IGF-II Receptor Research Articles

Major proteins of bovine seminal fluid bind to insulin-like growth factor-II.

Bovine seminal plasma (BSP) contains a family of four closely related proteins previously designated as BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins). They are secreted by seminal vesicles and they bind to the choline phospholipids composing the sperm plasma membrane upon ejaculation. these proteins contain two homologous domains that are similar to the type II structure present in the putative insulin-like growth factor II (IGF-II) binding domain of the IGF-II receptor. Thus, it was of interest to verify the interaction between the IGF-II and these proteins. The interaction between the IGF-II and BSP proteins was demonstrated by solid phase assay, analytical gel filtration, affinity cross-linking, and gel overlay binding technique. All techniques showed that 125I-IGF-II interacted specifically with BSP proteins and that the binding could be inhibited with an excess of unlabeled IGF-II. No structural homology has been found between BSP proteins and any IGF-binding proteins (IGFBP) identified to date, and therefore, they represent a new family of IGFBP. Since BSP cross-reacting proteins are ubiquitous, we propose that the specific binding of these proteins to IGF-II could modulate the activity of this growth factor.

Partition of insulin-like growth factor (IGF)-binding sites between the IGF-I and IGF-II receptors and IGF-binding proteins in the human kidney.

Quantitative ligand binding autoradiography and in situ hybridization were employed to analyze [125I]insulin-like growth factor-I ([125I] IGF-I) and [125I]IGF-II-binding sites in human kidney sections. Binding sites for both ligands were concentrated in the inner medulla and glomeruli, with low levels present in the tubulo-interstitial cortex. Competition with cold IGF-I, IGF-II, and insulin was used to determine nonspecific binding and differentiate binding of ligands to the IGF-I and IGF-II receptors and IGF-binding proteins (IGFBPs). Nonspecific binding was less than 20% of the total for both ligands. Insulin (10(-5) mol/L), which binds to the IGF-I receptor, but not to the IGF-II receptor or IGFBPs, displaced 39 +/- 8% of [125I]IGF-I binding in glomeruli, 60 +/- 7% in the tubulo-interstitial cortex, and 32 +/- 7% in the medulla. Insulin produced no detectable decrease in [125I]IGF-II binding in any region. IGF-I (10(-8) mol/L), which binds strongly to IGFBPs, but not appreciably to the IGF-II receptor, produced reductions of 46 +/- 9%, 35 +/- 8%, and 39 +/- 12% in [125I]IGF-II binding in glomeruli, tubulo-interstitial cortex, and medulla, respectively. In situ hybridization showed that IGFBP-1-5 mRNAs were all expressed in glomeruli. IGFBP-2 mRNA was abundant in medullary collecting duct epithelium, whereas IGFBP-3, -4, and -5 mRNAs were localized in interstitial and vascular cells throughout the kidney. IGF-I and -II receptor mRNAs were widely distributed in renal epithelium. The abundance of local IGFBP gene expression was positively correlated with insulin-nondisplaceable IGF binding in specific kidney regions. In summary, [125I]IGF-I binding appears to be partitioned largely to IGFBPs in glomeruli and largely to the IGF-I receptor in the tubulo-interstitial cortex, with binding in the medulla more evenly divided. The proportion and regional distribution of [125I]IGF-II binding to IGFBPs are similar, but the balance appears to be primarily associated with the IGF-II, rather than the IGF-I, receptor. Finally, this study shows that [125I]IGF binding autoradiography combined with in situ hybridization can be used to localize and potentially quantitative expression of IGFBPs in tissue sections.

Partition of insulin-like growth factor (IGF)-binding sites between the IGF-I and IGF-II receptors and IGF-binding proteins in the human kidney

Partition of insulin-like growth factor (IGF)-binding sites between the IGF-I and IGF-II receptors and IGF-binding proteins in the human kidney

Analysis of the IGF-II receptor gene copy number in breast carcinoma

Insulin and the insulin-like growth factors (IGFs) may be important regulators of breast cancer growth. The IGF-II receptor is identical to the mannose 6-phosphate (Man-6-P) receptor, which is involved in lysosomal enzyme pathways. In order to determine whether the Man-6-P/IGF-II receptor gene copy number is altered in breast cancer we analysed specimens of invasive breast carcinoma from 51 patients by Southern blotting. No amplification of the receptor gene was observed whatever the clinical presentation of the tumour and irrespective of a concomitant amplification of c-erbB2 or int-2 genes in several tumours. As indicated by Northern blotting, the gene is transcribed in breast tumour tissues and non-tumour breast tissue. These results suggest that the receptor gene is stable in breast carcinoma and that, if anything, the receptor involvement in breast cancer progression may be the result of a disregulation of its expression at a post-transcriptional or post-translational level.

