insulin-like growth factor-I hybrid receptors in human tissues

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Molecular and Cellular Endocrinology 129 (1997) 121 126

Distribution of insulin/insulin-like growth factor-I hybrid receptors in human tissues M.

Federici, O.

Porzio, L. Zucaro, A. Fusco, P. Borboni, D. Lauro, G. Sesti *

Laboratory of Molecular Medicine, Department of Internal Medicine, University oJ Rorne-'Tor Vergata', Via Orazio Raimondo, 00173 Rome, Italy

Received 4 December 1996; received in revised form 19 February 1997; accepted 19 February 1997

Abstract Insulin receptors (IR) and type 1 IGF receptors (IGF-IR) have been shown to form insulin/IGF-I hybrid receptors in tissues expressing both molecules. The biological function of hybrid receptors is still undefined. To date there is no information about the distribution of hybrid receptors in human tissues. We have applied two microwell-based immunoassays which are capable of quantitating hybrid receptors in small samples of human tissues and cells. Results demonstrated that the proportion of total IGF-IR assembled as hybrids varied between 40 and 60%, thus indicating that hybrid receptors account for a large fraction of total IGF-I binding in human tissues. A significant fraction of total IR was assembled as hybrids in the tissues examined, varying from 37% in placenta to 45% in hepatoma, with the exception of adipose tissue where the fraction of insulin receptors forming hybrids was 17%. Because hybrid receptors bind IGF-I, but not insulin, with high affinity, it is likely that in human tissues hybrid receptors may be primarily activated by 1GF-I rather than insulin under physiological conditions. Therefore, differences in hybrid receptors distribution may contribute to regulate tissue sensitivity to insulin and IGF-I by sequestering insulin receptor c~fl-heterodimer in an IGF-I responsive form. © 1997 Elsevier Science Ireland Ltd. Key~eords: Insulin receptor; Type 1 IGF receptor; Hybrid receptor

1. Introduction Type 1 insulin-like growth factor (IGF) receptors ( I G F - I R ) and insulin receptors (IR) are highly homologous tyrosine kinase receptors, each consisting of two ~fl-heterodimers linked together by disulfide bonds to yield the mature ~2fl2 heterotetrameric receptor [1-3]. The c~-subunits are entirely extracellular and contain the high affinity hormone binding site(s). The transmembrane fl-subunits possess the hormone-stimulated tyrosine kinase activity in their cytoplasmic domain which plays a crucial role in signal transduction. There is evidence that hybrid receptors comprised of an insulin receptor ~fl-heterodimer and a type 1 I G F *Corresponding author. Tel.: +39 6 72596528; fax: +39 6 72596538; e-mail: [email protected]

receptor ~fl-heterodimer are formed in tissues co-expressing both molecules [4 6]. Insulin/IGF-I hybrid receptors are also assembled in vitro under defined ligand incubation conditions [7]. The functional significance of hybrid receptors is yet unclear. Studies with transfected cells overexpressing the h u m a n insulin receptor or with affinity-purified hybrid receptors have shown that hybrid receptors bind I G F - I with an affinity similar to that of type 1 I G F receptors, but bind insulin with lower affinity than classic insulin receptors [8,9]. Furthermore, hybrid receptors behave as type 1 I G F receptors rather than an insulin receptor in terms of receptor autophosphorylation, hormone internalization and degradation [9-11]. Therefore, the presence of insulin/IGF-I hybrid receptors would be expected to affect insulin binding, and, thereby, insulin sensitivity in tissues co-expressing both receptors. To date there is

0303-7207/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S0303-7207(97)04050-1

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M. Federici et al./ Molecular and Cellular Endocrinology 129 (1997) 121-126

limited information about the distribution of hybrid receptors in human tissues. To address this, we have developed and applied two microwell-based immunoassays which are capable of determining the distribution of insulin/IGF-I hybrid receptors in various human tissues and cells.

