayward AC, Siddle K 1997 Insulin receptor/IGF-I receptor hybrids are widely distributed in mammalian tissues: quantification of individual receptor species by selective immunoprecipitation and immunoblotting. Biochem J 327:209–215
  20. Jonas HA, Cox AJ, Harrison LC 1989 Delineation of atypical insulin receptors from classical insulin and type I insulin-like growth factor receptors in human placenta. Biochem J 257:101–107[Medline]
  21. Alexandrides TK, Smith RJ 1989 A novel fetal insulin-like growth factor (IGF) I receptor. Mechanism for increased IGF I- and insulin-stimulated tyrosine kinase activity in fetal muscle. J Biol Chem 264:12922–12930[Abstract/Free Full Text]
  22. Alexandrides TK, Chen JH, Bueno R, Giorgino F, Smith RJ 1993 Evidence for two insulin-like growth factor I receptors with distinct primary structure that are differentially expressed during development. Regul Pept 48:279–290[CrossRef][Medline]
  23. Quiroga S, Garofalo RS, Pfenninger KH 1995 Insulin-like growth factor I receptors of fetal brain are enriched in nerve growth cones and contain a beta-subunit variant. Proc Natl Acad Sci USA 92:4309–4312[Abstract/Free Full Text]
  24. Havrankova J, Roth J, Brownstein M 1978 Insulin receptors are widely distributed in the central nervous system of the rat. Nature 272:827–829[CrossRef][Medline]
  25. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  26. Kasuga M, Sasaki N, Kahn CR, Nissley SP, Rechler MM 1983 Antireceptor antibodies as probes of insulin-like growth factor receptor structure. J Clin Invest 72:1459–69
  27. Herrera R, Petruzzelli L, Thomas N, Bramson HN, Kaiser ET, Rosen OM 1985 An antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity. Proc Natl Acad Sci USA 82:7899–7903[Abstract/Free Full Text]
  28. Kamps MP, Sefton BM 1988 Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-b oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene 2:305–315[Medline]
  29. Holzenberger M, Lapointe F, Leibovici M, Lievre CA 1996 The avian IGF type 1 receptor: cDNA analysis and in situ hybridization reveal conserved sequence elements and expression patterns relevant for the development of the nervous system. Brain Res Dev Brain Res 97:76–87[CrossRef][Medline]
  30. Ou SK, Hwang JM, Patterson PH 1993 A modified method for obtaining large amounts of high titer polyclonal ascites fluid. J Immunol Methods 165:75–80[CrossRef][Medline]
  31. Marchanolis JJ 1969 An enzymatic method for the trace iodination of immunoglobulins and other proteins. Biochem J 113:299–305[Medline]
  32. Munson PJ, Rodbard D 1980 Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220–239[CrossRef][Medline]
  33. Scatchard G 1949 The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660–672[CrossRef]
  34. Bassas L, De Pablo F, Lesniak MA, Roth J 1987 The insulin receptors of chick embryo show tissue-specific structural differences which parallel those of the insulin-like growth factor I receptors. Endocrinology 121:1468–1476[Abstract/Free Full Text]
  35. Marin-Hincapie M, Garofalo RS 1995 Drosophila insulin receptor: lectin-binding properties and a role for oxidation-reduction of receptor thiols in activation. Endocrinology 136:2357–2366[Abstract]
  36. Hedo JA, Kasuga M, Van Obberghen E, Roth J, Kahn CR 1981 Direct demonstration of glycosylation of insulin receptor subunits by biosynthetic and external labeling: evidence for heterogeneity. Proc Natl Acad Sci USA 78:4791–4795[Abstract/Free Full Text]
  37. Waldbillig RJ, Chader GJ 1988 Anomalous insulin-binding activity in the bovine neural retina: a possible mechanism for regulation of receptor binding specificity. Biochem Biophys Res Commun 151:1105–1112[CrossRef][Medline]
  38. Alemany J, Girbau M, Bassas L, De Pablo F 1990 Insulin receptors and insulin-like growth factor I receptors are functional during organogenesis of the lens. Mol Cell Endocrinol 74:155–162[CrossRef][Medline]
  39. Freychet P 1976 Insulin receptors. In: Blecher M (ed) Methods in Receptor Research. M. Dekker, New York, vol 2:385–426
  40. Bassas L, Lesniak MA, Serrano J, Roth J, De Pablo F 1988 Developmental regulation of insulin and type I insulin-like growth factor receptors and absence of type II receptors in chicken embryo tissues. Diabetes 37:637–644[Abstract]
  41. Waldbillig RJ, Arnold DR, Fletcher RT, Chader GJ 1991 Insulin and IGF-I binding in developing chick neural retina and pigment epithelium: a characterization of binding and structural differences. Exp Eye Res 53:13–22[CrossRef][Medline]
  42. Waldbillig RJ, Pfeffer BA, Schoen TJ, Adler AA, Shen-Orr Z, Scavo L, LeRoith D, Chader GJ 1991 Evidence for an insulin-like growth factor autocrine-paracrine system in the retinal photoreceptor-pigment epithelial cell complex. J Neurochem 57:1522–1533[Medline]
  43. Díaz B, Pimental B, De Pablo F, De la Rosa EApoptotic cell death affecting proliferating neuroepithelial cells in the embryonic retina is prevented by insulin. Eur J Neurosci, in press
  44. Serna J, González-Guerrero PR, Scanes CG, Pratti M, Morreale G, De Pablo F 1996 Differential and tisue-specific regulation of (pro)insulin and insulin-like growth factor-I mRNAs and levels of thyroid hormones in growth-retarded embryos. Growth Regul 6:73–82[Medline]
  45. Yang YW, Brown DR, Robcis HL, Rechler MM, De Pablo F 1993 Developmental regulation of insulin-like growth factor binding protein-2 in chick embryo serum and vitreous humor. Regul Pept 48:145–155[CrossRef][Medline]
  46. Peterson SW, Kyriakis JM, Hausman RE 1986 Changes in insulin binding to developing embryonic chick neural retina cells. J Neurochem 47:851–855[CrossRef][Medline]
  47. Garrouste FL, Remacle-Bonnet MM, Lehmann MM, Marvaldi JL, Pommier GJ 1997 Up-regulation of insulin/insulin-like growth factor-I hybrid receptors during differentiation of HT29–D4 human colonic carcinoma cells. Endocrinology 138:2021–2032[Abstract/Free Full Text]
  48. Federici M, Lauro D, D’Adamo M, Giovannone B, Porzio O, Mellozzi M, Tamburrano G, Sbraccia P, Sesti G 1998 Expression of insulin/IGF-I hybrid receptors is increased in skeletal muscle of patients with chronic primary hyperinsulinemia. Diabetes 47:87–92[Abstract]
  49. Chan SJ, Plisetskaya EM, Urbanati E, Steiner DF 1997 Expression of multiple insulin and insulin-like growth factor receptor genes in salmon gill cartilage. Proc Natl Acad Sci USA 94:12446–12451[Abstract/Free Full Text]
  50. LeRoith D, Werner H, Beitner-Johnson D, Roberts CT 1995 Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr Rev 16:143–163[Abstract/Free Full Text]
  51. Schlessinger J 1997 Direct binding and activation of receptor tyrosine kinases by collagen. Cell 91:869–872[CrossRef][Medline]
  52. Morales AV, Serna J, Alarcón C, De la Rosa EJ, De Pablo F 1997 Role of prepancreatic (pro)insulin and the insulin receptor in prevention of embryonic apoptosis. Endocrinology 138:3967–3975[Abstract/Free Full Text]
  53. Alarcón C, Serna J, Pérez-Villamil B, De Pablo F 1998 Synthesis and differentially regulated processing of proinsulin in developing chick pancreas, liver and neuroretina. FEBS Lett 361–366AU: Please supply all names and initials.