IGF-II in the pathogenesis of rhabdomyosarcoma: a prototype of IGFs involvement in human tumorigenesis.

In recent years there has been a growing interest in the role of peptide growth factors in the regulation of the biologic behavior of several human malignancies. Among those peptides that might have importance in neoplastic pathogenesis are the insulin-like growth factors I and II (IGF-I and IGF-II). These proteins have been shown to stimulate myoblast proliferation and differentiation (1), to promote nutrient uptake and to inhibit proteolysis (2, 3). IGF-II mRNA is highly expressed in human skeletal fetal muscle tissue, but it is not detectable in normal adult skeletal muscle by standard Northern analysis and in situ hybridization (4). In mouse myoblasts, IGF-I and IGF-II mRNA levels increase transiently (IGF-II > > IGF-I) within 48-72 hours of the beginning of the myogenic differentiation process. The expression of mRNA is accompanied by secretion of the peptides in the culture medium that peaks at hour 92. IGF-I receptors increase transiently, doubling by 48 hours after the onset of differentiation, while IGF-II receptors increase and remain at a higher number throughout the differentiated state (5, 6). The production of IGF-binding proteins of Mr 29,000 to 32,000 is also induced throughout differentiation (7).

Conversion of G-protein specificity of insulin-like growth factor II/mannose 6-phosphate receptor by exchanging of a short region with beta-adrenergic receptor.

The 14-residue peptide (peptide 14) corresponding to Arg2410-Lys2423 of the insulin-like growth factor II receptor (IGF-IIR) can activate the adenylate cyclase-inhibitor guanine nucleotide-binding protein Gi, and the 15-residue beta III-2 peptide Arg259-Lys273 of the beta 2-adrenergic receptor (beta 2AR) can activate the stimulatory protein Gs. In phospholipid vesicles, IGF-IIR and beta 2AR activate Gi and Gs in response to IGF-II and isoproterenol, respectively. We constructed a chimeric IGF-II receptor (beta III-2/IGF-IIR) by converting its native peptide 14 sequence to the beta III-2 sequence. In cells expressing beta III-2/IGF-IIR, membrane adenylate cyclase activity markedly increased without IGF-II and was further promoted by IGF-II. This was verified by measuring chloramphenicol acetyltransferase (CAT) activity in beta III-2/IGF-IIR cells with cotransfection of a cAMP response element-CAT construct. This study shows not only the conversion of G-protein specificity of a receptor from Gi to Gs but also the simulation of G protein-coupled receptor signals by using a short receptor region and intact cells. These findings indicate that the G protein-activation signals are interchangeable, self-determined structural motifs that function in the setting of either a single-spanning or multiple-spanning receptor.

Expression, purification and binding to the receptor of human insulin-like growth factor II

Human insulin-like growth factor II (IGF-II) was expressed as a fused protein with 14 additive amino acids in Escherichia coli with a high yield by an expression system using T7 RNA polymerase. Purification of the expressed protein was simply performed using only differential ultrafiltrations, giving a homogeneous preparation upon polyacrylamide gel electrophoresis and high-performance liquid chromatography. The expressed peptide was reacted with a monoclonal antibody raised against native IGF-II on a blotted membrane. Furthermore, the peptide was bound to IGF-II receptor in solubilized rat fetus membrane, though the affinity was slightly inferior to that of native IGF-II. In addition, fusion IGF-II immobilized on a gel matrix was useful for one-step purification of the IGF-II receptor with a high yield from solubilized rat fetus membranes.