2. Materials and methods

2. I. Materials Human [125I]A14-monoiodoinsulin (290-320,/~Ci//zg) and [125I]IGF-I (280-310 #Ci//~g) were purchased from Amersham (Buckinghamshire, UK). Recombinant human insulin was kindly provided by Novo-Nordisk A/S (Bagsva~rd, Denmark). Recombinant human IGF-I was purchased from Boehringer Mannheim (Mannheim, Germany). c~-IGF-IR-PA, an an~ti-IGF-IR polyclonal antibody which does not cross-react with the insulin receptor, was raised in rabbit against a synthetic peptide corresponding to residues 642-661 of the type 1 IGF-I receptor c~-subunit sequence [3], according to previously described methods [12]. MA-20 and MA-10 anti-insulin receptor monoclonal antibodies which do not cross-react with type 1 IGF receptors were produced as previously described [13]. eIR3 anti-IGF-IR monoclonal antibody was purchased from Oncogene Science (Manhasset, NY). Antibodies were radioiodinated using the I O D O G E N method (Pierce Chemical, Rockford, IL) as previously described [14]. All other chemicals were from Sigma (St. Louis, MO). 2.2. Subjects Fresh human placentae were obtained from normal women after full term vaginal deliveries. Samples of subcutaneous adipose tissue and rectus abdominus skeletal muscle were obtained from nondiabetic subjects who underwent surgery for uncomplicated gallstone disease. Samples of human hepatoma were obtained from patients who underwent surgery for subtotal hepatectomy. Tissue samples were cleaned of all connective tissue and blood, immediately frozen in liquid nitrogen, and stored at - 7 0 ° C until use. Mononuclear leukocytes and erythrocytes were obtained from 100 ml blood of normal subjects using the Ficoll Hypaque procedure for separation. Fibroblasts from normal subjects were obtained by forearm skin biopsies. All subjects had normal fasting plasma glucose levels, normal blood pressure, and no family history of diabetes, and had not taken any other medication known to alter insulin or glucose metabolism. Consent was obtained from all subjects after the nature of the procedure was explained.

2.3. Tissue and cell solubilization Extracts from tissue specimens and mononuclear leukocytes were prepared by homogenization with polytron for 30 s at 4°C in 50 mM HEPES buffer, pH 7.6, containing 150 mM NaC1, 1 mg/ml bacitracin, 2 mM PMSF, 1000 U/ml aprotinin. Homogenates were centrifuged at 15 000 x g for 20 min at 4°C, and supernatants were solubilized in the same buffer containing 1% Triton X-100, for 60 min at 4°C. Insoluble material was removed by centrifugation at 100 000 x g for 60 min at 4°C, and soluble fractions were diluted to 0.2% Triton X-100 and immediately assayed. Membranes and solubilized receptors from erythrocytes were prepared as previously described [15]. Protein content of tissue and lysates was determined by the Bradford method [16].

2.4. Microwell immunoassay The assay was performed as previously described [17]. Briefly, 96 well microwells were coated with 50/~1 of e-IGF-IR-PA (10 /~g/ml) or MA-20 antibody (4 /~g/ml) in 20 mM NaHCO3, pH 9.6, and incubated for 16 h at 4°C. The wells were washed three times with binding buffer containing 50 mM HEPES buffer, pH 7.6, 150 mM NaC1, 0.1% Triton X-100, 1 mg/ml bacitracin, 2 mM phenylmethylsulfonyl fluoride (PMSF), 1000 U/ml aprotinin, 0.1% bovine serum albumin (BSA), and incubated with tissue and cell lysates (500 #g in 40 /~1) for 16 h at 4°C. The wells were then washed three times with binding buffer, and immoadsorbed receptors were incubated in the same buffer with [125I]IGF-I (60 pM) or [125I]insulin (60 pM) for 16 h at 4°C in the presence or absence of various concentrations of unlabeled ligands. Thereafter, the wells were washed three times with binding buffer to remove unbound ligands. Radioactivity bound to immoadsorbed receptors was collected by adding 2% SDS for 30 min at 24°C to the wells, and counted in a y-counter.