This article has been cited by other articles:


Home page
 IOVSHome page
S. Corrochano, R. Barhoum, P. Boya, A. I. Arroba, N. Rodriguez-Muela, V. Gomez-Vicente, F. Bosch, F. de Pablo, P. de la Villa, and E. J. de la Rosa
Attenuation of Vision Loss and Delay in Apoptosis of Photoreceptors Induced by Proinsulin in a Mouse Model of Retinitis Pigmentosa
Invest. Ophthalmol. Vis. Sci., September 1, 2008; 49(9): 4188 - 4194.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
T. T Nguyen, A. M Sheppard, P. L Kaye, and P. G Noakes
IGF-I and insulin activate mitogen-activated protein kinase via the type 1 IGF receptor in mouse embryonic stem cells
Reproduction, July 1, 2007; 134(1): 41 - 49.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
C. Hernandez-Sanchez, E. Rubio, J. Serna, E. J. de la Rosa, and F. de Pablo
Unprocessed Proinsulin Promotes Cell Survival During Neurulation in the Chick Embryo
Diabetes, March 1, 2002; 51(3): 770 - 777.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
P. F. Hitchcock, D. C. Otteson, and P. F. Cirenza
Expression of the Insulin Receptor in the Retina of the Goldfish
Invest. Ophthalmol. Vis. Sci., August 1, 2001; 42(9): 2125 - 2129.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
B Diaz, J Serna, F De Pablo, and E. de la Rosa
In vivo regulation of cell death by embryonic (pro)insulin and the insulin receptor during early retinal neurogenesis
Development, January 4, 2000; 127(8): 1641 - 1649.
[Abstract] [PDF]

Home page
 J. Biol. Chem.Home page
E. Bieberich, S. MacKinnon, J. Silva, and R. K. Yu
Regulation of Apoptosis during Neuronal Differentiation by Ceramide and b-Series Complex Gangliosides
J. Biol. Chem., November 21, 2001; 276(48): 44396 - 44404.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
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 García-de Lacoba, M.
Right arrow Articles by de Pablo, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by García-de Lacoba, M.
Right arrow Articles by de Pablo, F.

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