Anatomy of the insulin-like growth factor system in the human testis

To study the cellular patterns of gene expression for insulin-like growth factors I and II (IGF-I and IGF-II), their receptors and specific high-affinity binding proteins (IGFBPs) in the human testis. In situ hybridization histochemistry was used to localize messenger ribonucleic acids (mRNAs) for IGF-I and IGF-II, the IGF-I and IGF-II receptors and IGFBP-1 to 6 in fresh-frozen testis sections from healthy young men who died of trauma. Insulin-like growth factor I mRNA was not detected. Insulin-like growth factors II mRNA was abundant in testicular blood vessels and peritubular connective tissue. Both IGF-I and IGF-II receptor mRNAs were most abundant in the germinal epithelium. Insulin-like growth factor binding protein 1 mRNA was not detected. Binding protein-2 mRNA was expressed in both Leydig and Sertoli cells. Binding protein-3 mRNA was detected only in the endothelium of testicular blood vessels. Binding protein-4 mRNA was also localized in endothelium but, in addition, was present in Leydig cells and interstitial connective tissue. Binding protein-5 mRNA was abundant in connective tissue and was detected at low levels in Leydig cells. Binding protein-6 mRNA was present in some of the peritubular cells and in some cells of the interstitial compartment. Our results suggest that IGFs may play significant roles in testicular function and germ cell development.

Benign prostatic hyperplasia and normal prostate aging: differences in types I and II 5 alpha-reductase and steroid hormone receptor messenger ribonucleic acid (mRNA) levels, but not in insulin-like growth factor mRNA levels.

Benign prostatic hyperplasia (BPH) is so common in elderly men that the development of adenomatous nodules in this organ can be seen as a normal age-dependent process. In this work, we used Northern blotting to compare the levels of androgen, estrogen, and insulin-like growth factor-I (IGF-I) receptor in young (age range, 23-33; n = 3), old normal (age range, 52-80; n = 3), and BPH-affected subjects (age range, 66-87; n = 15). We have also investigated in these groups the expression of genes coding for the two 5 alpha-reductases (types I and II), aromatase, IGF-I, and IGF-II. Our results show significantly increased levels of IGF mRNA in old healthy and BPH-affected subjects; the respective rises for IGF-I, IGF-II, and IGF-I receptor mRNAs were 3.0-, 2.9-, and 1.5-fold (BPH) and 2.7-, 2.4-, and 1.8-fold (old normal controls). For estrogen receptor, androgen receptor, and type I and II 5 alpha-reductase mRNAs, a marked but opposite effect was observed in adenomatous tissues only; the respective levels were 2.2-, 1.8-, 3.9-, and 1.7-fold lower than those in young adult subjects, whereas no significant differences were recorded between the two normal groups. Morphometric analysis of each tissue specimen confirmed the significantly lower epithelium/stroma ratio in adenomas compared to young or old healthy tissues. Together, these observations suggest that prostatic adenomas may result from at least two conjugate processes: one characterized by a drop in the mRNA levels of steroid hormone receptors, which might be associated with a lower epithelium/stroma ratio, and another characterized by normal aging phenomena, of which the increased production of IGFs and IGF-I receptor transcripts could be biochemical markers.

Expression of insulin-like growth factor-I (IGF-I) and IGF-II and the IGF-I, IGF-II, and insulin receptor genes and localization of the gene products in the human ovary.

