Interaction of insulin-like growth factor I with porcine thyroid cells cultured in monolayer

The interaction of insulin-like growth factor I (IGF-I) with porcine thyroid cells cultured in monolayer was studied. Specific binding of [125I]iodo-IGF-I to thyroid cells was a reversible process dependent on the time and temperature of incubation. A steady state was achieved in 18 h at 4 C and averaged 14.2 +/- 2% (mean +/- SD)/10(6) cells. Binding of [125I]iodo-IGF-I was inhibited by unlabeled IGF-I; half-maximal inhibition occurred at concentrations of 2-5 ng/ml. Multiplication-stimulating activity (rat IGF-II) and pork insulin had relative potencies of 1:20 and 1:300 compared with IGF-I. Scatchard analysis of binding data revealed a single class of IGF-I receptors with a Ka of 4.3 X 10(10) M-1, 49,000 binding sites were estimated per cell. Affinity cross-linking and autoradiography demonstrated the presence of type I IGF receptors. Thyroid cells also had specific receptors for insulin, but specific binding of [125I]iodoinsulin (2.03 +/- 0.03%/10(6) cells) was much lower than that of [125I]iodo-IGF-I. Preincubation of thyroid cells with IGF-I or insulin caused a concentration-dependent decrease in [125I]iodo-IGF-I binding due to an apparent loss of receptors. Preincubation with epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, or TSH did not alter subsequent binding of [125I]iodo-IGF-I. Low concentrations of IGF-I stimulated DNA synthesis and proliferation of thyroid cells and acted synergistically with epidermal growth factor. Multiplication-stimulating activity and insulin had relative potencies in stimulating DNA synthesis comparable to their abilities to inhibit the binding of [125I]iodo-IGF-I to thyroid cells, suggesting that their effects are mediated primarily by IGF-I receptors. Preincubation with IGF-I did not alter cAMP responsiveness to TSH. We, thus, demonstrated the presence of functional and regulated IGF-I receptors on porcine thyroid cells.     

Characterization of high affinity receptors for insulin-like growth factors I and II on rat anterior pituitary cells

Primary cultures of rat anterior pituitary cells were assessed for the presence of specific receptors for insulin and for the somatomedin peptides, insulin-like growth factors I and II (IGF-I and IGF-II). Specific binding per 100,000 pituitary cells averaged 9.45 +/- 1.69% (mean +/- SD) for [125I]IGF-II, 0.83 +/- 0.06% for [125I]IGF-I, and only 0.11% for [125I]insulin, IGF-II was twice as potent as IGF-I in displacing [125I]IGF-II, while insulin was totally nonreactive, IGF-I was 5-fold more potent than IGF-II at displacing [125I]IGF-I and 1000-fold more potent than insulin. Scatchard analysis of [125I]IGF-II binding revealed a curvilinear plot, which could be resolved into a high affinity receptor with a Ka of 7.0 X 10(8) M-1 and 120,000 receptor sites/cell, and a low affinity receptor with a Ka of 1.1 X 10(8) M-1 and 720,000 receptor sites/cell. The existence of abundant high affinity somatomedin receptors (especially for IGF-II) on rat anterior pituitary cells is consistent with a potential role for these peptides in the regulation of GH secretion.     

Characterization of type I and type II insulin-like growth factor receptors in an intestinal epithelial cell line