2.5. Two Antibody Sandwich Assay Ninety six-well microwells coated with either ~-IGFIR-PA or MA-20 antibody were incubated with tissue and cell lysates (500/~g in 40 #1) for 16 h at 4°C. The wells were washed three times with binding buffer, and immoadsorbed receptors were incubated with [125I]cdR3 (30 pM) or [125I]MA-10 antibody (30 pM) for 16 h at 4°C. After washing three times the wells with binding buffer, radioactivity bound to immoadsorbed receptors was collected by adding 2% SDS for 30 min at 24°C to the wells, and counted. Nonspecific binding was determined by omitting receptor preparations.

M. Federici et al./Molecular and Cellular Endocrinology 129 (I997) 121-126

3. Results and discussion

3.1. Characterization of microwell-based immunoassay A previously validated microwell-based immunoassay was used to measure insulin and IGF-I binding to immunoadsorbed receptors from various human cells and tissues [17]. Microwells coated with either MA-20 or c~-IGF-IR-PA antibody were incubated with tissue and cell lysates, and ligand binding characteristics of immoadsorbed receptors were analyzed by inhibition binding studies. Fig. 1 shows representative competition-inhibition curves of insulin (A) and IGF-I (B) binding to immunoadsorbed receptors isolated from skeletal muscle, adipose tissue, and placenta. Binding of I Off A

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insulin and IGF-I to receptors displayed properties appropriate for each specific receptor. Insulin and IGFI binding to immunoadsorbed receptors displayed binding properties similar to those observed with their respective receptors in solution, as detemined by immunoprecipitation assay [17,18]. The mean values for total [125I]insulin and [125I]IGF-I binding are reported in Table 1. As expected, maximal specific insulin and IGF-I binding to immunoadsorbed receptors varied between tissues and cells. Thus, placenta, skeletal muscle, and hepatoma have higher levels of insulin binding as compared with adipose tissue, fibroblasts, mononuclear leukocytes, and erythrocytes. Receptor binding affinity, estimated as the concentration of unlabeled insulin required for half-maximal inhibition of [125I]insulin binding to immunoadsorbed receptors (ICs0), was similar in all cells and tissues tested (ICs0 = 0.4-0.6 nM insulin). Maximal specific IGF-I binding was higher in placenta, skeletal muscle, and hepatoma as compared with adipose tissue, mononuclear leukocytes, erythrocytes, and fibroblasts. ICso values for IGF-I binding to receptors was similar in all cells and tissues studied ranging from 0.4 to 0.7 nM IGF-I.

3.2. Quantitation of insulin/IGF-I hybrid receptors in various human tissues and cells

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IGF-I (M) Fig. 1. Inhibition of [125I]insulin or [125I]IGF_ I binding to imnmnoadsorbed receptors. Tissue extracts prepared from skeletal muscle (open circles), adipose tissue (close squares), and placenta (open triangles) were added to microwell coated with MA-20 anti-insulin receptor antibody (A) or c~-IGF-IR-PA (B). After washing, ligand binding to imnmnoadsorbed receptors was assessed by incubating the wells with [125I]insulin (A) or [125I]IGF-I (B) in the presence or absence of varying concentrations of unlabeled ligands. Data of three experiments carried out in triplicate are shown. Results of insulin (A) or IGF-I (B) binding competition expressed as percent of maximal specific binding are presented as mean + S.E.M. Maximal specific [125I]insulin and [125I]IGF-I binding expressed as percentage of total added counts (B/T) were: 6.0 _+ 0.9 and 3.3 _+ 0.7% for muscle, 4.1 _+ 0.7 and 0.9 ± 0.3% for adipose tissue, and 8.0 +_ 0.6 and 3.7 _+ 0.5% for placenta, respectively.