We examined the expression of the genes encoding the insulin-like growth factors (IGFs) and their receptors (r) and the localization of their gene products in specific cellular compartments of the human ovary. mRNA was localized by in situ hybridization with specific human 35S-labeled antisense RNA probes, and protein was detected by immunocytochemistry with specific antisera. We studied 34 follicles (10 ovaries), which included both dominant and small antral follicles. In dominant follicles, no IGF-I mRNA was seen in either thecal or granulosa cells (GC), but IGF-Ir mRNA was expressed in GC. In contrast, abundant IGF-II mRNA was found exclusively in GC, whereas the IGF-IIr gene was expressed in both thecal cells and GC. Insulin receptor mRNA was widely distributed and expressed in all cell types, including stromal cells. Small antral follicles contained both IGF-I and IGF-II mRNA, which was restricted to thecal cells. Although IGF-Ir message was detected only in GC, IGF-IIr mRNA was expressed in both granulosa and thecal cells. As in dominant follicles, insulin receptor mRNA was found in thecal, granulosa, and stromal cells. No IGF-I immunoreactivity was seen in either dominant or small antral follicles; however, immunostaining for the other gene products demonstrated that each of these proteins colocalized with its corresponding mRNA. Thus, the relative distribution of ligand and receptor transcripts and protein in cellular compartments of the human ovary observed in this study supports the presence of an intraovarian IGF system and suggests that both autocrine and paracrine mechanisms of IGF action occur between GC and thecal cells. We conclude that 1) IGF-II, rather than IGF-I, is the principal IGF in human ovarian follicles, being synthesized in thecal cells in small antral follicles and in GC in dominant follicles; 2) in small antral follicles, IGF-II acts in an autocrine fashion in thecal cells and in a paracrine fashion in GC; 3) in dominant follicles, granulosa-derived IGF-II acts in an autocrine manner in GC; and 4) the presence of transcripts and proteins corresponding to the IGF and insulin receptors in cellular compartments of human ovaries may also provide target sites for the action of circulating ligands with a potential extraovarian role in the regulation of folliculogenesis.

Expression of insulin-like growth factor-I (IGF-I) and IGF-II and the IGF-I, IGF-II, and insulin receptor genes and localization of the gene products in the human ovary

Expression of insulin-like growth factor-I (IGF-I) and IGF-II and the IGF-I, IGF-II, and insulin receptor genes and localization of the gene products in the human ovary

Inhibition of internalization of glucose transporters and IGF-II receptors. Mechanism of action of MHC class I-derived peptides which augment the insulin response in rat adipose cells.

Peptides from the alpha 1 domain of the major histocompatibility complex class I antigen (MHC class I), e.g. Dk-(61-85) and Dk-(62-85), have been shown previously to augment glucose uptake in insulin-stimulated cells and to inhibit insulin receptor internalization (Stagsted, J., Reaven, G. M., Hansen, T., Goldstein, A., and Olsson, L. (1990) Cell 62, 297-307). We now report that these peptides inhibit by 80-100% the internalization of glucose transporters (GLUT4) and insulin-like growth factor II (IGF-II) receptors in insulin-stimulated cells and correspondingly double insulin-stimulated glucose transport activity and the number of GLUT4 and IGF-II receptors on the cell surface. In addition, the peptides enhance the apparent affinity about 3-fold of IGF-II binding to its receptor. It is concluded that the effects of the peptides on glucose transport and IGF-II binding are a consequence of the peptide-mediated inhibition of internalization of GLUT4 and IGF-II receptor. The active peptides are derived from the alpha 1 domain of a MHC class I molecule, suggesting that the latter is involved in regulation of internalization of cell surface integral membrane proteins such as the GLUT4 and IGF-II and insulin receptors.

Insulin-like growth factor (IGF) binding proteins and insulin-like growth factor secretion by cultured chondrocyte cells: identification, characterization and ontogeny during cell differentiation.

Insulin-like growth factor binding proteins (IGF BP) and insulin-like growth factors (IGF) secretion by differentiating chondrocytes, derived from mouse embryonic limb bud and responsive to both IGF-I and -II [23], was investigated. The Western ligand blot analysis of the conditioned medium (CM) from days 1, 3, 5 and 7 of culture revealed the secretion of IGF BP of approx. 35-40, 28-30 and 24-26 kDa. The 35-40 kDa protein which comigrated with the 40 kDa protein in CM of trophoblast cells identified as IGF BP-3. The 28-30 kDa protein was identified as IGF BP-2 by Western immunoblotting with alpha-IGF BP-2 antisera. The 24-26 kDa protein was consistent with the nonglycosylated form of IGF BP-4. Secretion of three IGF BPs were increased with the age of the culture. This suggested that the major IGF BP secreted by differentiating chondrocytes in culture are IGF BP-2, -3 and -4. All three of these IGF BPs were stimulated by both IGF-I and -II. IGF-I was approx. 2-fold more potent than IGF-II. The investigation of the localized production of IGF revealed that chondrocytes, similar to IGF BP, secreted IGF-II in differentiation dependent manner. No IGF-I secretion was identified. Examination of the secretion of solubilized IGF-II receptor by the chondrocytes, in contrast to trophoblasts, failed to reveal the presence of IGF-II receptor in the CM. This suggested that, unlike many other cells, including trophoblasts, chondrocytes do not secrete solubilized IGF-II receptor. In summary, the present results suggested an interactive autocrine/paracrine action of IGF BP and IGF-II in the chondrocytes, while the IGF-I action is predominantly endocrine.