Insulin-like growth factors (IGFs) and insulin stimulate DNA and protein synthesis in IEC-6 cells (an intestinal epithelial cell line) grown in a chemically defined medium. IGF-I stimulates proliferation of IEC-6 cells at a lower concentration (ED50 = 1.6 nM) than either insulin or IGF-II. To gain insight into the mechanisms by which IGFs stimulate IEC-6 cell growth, we have examined the characteristics of specific IGF receptors on IEC-6 cells. Binding of 125I-IGF-I and 125I-IGF-II to IEC-6 monolayers was analyzed by incubation with various concentrations (0.2 nM to 0.5 microM) of radiolabeled IGFs for 16 h at 3 C. Scatchard plots of 125I-IGF-I binding were linear, suggesting a single class of binding sites with KD = 3.1 +/- 0.35 nM and Bmax = 50.7 +/- 6 fmol/10(6) cells. IGF-II was potent in displacing 125I-IGF-I (KI = 8.1 +/- 0.85 nM), but insulin had little effect. Affinity cross-linking with 125I-IGF-I followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed three bands with Mr of 270,000, 245,000, and 133,000, and the major band was the 133,000 species. Labeling of the 133,000 and 270,000 bands was greater than or equal to 80% inhibited by 10(-7) M unlabeled IGF-I, less potently inhibited by IGF-II and not at all by insulin. These results suggest that the 133,000 band represents the alpha-subunit of the type I IGF receptor. Scatchard plots of 125I-IGF-II binding to IEC-6 cell monolayers were curvilinear, suggesting two classes of binding sites: high affinity, low capacity sites, KD = 0.87 +/- 0.08 nM and Bmax = 28 +/- 2.5 fmol/10(6) cells; low affinity, high capacity sites, KD = 60 = +/- 8.8 nM and Bmax = 1780 +/- 230 fmol/10(6) cells. Neither IGF-I nor insulin was effective in inhibiting 125I-IGF-II binding. Affinity cross-linking with 125I-IGF-II labeled predominantly a 245,000 band, suggesting that this species is the type II receptor. A band with Mr 131,000 was barely detectable with 125I-insulin. These results indicate that IEC-6 cells have abundant quantities of the type I and II IGF receptors and few insulin receptors, suggesting that the mitogenic effect of IGFs is mediated through the type I IGF receptor.     

Immunoreactive insulin-like growth factor I (IGF-I) and IGF-I-binding protein content in human thyroid tissue

We measured immunoreactive insulin-like growth factor I (IGF-I) in extracts of normal and nodular thyroid tissue obtained at surgery from patients with nontoxic goiter. The nodular tissues contained a higher concentration [mean, 279.0 +/- 69.7 (+/- SE) mU/g] than paired normal tissues (115.5 +/- 17.9 mU/g; P = 0.024; n = 12); a difference was evident in all but one patient. Sephadex G-50 gel filtration of tissue extracts revealed two immunoreactive peaks, the first in the void volume of the column, and the second in the elution volume of authentic IGF-I. The first peak was identified as IGF-I-binding protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after cross-linking with iodinated IGF-I. Isolated thyroid cell membranes contained high affinity IGF-I-binding sites of similar affinity and numbers in both normal and nodular thyroid tissue. The IGF-I content of six thyroid cancer extracts was higher than that of normal thyroid tissue, but the IGF-I content of thyroid tissue from six patients with Graves' disease and five patients with Hashimoto's thyroiditis was similar to that in normal thyroid tissue. These data suggest that the stimulatory effect of TSH on thyroid cell proliferation could be mediated through IGF-I action and suggest that an increase in IGF-I production could sustain the goitrogenic process.     

Thyrotropin receptors in normal and pathological human thyroid tissues.

The properties of TSH receptors in normal and pathological human thyroid tissues were studied. Highly purified bovine TSH after lactoperoxidase iodination retained full biological activity, as assessed by radioreceptor assay. Binding of bovine [125I]TSH to 1000 x g pellets of human thyroid homogenate was specific with respect to hormone and tissue. Total binding amounted to 50-60% of total radioactivity using 10 mg (wet weight) normal thyroid tissue. Nonspecific binding was only 6% of total radioactivity. Normal thyroid tissue contained two orders of binding sites, which were shown to be independent of each other by Hill plot analysis. The high affinity sites [equilibrium dissociation constant (Kd), 0.015-0.16 x 10-9 M] were present in concentrations of 1.05-9.30 pmol/mg protein and concentrations of low affinity sites (Kd, 1.2-2.4 x 10-9 M) were 35.9-213 pmol/mg. In all pathological thyroid tissue studied, two orders of binding sites were found with dissociation constants similar to those of normal tissues, but the number of binding sites was markedly reduced. Both orders of binding sites in solitary "cold" adenomas and only the low affinity sites in thyroid tissue from patients with Graves' disease were significantly reduced in number (P less than 0.01). There was a questionable decrease in high affinity sites in the Graves' tissue (P = 0.05). We have found the definite presence but a decreased number of binding sites in both orders of receptors in papillary and follicular carcinomas. There were few or no binding sites in medullary carcinoma.     