This assay was based on the ability of insulin/IGF-I hybrid receptors to bind IGF-I, but not insulin, with high affinity, and react with specific anti-insulin receptor antibody [8,9]. Tissue and cell lysates were added to microwell coated with MA-20 anti-insulin receptor antibody. MA-20-adsorbed receptors were then incubated with [125I]IGF-I (60 pM) in the presence or absence of increasing amounts of unlabeled IGF-I or insulin. Fig. 2 shows representative competition-inhibition curves of [125I]IGF-I binding to immunoadsorbed hybrid receptors isolated from skeletal muscle (A) and placenta (B). Binding of [125I]IGF-I to hybrid receptors was inhibited by low concentrations of unlabeled IGF-I (ICso = 0.3 _+0.05 riM), similar to those observed with type 1 IGF receptors. By contrast, low concentrations of unlabeled insulin were unable to compete for [125I]IGF-I binding to hybrid receptors (Fig. 2), thus indicating that no significant [125I]IGF-I binding to the insulin receptor occurred at the concentration of tracer used. Relative abundance of hybrid receptors quantified as the fraction of [125I]IGF-I binding immobilized with MA-20 antibody and expressed as percentage of total IGF-IR (type 1 + hybrids) immobilized with ~-IGF-IRPA antibody is reported Table 1. A high percentage of type 1 IGF receptors was assembled as hybrid receptors varying from 36% in erythrocytes to 55% in placenta. These data indicate that hybrid receptors account for a significant fraction of total IGF-I binding in human cells and tissues. However, there was no clear relation-

M. Federici et al./Molecular and Cellular Endocrinology 129 (1997) 121-126

124

Table 1 Distribution of insulin/IGF-I hybrid receptors in various h u m a n tissue and cells Tissue

Maximal specific insulin binding cpm/mg protein

Maximal specific IGF-I binding cpm/mg protein

Hybrid receptors % of total IGF-IR

Placenta Skeletal muscle Hepatoma Adipose tissue Mononuclear leukocytes Erythrocytes Fibroblasts

8000 6040 6600 4160 1590

3760 _+ 500 3300 _+ 700 3370 + 400 980 _+ 350 510 + 60

55 46 45 38 48

_+ 600 _+ 900 _+ 550 _+ 720 + 280

1600 _+ 300 440 _+ 60

590 _+ 50 390 _+ 80

_+ 2 _+ 4 _+ 3 + 3 _+ 2

36 _+ 5 53 + 5

Tissue and cell extracts were added to microwell coated with either ~ - I G F - I R - P A anti-IGF-I receptor or MA-20 anti-insulin receptor antibody. After washing, immoadsorbed receptors were incubated with [~25I]IGF-I or [~25I]insulin in the presence or absence of increasing concentrations of unlabeled ligand, and radioactivity b o u n d to immoadsorbed receptors was collected and counted. Nonspecific binding, defined as the binding in the presence of 10 -6 M insulin or 10 7 M IGF-I, was determined and subtracted. Relative abundance of hybrid receptors was quantified as the fraction of [125I]IGF-I maximal specific binding to immobilized-MA-20 anti-insulin receptor antibody and expressed as percentage of total I G F - I R (type 1 + hybrids) immobilized with ~ - I G F - I R - P A antibody. Results of three experiments carried out in triplicate are presented as mean _+ S.E.M.

ship in the various tissues and cells between the fraction of type 1 IGF receptors assembled as hybrid receptors and the ratio of IGF-I to insulin binding. This may in part reflect cellular heterogeneity of the tissues examined, and uneven distribution of type 1 IGF receptors and insulin receptors among different cell types. Further studies are required to address the question of whether the proportion of hybrid receptors is a simple function of the relative number of type 1 IGF receptors and insulin receptors expressed in tissues, or whether hybrid receptors formation is regulated by metabolic and hormonal factors.

3.3. Two antibody sandwich assay of insulin/IGF-I hybrid receptors Most of the tissues examined express higher levels of insulin receptors than type 1 IGF receptors, and therefore it is likely that the fraction of insulin receptors assembled as hybrids is lower than the fraction of type 1 IGF receptors. However, it is not possible to directly quantitate insulin receptor ~fi-heterodimers assembled as hybrids using the above described immunoassay because of differences in the ligand binding affinity between insulin receptors and hybrids. Therefore, to quantitate the fraction of insulin receptors assembled as hybrid receptors, we developed a two antibody sandwich assay which is based on the use of ~2SI-labeled anti-IGF-I or anti-insulin receptor antibody as tracer in place of their respective ligands. ~-IGF-IR-PA-adsorbed receptors were incubated with [125I]~IR3 antiIGF-I receptor antibody or 125I-MA-10 antiinsulin receptor antibody, and relative abundance of hybrids vs. total IGF-I receptors (type 1 +hybrids) was quantified as the ratio between [125I]MA-10 binding and [125I]cdR3 binding to c~-IGF-IR-PA-adsorbed receptors. As shown in Table 2, results were in close agreement