The insulin-like growth factors.

The recent availability of reagents to study the IGFs, their receptors, and binding proteins has led to an explosive growth in the study of IGF physiology. However, most studies to date have been descriptive, and studies delineating mechanisms of action are limited. It is apparent that most organ systems synthesize several components of the IGF system necessary for IGF to function in an autocrine or paracrine fashion and that regulation of this system occurs at the local level. However, the relative importance of locally produced IGF vs circulating IGF remains unclear. The mechanisms by which the IGFBPs modulate IGF activity are crucial to understanding this system, and identification of specific roles for each of these proteins will be required. Of critical importance is the identity of the intracellular signal transduction system by which the IGF receptor mediates the effects of the IGFs, and the delineation of mechanisms by which the IGFBPs interact with the receptor at the cellular level. It is also of interest to determine what role, if any, the IGF-II receptor plays in mediating the growth-promoting effects of the IGFs. The ubiquitous distribution of the IGFs, IGFBPs, and IGF receptors indicates that they may play a role in the regulation of coordinate growth among several tissues and cell types. Understanding the mechanisms by which these components interact to coordinate growth responses between different cell types should greatly enhance our understanding of normal growth and development.

Phylogeny of the insulin‐like growth factors (IGFS) and receptors: A molecular approach

The IGFs (IGF-I and IGF-II) are essential for normal mammalian growth and development. Their actions are mediated primarily by their interactions with the type I IGF receptor (IGF-I receptor), a transmembrane tyrosine kinase. The ligands and the IGF-I receptor are structurally related to insulin and to the insulin receptor, respectively. Analysis of evolutionary conservation has often provided insights into essential regions of molecules such as hormones and their receptors. The genes for insulin and IGFs have been partially characterized in a number of vertebrate species extending evolutionarily from humans as far back as fish. The sequences of the exons encoding the mature insulin and IGF peptides are highly conserved among vertebrate species, and IGF-I-like molecules are found in species whose origins extend back as much as 550 million years. The insulin receptor is also highly conserved in vertebrate species, and an insulin-receptor-like molecule has been characterized in Drosophila. In contrast, IGF-I receptors have only been characterized in mammalian species and partially studied in Xenopus, in which the tyrosine kinase domain is highly conserved. Studies are presently being undertaken to analyze in more detail the regulation of the genes encoding this important family of growth factors and the structure/function relationships in the gene products themselves.

A developmentally regulated form of insulin-like growth factor receptor beta-subunit in C2 myoblasts exhibiting altered requirements for differentiation.

Ligand-dependent autophosphorylation and immunoprecipitation have been used to distinguish insulin and insulin-like growth factor-I (IGF-I) receptor beta-subunits in the permissive and inducible subclones of the C2 myoblast cell line. Permissive myoblasts differentiate spontaneously, whereas myoblasts of the inducible subclone require exogenous IGFs to undergo terminal differentiation. Permissive myoblasts contain beta-subunits of 95 and 101 kilodalton (kDa) mol wt. The 95-kDa subunits are immunoprecipitated with antipeptide antibodies directed against tyrosine kinase (AbP2), juxtamembrane (AbP4), and carboxy-terminal (AbP5) domains of the insulin receptor and insulin receptor monoclonal antibody 29B4. The tryptic phosphopeptide map of the 95-kDa band suggests that it contains both insulin and IGF-I receptor beta-subunits. The 101-kDa subunit is immunoprecipitated by AbP2, AbP4, and AbP5, because it forms a hybrid complex with the 95-kDa protein, but it does not react directly with AbP4, AbP5, or antibody 29B4. Phosphorylation of the 101-kDa subunit is more responsive to IGF-I than to IGF-II or insulin, indicating that it is a second IGF-I receptor beta-subunit. Inducible myoblasts exhibit a single major beta-subunit of 106 kDa mol wt. Its immunoreactivity and phosphopeptide map are virtually identical to those of the 101-kDa IGF-I receptor beta-subunit from permissive cells. However, unlike the 101-kDa beta-subunit, phosphorylation of the 106-kDa protein appears to be more responsive to IGF-II than to either IGF-I or insulin. It is lost upon differentiation of myoblasts into myotubes concomittant with the appearance of 95- and 101-kDa beta-subunits. These data demonstrate 1) an alpha 2 beta 2 IGF receptor that has high sensitivity for IGF-II in inducible, but not in permissive, myoblasts; 2) the beta-subunit of this receptor exhibits different migration in sodium dodecyl sulfate-polyacrylamide gels from either of those found in permissive cells; and 3) expression of this beta-subunit is developmentally regulated. This suggests that the inducible cell beta-subunit is a component of a stage-specific alpha 2 beta 2 IGF receptor subtype that functions as an IGF-II receptor.