Epidermal growth factor receptors in plasma membranes of normal and diseased human thyroid glands

We studied the characteristics of epidermal growth factor (EGF) receptors in plasma membrane fractions derived from normal and diseased human thyroid tissues. The mean maximal specific binding of EGF to membrane fractions of normal thyroid tissue (n = 25) was 1.46 +/- 0.47 (+/- SD) fmol/mg protein. The maximal specific binding was higher than the upper limit of the normal range (2.40) in 12 of the 39 (31%) differentiated carcinomas, 2 of the 3 (67%) undifferentiated carcinomas, and 1 squamous cell thyroid carcinoma. In contrast, the maximal specific binding in samples derived from adenomas (1.13 +/- 0.91), adenomatous goiters (0.92 +/- 0.56), and hyperplastic (Graves') thyroids (1.57 +/- 0.61) was not different from that in normal thyroid tissue. Scatchard plot analysis revealed that all thyroid membrane fractions had two classes of specific receptors for EGF. The mean association constant for the high affinity EGF receptors in normal thyroid tissue was 7.9 +/- 2.9 (+/- SD) X 10(9) mol/L-1, and the capacity was 22.9 +/- 7.0 fmol/mg protein. The capacity of the high affinity receptors was higher (P less than 0.05) in differentiated carcinoma (37.2 +/- 25.5) and undifferentiated carcinoma (32.7 +/- 11.6) than in normal thyroid tissue. In one squamous cell carcinoma, the capacities for the two classes of binding sites were about 15-fold greater than in normal thyroid tissue. In contrast, the association constants of the high affinity receptors from carcinomas (differentiated, 6.9 +/- 2.8; undifferentiated, 11.8 +/- 4.1; squamous cell, 8.2) were similar to that of normal thyroid tissue. In the thyroid tissues from eight patients with Graves' disease the capacity of the high affinity binding sites (37.5 +/- 12.3 fmol/mg protein) was higher than that in normal tissue, but the affinity (4.4 +/- 1.6 X 10(9) mol/L-1) was less, and the maximal specific binding was similar in the two types of tissue. These results suggest that a significant increase in the number of high affinity EGF receptors may play a role in the pathogenesis of human thyroid carcinoma.     

Expression of type I insulin-like growth factor receptors on human peripheral blood mononuclear cells.

Insulin-like growth factor-I and -II (IGF-I and IGF-II), both of which bind to type I IGF receptors, can modulate certain functions of the immune system. We, therefore, studied the expression of type I IGF receptors on purified subpopulations of peripheral blood mononuclear cells. Using two-color flow cytometry, specific binding of a monoclonal antibody directed against the type I IGF receptor (alpha IR3) was found in every subpopulation. Relatively high numbers of receptors were detected on monocytes, natural killer cells, and CD4+ T-helper cells, an intermediate number of receptors on CD8+ suppressor/cytotoxic T-cells, and a relatively low number of receptors on B-cells. The presence of these receptors was confirmed by specific binding of [125I] IGF-I to purified subpopulations. alpha IR3 inhibited the binding of [125I] IGF-I. The specific binding of [125I]IGF-I to monocytes could be completely inhibited by IGF-II and insulin, but higher doses of these peptides were needed than of IGF-I. Scatchard analysis revealed the presence of 734 +/- 426 receptors/monocyte, with a Kd of 0.23 +/- 0.05 nM. A lower number of receptors (230 +/- 52), but with a higher affinity (Kd = 0.05 +/- 0.02 nM), was found on purified T-cells. The positive effect of IGF-I on natural killer cell cytotoxicity indicates that the type I IGF receptors on this cell type are functional. The possibility that IGF-I mediates hormonal effects on the immune system is discussed.     

Uterine insulin-like growth factor-I receptors: regulation by estrogen and variation throughout the estrous cycle

This study was undertaken to identify uterine insulin-like growth factor-I (IGF-I) receptors and examine the regulation of these receptors throughout the estrous cycle and after 17 beta-estradiol (E2) administration to immature rats. We have demonstrated type I IGF receptors in crude uterine membranes by binding and cross-linking experiments. The characteristics of the uterine IGF-I receptor are similar to those reported for other tissues, with a high affinity component (Kd = 0.12 nM; binding capacity = 0.028 pmol/mg protein) and a low affinity component (Kd = 0.98 nM; binding capacity = 0.041 pmol/mg protein). Autoradiographic visualization of [125I]IGF-I binding to uterine sections localized the IGF-I receptors to the smooth muscle cells of the myometrial layer of the uterus, with a higher density of IGF-I receptors in the outer longitudinal layer than in the inner circular layer. In immature rats administration of E2 significantly increased total [125I]IGF-I binding per uterus as early as 6 h after E2 injection. Although [125I]IGF-I binding was significantly increased per mg DNA, because of a more marked increase in membrane protein after E2, [125I]IGF-I binding, when expressed per mg membrane protein, decreased. This change in [125I]IGF-I binding resulted from a change in receptor number with no change in receptor affinity. In mature cycling rats, the proestrous uteri showed the lowest level of [125I]IGF-I binding per mg membrane protein, although because of the greater yield of protein from proestrous uteri, the total [125I]IGF-I-binding capacity of these uteri were greater than that of uteri from other stages of the estrous cycle. The lowest [125I]IGF-I binding was seen in the diestrous uteri. These studies demonstrate the presence of authentic type I IGF-I receptors in the rat uterus localized predominantly to the myometrial smooth muscle cells. In addition, E2 appears to regulate the uterine IGF-I receptor in the immature rat, and the cyclical changes in the mature rat are consistent with a role of E2 in regulation of this receptor in vivo.     

Insulin-like growth factor-I receptors in nonfunctioning thyroid nodules

We have recently demonstrated the production of insulin-like growth factor-I (IGF-I) as well as the presence of type I IGF receptors in human thyroid cells in primary culture. The role of IGF-I in the control of thyroid cell growth has been well established. In order to investigate the involvement of IGF-I in abnormal thyroid growth, the density of IGF-I receptors in solitary, cold, micro- and macro-follicular thyroid adenomas, and in extranodular histological normal tissue was studied. Forty-three euthyroid patients with isolated cold nodules were selected for the study. In 30 patients the presence of IGF-I receptors was evaluated by using quantitative immunohistochemistry; in 10 patients by using radioligand binding studies, and in 3 patients by using affinity labeling. Cross-linking and binding studies clearly demonstrated the presence of a homogeneous class of binding sites for type I IGF receptors. Furthermore, radioligand studies did not show any significant differences in receptor density between the 2 types of thyroidal tissues. Conversely, the computerized analysis of 900 fields of nodular and normal thyroid tissues immunostained with the monoclonal antibody alpha-IR3, strongly indicated that higher concentrations of IGF-I receptors were present in the epithelial cells of non-functioning thyroid nodules than in the adjacent extranodular thyroid tissues. These studies strongly suggest that the same type I IGF receptor is present in thyroid follicular adenomas as in histological normal thyroid tissue removed from the same patient. The higher concentration of IGF-I receptors as documented by immunostaining in the adenomas suggests that IGF-I may contribute to the abnormal growth of the neoplasms.     

Distribution and characterization of insulin and insulin-like growth factor I receptors in normal human ovary

Insulin-resistant hyperinsulinemic states are now widely known to be associated with ovarian hyperandrogenism, and this is thought to be due to an action of insulin on the ovary. However, the identity of the receptor that is responsible for insulin action in these patients, whose insulin receptors on classical target tissues are severely impaired, is unclear. We now report the presence of insulin receptors in stromal and follicular compartments as well as in granulosa cells obtained from normal ovaries. After 15-h incubations at 4 C with [125I]insulin and tissue fragments, specific insulin binding was 6-19% and 7-13%/mg protein (n = 8) to stroma and theca, respectively. Granulosa cells obtained in the course of in vitro fertilization were separated from red cells on a Percoll gradient; specific insulin binding ranged from 9-15%/10(6) cells. Insulin binding was characterized by sensitive insulin competition (half-maximal, 10 ng/ml), appropriately shifted proinsulin competition (20 times to the right), and complete inhibition by specific anti-insulin receptor antibodies (B-2). An antibody to the insulin-like growth factor I (IGF-I) receptor (alpha IR-3) that inhibits IGF-I binding to IGF-I receptors in other cell systems had no effect on insulin binding. Further proof that this binding is to classic insulin receptors was obtained from measurement of insulin-stimulated receptor autophosphorylation. When insulin receptors from stroma were extracted with Triton X-100 and incubated with [gamma-32P]ATP and Mn, insulin increased the incorporation of 32P into the beta-subunit of the receptor 5-fold. In parallel studies with [125I-]IGF-I and specific blocking antibodies to its receptor, no detectable IGF-I binding to stroma or follicles was found. We conclude that specific high affinity insulin receptors possessing tyrosine kinase activity are widely distributed in normal human ovary. IGF-I receptors in normal ovary are either absent or present at very low density. Binding of insulin to its own receptor (as opposed to IGF-I receptors) appears to be the most likely first step in the stimulation of ovarian steroidogenesis by insulin in normal ovaries and possibly in insulin-resistant states as well.     