Expression of IGF ligand and receptor genes during preimplantation mammalian development.

The temporal patterns of expression of genes encoding insulin-like growth factor (IGF) ligands and receptors during very early development have been investigated in several laboratories in several different mammalian species. Both reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemical techniques have been used to identify the time of appearance of gene transcripts or end-products. In preimplantation mouse embryos, IGF-II ligand and receptor gene activity is detectable as early as at the two-cell stage, the time when transcription from the embryonic genome is activated, but receptors for insulin and IGF-I are not detectable until the compacted eight-cell stage. Transcripts for insulin or IGF-I are not detectable in preimplantation mouse embryos, although the ligands are present in the reproductive tract. The pattern of IGF gene expression is not, however, identical in all mammalian species. In cow embryos, for example, transcripts for IGF-I and IGF-II ligands and receptors and insulin receptors have been detected at all stages of preimplantation development from mature oocyte to blastocyst (Watson et al., 1992). Attempts to quantitate transcript abundance in these early embryos are in progress in our laboratory. In the preimplantation mouse embryo, transcripts for several different IGF-binding proteins (IGFBP-2, -3, -4, and -6) have been detected by RT-PCR procedures. In addition, transcripts for IGFBPs have been identified in RNA derived from cumulus cells, the ovary, the oviduct, the uterus, and the decidua. These findings suggest that the interactions of IGF ligands and receptors in preimplantation development might, indeed, be modulated by IGFPs.(ABSTRACT TRUNCATED AT 250 WORDS)

The IGF‐II receptor system: A G protein‐linked mechanism

Based on the finding that stimulation of the IGF-II receptor (IGF-IIR) is capable of activating Gi2 and calcium channels in BALB/c 3T3 fibroblasts, it was found that purified IGF-IIR can couple directly to purified Gi2 in phospholipid vesicles. IGF-IIR-Gi2 coupling can be characterized as follows. IGF-IIR directly couples to Gi2 in response to IGF-II in a stoichiometrical manner, suggesting that IGF-IIR works as a transmembrane signaling molecule and that the seven-transmembrane structure is not essential for receptor-G protein coupling. The mode of IGF-IIR-G12 interaction is similar to that of conventional receptor-G protein coupling, suggesting that a common G protein recognition mechanism is shared by IGF-IIR and conventional G-coupled receptors. The action of IGF-IIR is specific on Gi2 among various G proteins. Finally, the activity of IGF-IIR on Gi2 is similarly potent across the species of the proteins. These characteristics led to the discovery of a 14-amino-acid region in IGF-IIR that can directly interact with and activate Gi2, and is located at residues 2410-2423 of the human receptor. Subsequent work has indicated that this region is responsible for Gi-coupling function of intact IGF-IIR. The most important extensions of this discovery are the following: (1) The structure-function relationship for the Gi-activating function of this 14-amino-acid sequence, (2) the prediction of G protein-coupled functions of receptors based on the results obtained from 1), and (3) clarification of the detailed mechanism whereby ligand-receptor complex recognizes G proteins. This paper reviews what we have learned from IGF-IIR in terms of receptor-G protein interfaces and discusses future prospects.

Okadaic acid stimulates IGF-II receptor translocation and inhibits insulin action in adipocytes.