Characterization of insulin-like growth factor I receptor on human erythrocytes

[125I]Insulin-like growth factor I (IGF-I) specifically bound to erythrocytes; the binding was saturable, and time and temperature dependent. Steady state binding was reached at 16 h at 4 C, and specific binding averaged 14.3 +/- 0.7% (+/- SEM) at a concentration of 3.6 X 10(9) cells/ml in seven normal subjects. [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose-dependent manner. Scatchard analysis indicated a linear plot, and Ka and number of binding sites/cell were 1.43 +/- 0.07 X 10(9) M-1 and 20.7 +/- 2.2, respectively. Compared to IGF-I, the relative potencies of multiplication-stimulating activity and insulin for displacing [125I]IGF-I binding were 20% and 1%, respectively. [125I]IGF-I binding to erythrocytes from patients with acromegaly was lower than binding to cells from pituitary dwarfs. An inverse correlation between plasma IGF-I level and the number of IGF-I-binding sites per cell was found (r = -0.75; P less than 0.005). These results demonstrate that [125I]IGF-I binding to erythrocytes can be used for clinical measurement of the IGF-I receptor.     

Insulin-like growth factor-I receptors in human glial tumors

Insulin and insulin-like growth factors (IGFs) are anabolic effectors in many tissues and cultured cells, including astrocytes and neurons. Receptors for insulin and IGFs are found throughout the human brain. We examined the level of insulin and IGF receptors on membranes prepared from surgical specimens of tumor (astrocytomas and glioblastomas) and normal human brain. Specific binding (per 100 micrograms membrane protein) of insulin was less than 5% in all normal and tumor samples. Specific binding of IGF-I to 12 normal brain specimens ranged from 1-8%. IGF-I binding to 18 glioma specimens ranged from 2-25%. Scatchard analyses of IGF-I binding confirmed increased IGF-I-binding sites in some glial tumors vs. normal brain, but detected no difference in affinity characteristics. Cross-linking of [125I]IGF-I demonstrated that glioma tissue expressed the same lower mol wt (approximately 118 kDa) alpha-subunit as the normal brain confirming the neural origin of the cells expressing the IGF-I receptor. IGF-binding proteins (approximately 40 kDa) were also found in the membranes of some of the glioma but none of the normal brain specimens. In cell lines derived from glioma specimens, IGF binding was readily detectable (4-10% specific binding), but insulin binding was barely detectable (0-03%) in every line examined. The size of the IGF-I alpha-subunit in the cultured cells was larger (approximately 133 kDa) than that in the original tissue. Most glioma cell lines exhibited an IGF-I dose-dependent stimulation of thymidine incorporation into DNA, and partially purified IGF-I receptors from these cells exhibited a dose-dependent stimulation of the autophosphorylation of the beta-subunit. We conclude that human glioma cells have functional IGF-I receptors and suggest a role for this receptor in glioma cell growth.     

Identification of endothelin receptor subtypes in human renal cortex and medulla using subtype-selective ligands.

High affinity and high density endothelin (ET)-binding sites were identified in membranes prepared from human kidney cortex and medulla. Saturation binding experiments performed in membranes prepared from cortex and medulla using [125I]ET-1 and [125I]ET-3 revealed that the proportion of [125I]ET-3-binding sites was 30-35% less than that of [125I]ET-1-binding sites. The apparent dissociation constants and maximum binding for [125I]ET-1 and [125I]ET-3 to membranes from cortex were 91 +/- 5 pM and 165 +/- 10 fmol/mg protein, and 117 +/- 9 pM and 110 +/- 7 fmol/mg protein, respectively, whereas in medulla they were 139 +/- 10 pM and 360 +/- 11 fmol/mg protein, and 142 +/- 11 pM and 245 +/- 15 fmol/mg protein, respectively. In the presence of 10 nM sarafotoxin-6c, which is selective for ETB receptors, [125I]ET-1 binding was decreased by 65-70%, whereas [125I]ET-3 binding was totally abolished, suggesting that 65-70% of [125I]ET-1 binding and 100% of [125I]ET-3 binding was to ETB receptors. This was further confirmed by the use of a cyclic pentapeptide [cyclo(D-Trp,D-Asp,L-Pro, D-Val,L-Leu)] (BQ123), which is selective for ETA receptors. In the presence of 1 microM BQ123, [125I]ET-1 binding was decreased by 25-30%, whereas [125I]ET-3 binding was unaffected, confirming that 30-35% of ET receptors belong to the ETA subtypes, and that [125I]ET-1 bound to both ETA and ETB receptors with the same high affinity, but [125I]ET-3 bound only to ETB receptors with high affinity. These results suggest that human kidney cortex and medulla contain ETA and ETB receptors in a ratio of 30:70, and that sarafotoxin-6c and BQ123 are valuable tools in identifying the subtype of ET receptors in various tissues.     

Soluble 34K binding protein inhibits the binding of insulin-like growth factor I to its cell receptors in human secretory phase endometrium: evidence for autocrine/paracrine regulation of growth factor action

The human secretory phase endometrium synthesizes and secrets a 34K insulin-like growth factor (IGF)-binding protein designated placental protein 12. We now report that membrane preparations of human endometrium possess IGF receptors that complete with the soluble binding protein for binding to IGF-I. Multiplication-stimulating activity and insulin were 1% and 0.1% as potent as recombinant (Thr59)IGF-I in inhibiting the binding of [125I](Thr59)IGF-I to endometrial membranes. Scatchard plots for the IGF-I binding data were curvilinear, and the apparent affinities [Ka = 1.4 +/- 0.2 ( +/- SEM) X 10(9) M-1] for (Thr59)IGF-I (high affinity site) did not change during the menstrual cycle. Affinity cross-linking of [125I](Thr59)IGF-I to endometrial membranes revealed two major bands with mol wt of 130K and 260K on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The 130K band is consistent with the alpha-subunit of the type I IGF receptor. The 260K band is either the type II IGF receptor or represents cross-linking (dimer) of two alpha-subunits of the type I receptor. A less intense band at about 40K was also seen in all membrane preparations. It comigrated with the cross-linked purified 34K binding protein. The band was more intensely labeled when the tracer was cross-linked to proteins in the cytosol fractions of late secretory phase endometria. By specific RIA, the 34K binding protein was detected in the cytosol of late secretory phase endometria only. Newly synthesized binding protein, which contaminated membrane preparations, caused an apparent increase in the binding of (Thr59)IGF-I to the membranes prepared from late secretory phase endometria when studied by competitive binding assay. In contrast, purified binding protein prevented the binding of [125I](Thr59)IGF-I to membrane receptors, as confirmed by affinity cross-linking. These results suggest that the 34K IGF-binding protein, secreted by the human endometrium in a cyclic fashion, has a significant role in inhibiting the receptor binding and, thus, the possible biological action of IGF-I in the endometrium in an autocrine/paracrine manner.     

Characterization and regulations of the receptor for insulin-like growth factor-I in the FRTL-5 rat thyroid follicular cell line

In previous studies we have shown that insulin-like growth factor I (IGF-I) has a mitogenic effect in a line of rat thyroid follicular cells, the FRTL-5. In view of this effect, we undertook studies to identify and characterize some physicochemical and binding properties of the receptor for IGF-I in these cells and to determine what role it plays in the mitogenic activity of insulin and insulin-like growth factors in the FRTL-5 cell. Binding of 125I-labeled IGF-I (biosynthetic Thr59-IGF-I) to FRTL-5 was a function of time, temperature, and pH and was completely inhibited by high concentrations of unlabeled IGF-I. Scatchard plots of four saturation studies revealed a single apparent binding site with an average Ka of 4.2 +/- 0.6 X 10(9) M-1 (mean +/- SD) and an average maximum binding capacity of 20 +/- 2 pm/100 micrograms cellular protein. Rat IGF-II (rIGF-II) and insulin were far less potent that IGF-I in inhibiting the binding of [125I] IGF-I, and bovine TSH was without effect. 125I-Labeled IGF-II also bound to FRTL-5 cells. Binding was completely inhibited by unlabeled rIGF-II and, with lesser potency, by IGF-I. Even at high concentrations, insulin failed to inhibit the binding of [125I]IGF-II. Disuccinimidyl suberate cross-linked [125I]IGF-I to a moiety in FRTL-5 that had an apparent mol wt of approximately 135,000, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Cross-linking of [125I]IGF-I was inhibited in a dose-dependent manner by unlabeled IGF-I and, with far lower potency, by rIGF-II and insulin. All three peptides stimulated the incorporation of [3H]thymidine into the DNA of FRTL-5 cells, IGF-I being the most potent, followed in decreasing order of potency of rIGF-II and insulin. The mitogenic activities of these polypeptides correlated well with their abilities to inhibit the binding of [125I]IGF-I. These data indicate that the FRTL-5 cell possesses a receptor for IGF-I that resembles in its binding and physicochemical properties the receptor for IGF-I in other tissues (type I IGF receptor) and that mediates the mitogenic response to IGF-I and insulin in these cells. FRTL-5 cells also contain a receptor for IGF-II (type II IGF receptor), but its role vis-à-vis that of the type I IGF receptor in relation to the mitogenic effect of IGF-II in these cells is uncertain.     