Okadaic acid, an inhibitor of protein phosphatases 2A and 1, stimulates glucose transport in muscle and fat cells, suggesting that serine/threonine phosphorylation steps are involved in the translocation of glucose transporters. Here we have investigated whether such phosphorylation events could also participate in another membrane-associated insulin-stimulated process: insulin-like growth factor II (IGF-II) receptor translocation in adipocytes. Maximally effective concentrations of insulin and okadaic acid stimulated deoxyglucose uptake by 5.5- and 2.5-fold, respectively, whereas IGF-II binding was increased 3.5-fold and 1.5-fold. Subcellular fractionation indicated that the okadaic acid-induced stimulation of IGF-II binding resulted from an increase in the number of IGF-II receptors in the plasma membrane with a concomitant disappearance from the low-density microsomal fraction. These changes occurred in parallel to those observed for the glucose transporter GLUT-4. Both insulin-stimulated glucose transport and IGF-II binding were prevented when cells were pretreated with okadaic acid. To understand the mechanism of this inhibitory effect, insulin receptor autophosphorylation and the tyrosine phosphorylation of endogenous proteins were studied. Insulin induced the tyrosine phosphorylation of its receptor beta-subunit and of proteins at 120 and 185 kDa, whereas okadaic acid alone had no effect. When okadaic acid and insulin were added together, the beta-subunit autophosphorylation was similar to that observed with insulin alone, but the tyrosine phosphorylation of substrates was prevented. Taken together, our data suggest that, in adipocytes, serine/threonine phosphorylation events mimicked by okadaic acid are required for the translocation of IGF-II receptors and glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of components of the insulin-like growth factor system in pig mammary glands and serum during pregnancy and pseudopregnancy: effects of oestrogen.

To gain insight into the involvement and interactions of the insulin-like growth factors (IGFs) and estrogen in mammary growth and differentiation, the temporal expression of mammary mRNAs encoding components of the IGF system in pregnant and pseudopregnant pigs was examined. Pseudopregnant pigs received 5 mg oestradiol valerate or vehicle daily from day 45 after oestrus and underwent mammary biopsy on days 60, 90 or 112. In mammary tissue of pregnant pigs, steady-state levels of the mRNAs encoding IGF-I, IGF-II and type-I IGF receptor as well as the levels of the membrane-associated type-II IGF receptor were higher during the early phase of mammogenesis (< or = day 45) than during the subsequent stages of mammary development. Mammary IGF-I, IGF-II and type-I receptor mRNAs were expressed at their lowest levels around day 90 of pregnancy (20-40% of those for day 30 of pregnancy) coincident with the onset of beta-casein mRNA accumulation. Mammary IGF-binding protein-2 (IGFBP-2) mRNA levels increased twofold during the latter half of pregnancy, whereas the amount of IGFBP-3 mRNA declined after day 30 to undetectable levels by mid-pregnancy. Pseudopregnant pigs had reduced levels of these mRNAs (except for IGF-II) relative to their pregnant counterparts and this was associated with premature differentiation of mammary tissue as reflected by an earlier onset of beta-casein mRNA accumulation in the former. The administration of oestradiol valerate decreased the levels of IGF-I and type-I IGF receptor mRNAs by day 60 of pseudopregnancy, but the reverse was evident by day 112. Oestradiol administration increased beta-casein mRNA levels in pseudopregnant pigs, but had no effect on mammary IGFBP-2 and IGFBP-3 mRNA levels. Mammary IGF content was greater in late pregnancy (> or = day 90) and pseudopregnancy than at early pregnancy. Serum IGF-I and IGF-II levels declined steadily during pregnancy and this was similar to, but not correlated with, mammary IGF mRNA levels, whereas in pseudopregnant pigs, serum IGF concentrations did not change temporally or in response to oestradiol. Serum IGFBP-2 levels were unaltered during pregnancy or pseudopregnancy, but serum IGFBP-3 levels declined after day 60 of pregnancy. In pseudopregnant pigs, serum IGFBP-3 levels did not change temporally, but declined after oestradiol treatment. Results indicate that mammary IGF-I and type-I IGF receptor systems are down-regulated during pregnancy-associated differentiation of this tissue and in response to oestrogen.(ABSTRACT TRUNCATED AT 400 WORDS)