Expression of atypical and classical insulin receptors in Chinese hamster ovary cells transfected with cloned cDNA for the human insulin receptor

Human placenta and IM-9 lymphocytes contain subpopulations of atypical insulin receptors which differ from classical insulin receptors in their higher binding affinity for insulin-like growth factors I and II (IGF-I and IGF-II). Both types of insulin receptors may be derived from different but related genes, or may represent alternative post-translational modifications of the same gene product. To test these possibilities, we have examined the IGF binding characteristics of the human insulin receptors expressed in Chinese hamster ovary (CHO) cells which had been stably transfected with cloned human insulin receptor cDNA (CHO-T cells). The parent CHO cells contained 3 x 10(3) rodent insulin receptors/cell, and the CHO-T cells, 2.0 x 10(6) human insulin receptors/cell. Competition binding studies showed that the binding of [125I]IGF-I and [125I]multiplication stimulating activity (MSA/rat IGF-II) to parent CHO cells was primarily to type I and II IGF receptors, which cross-react poorly or not at all with insulin. However, competition binding studies with CHO-T cells showed that [125I]IGF-I binding was displaced 60-70%, and [125I]MSA binding, 50-55%, by low concentrations of insulin (20 ng/ml) and no further by higher concentrations of insulin (500 ng/ml). The insulin-insensitive IGF binding sites corresponded to the rodent type I and II IGF receptors; the insulin-sensitive IGF binding sensitive sites resembled the human atypical insulin receptors in that they bound IGF-I and MSA with moderately high affinity and reacted with insulin, MSA, and IGF-I in that order of potency. Atypical insulin receptors were also demonstrated by insulin-sensitive [125I]IGF-I and [125I]MSA binding to solubilized CHO-T proteins adsorbed to microtiter wells coated with monoclonal antibodies specific for the human insulin receptor. These results suggest that atypical human insulin receptors are generated by differential post-translational processing of the same gene product as classical human insulin receptors.     

Characterization of the thyrotropin receptor-adenylate cyclase system in neoplastic human thyroid tissue

Experiments were performed to determine whether the TSH receptor-adenylate cyclase (AC) system in benign and malignant thyroid neoplasms differs from the TSH receptor-AC system in normal thyroid tissue removed from the same patients. TSH binding and AC assays were performed using the same in vitro conditions. TSH binding was rapid, reversible, saturable, and hormone specific in particulate fractions from both normal and neoplastic thyroid tissue. A positive correlation existed between the equilibrium constants for [125I]bovine ([125I]bTSH) TSH binding and the concentration of TSH required to activate AC, suggesting that binding sites were coupled to AC in neoplastic thyroid tissue. Mean values for dissociation constants (Kd1 and Kd2), capacity (site 2), as determined by Scatchard analysis, and nonspecific binding (NSB) for the TSH receptors were lower in neoplastic thyroid. Some normal thyroid tissue appeared to lack a high affinity site, and some tumors lacked a low affinity binding site. Hormone specificities (bTSH, human (h) TSH, hLH, hFSH, hGH, hACTH, and glucagon) in normal thyroid and neoplastic tissue were virtually identical. hFSH, hACTH, hGH, and glucagon failed to inhibit [125I]bTSH binding or stimulate AC in either normal or neoplastic thyroid tissue, whereas hLH inhibited [125I]bTSH binding and stimulated AC, but required 10- to 100-fold higher concentrations than hTSH or bTSH. The specific binding and NSB of [125I]bTSH in both normal and neoplastic thyroid tissue was highest at pH 7.0 and lowest at pH 8.3. In contrast to bTSH binding, TSH stimulation of AC was lowest at pH 7.0 in both normal and neoplastic tissues and highest at pH levels of 7.5-8.0. TSH binding and TSH stimulation of AC activity were highest in the absence of NaCl and decreased progressively as the salt concentration was increased in both normal and neoplastic thyroid tissues. Increasing the sucrose concentration and, thus, the osmolarity of the system had a minimal effect on the binding of [125I]bTSH. Preincubation with ammonium sulfate did not significantly influence binding. Basal AC activity and the AC response to TSH were greater in neoplastic thyroid than in normal tissues. These studies demonstrate that changes in salt concentration and pH affect the TSH receptor-cyclase system in a comparable fashion in normal and neoplastic thyroid tissues. The discriminatory properties of the TSH receptor are also maintained in thyroid neoplasms. Thyroid tumors, however, have a higher affinity for TSH and display a greater AC response to TSH than normal thyroid tissue.     

An atypical insulin receptor with high affinity for insulin-like growth factors copurified with placental insulin receptors

Insulin receptors purified from human placenta by sequential affinity chromatography on wheat germ lectin-agarose and insulin coupled to 1,1'-carbonyldiimidazole-activated agarose (CDI-agarose) retained full binding activity but bound a greater than predicted amount of 125I-labeled insulin-like growth factor I (IGF-I). IGF-I and multiplication-stimulating activity (MSA; the rat homologue of IGF-II) were equipotent in displacing either 125I-labeled IGF-I or 125I-labeled MSA from the purified receptors; insulin was 5-15 times more potent. Competitive binding studies indicated that this IGF binding activity could not be explained by cross-reaction with classical insulin receptors or by coelution of IGF-I or IGF-II receptors. Instead, it was due to a minor population of discrete atypical insulin receptors (6-18% total insulin receptors) with moderately high affinity (Kd = 2-4 X 10(-9) M) for IGF-I and MSA. These receptors were not an artifact of insulin-CDI-agarose chromatography, since they were present in wheat germ lectin-agarose-purified preparations and could also be purified from insulin-succinyldiaminodipropylamino-agarose. Affinity labeling with 125I-labeled MSA revealed that these atypical receptors had the same binding subunit (Mr 140,000) as classical insulin and IGF-I receptors. They displayed intermediate reactivity with polyclonal and monoclonal antibodies to the insulin and IGF-I receptors. It is therefore likely that insulin receptors purified by immunoadsorption would also contain atypical insulin receptors. The finding of more than one type of insulin receptor might relate to the slight variations in the cDNA nucleotide sequences and the multiple mRNA species reported for the insulin receptor [Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J.-H., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A. & Rutter, W. J. (1985) Cell 40, 747-758].     

The interaction of proinsulin with the insulin-like growth factor-I receptor in human liver, muscle, and adipose tissue.

Because of the sequence homology and tertiary structure similarities between proinsulin (PI) and insulin-like growth factor-I (IGF-I), it is possible that PI interacts with the IGF-I receptor with higher affinity than insulin. To test this hypothesis in man, we have partially purified IGF-I receptors from liver, muscle, and adipose tissue and studied their interaction with PI, insulin, IGF-I, and IGF-II. With some tissue to tissue variation, [125I]insulin binding was 4- to 8-fold greater than IGF-I binding. Unlabeled IGF-I at about 1 x 10(-9 M, IGF-II at about 1 x 10(-8) M, and insulin at about 1 x 10(-6) M displace 50% the binding of [125I]IGF-I to its receptor, whereas PI at 1 x 10(-6) M displaces less than 20% of the binding of [125I]IGF-I to its receptor. We conclude that in human liver, muscle, and adipose tissue, PI does not interact with the IGF-I receptor at a higher affinity than insulin, and the affinity of IGF-I receptors is several-fold lower than that of insulin receptors. It is, therefore, unlikely that if PI were to be administered to man any of its biological effects would be by interacting with the IGF-I receptor.     

Functional insulin-like growth factor I receptors are expressed by neural-derived continuous cell lines

High affinity insulin-like growth factor I (IGF-I) receptors are expressed by two human neural derived cell lines, SK-N-SH and SK-N-MC. Specific [125I]IGF-I binding to crude membranes was 23.4% for SK-N-SH and 10.7% for SK-N-MC, with 50% inhibition of binding by unlabeled IGF-I between 0.6-0.7 nM. Scatchard analysis of crude membrane binding was linear, whereas Scatchard analysis after wheat germ agglutinin purification of the receptor became curvilinear. The IGF-I receptor alpha-subunits of SK-N-SH have an apparent Mr of 126K, whereas that for SK-N-MC is 132K. Despite these differences in alpha-subunit structure both cell lines demonstrate IGF-I-induced autophosphorylation of their own beta-subunits as well as specific IGF-I induced tyrosine kinase activity, suggesting normal coupling between the ligand-binding alpha-subunit and the tyrosine kinase-containing beta-subunit. Furthermore, IGF-I stimulated iododeoxyuridine uptake in both SK-N-SH and SK-N-MC in a dose-dependent manner, suggesting that these cells may be used to study the role of IGF-I action on neural tissues.