with those obtained using [~25I]IGF-I as tracer, and confirmed that a high percentage of IGF-I receptors was assembled as hybrids in human tissues. The fraction of insulin receptors assembled as hybrid receptors was determined by incubating MA-20-adsorbed receptors with [~25I]c~IR3 or [125I]MA-10 antibody. Relative abundance of hybrids vs. total insulin receptors was quantified as the ratio between [~25I]~IR3 binding and [125I]MA-10 binding to MA-20 adsorbed receptors. As shown in Table 2, a significant fraction of insulin receptors was assembled as hybrids in placenta, skeletal muscle, fibroblasts, and hepatoma, ranging from 37 to 45%, whereas the fraction of insulin receptors forming hybrids appears to be somewhat lower in adipose tissue (17%). It is possible that the two antibodies, MA-20 and e-IGF-IR-PA, used to quantitate hybrid receptors may modulate ligand binding to hybrids by inducing conformational changes. This possibility seems unlikely since both MA-20 and c~-IGF-IR-PA did not affect either [~25I]insulin or [125I]IGF-I binding to intact cells, particulate placental membranes or solubilized receptors at the concentrations used for the microwell-based immunoassay [13,17] (unpublished data). Furthermore, the affinity of both MA-20 and ~-IGF-IR-PA for hybrid receptors was similar to that for their respective receptors, as judged by the concentrations required for half-maximal immunoprecipitation of receptor[125I]hormone complexes (half-maximal precipitation of solubilized placental receptors affinity-labeled with either [125I]insulin or [125I]IGF-I occurring at 8 and 10 riM, respectively, with MA-20, and at 50 and 40 nM, respectively, with ~-IGF-IR-PA). In summary, we have developed a microwell-based immunoassay to assess the relative abundance of insulin/IGF-I hybrid receptors in small samples of human tissues. Using this method, we have provided the first information about the distribution of hybrid receptors

M. Federici et al./ Molecular and Cellular Endocrinology 129 (1997) 121 126

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in human tissues. Results demonstrate that hybrid receptors represent a high proportion of total type 1 IGF receptors, whereas the fraction of insulin receptors assembled as hybrids appears to be somewhat lower than the fraction of total type 1 IGF receptors. Several lines of evidence suggest that insulin binds to one c~-subunit through its dimer-forming surface and cross-links the other e-subunit through the hexamerforming surface [19]. IGF-I exhibits receptor binding properties similar to those of insulin including curvilinear Scatchard plots, 1:2 stoichiometry for high affinity binding, and ligand-accelerated tracer dissociation which are suggestive of negative co-operativity [19]. Table 2 Distribution of insulin/IGF-I hybrid receptors measured by two antibody sandwich assay Tissue

Hybrid receptors % of total IGF-IR

Hybrid receptors % of total IR

Placenta Skeletal muscle Adipose tissue Hepatoma Fibroblasts

52 49 39 40 61

37 -t- 6 42 _+ 3 17 -t- 4 45 _+ 2 40 _+ 2

_+ 8 _+4 _+ 6 + 9 _+ 7

Tissue and cell extracts were added to microwell coated with either c~-IGF-IR-PA anti-IGF-I receptor or MA-20 anti-insulin receptor antibody. After washing, immoadsorbed receptors were incubated with [1251]elR3 anti-IGF-I receptor or [I25I]MA-10 anti-insulin receptor antibody, and radioactivity bound to immoadsorbed receptors was collected and counted. Nonspecific binding was determined by omitting receptor preparations. Relative abundance of hybrids vs. total IGF-I receptors (type 1 +hybrids) was quantified as the ratio between [125I]MA-10 binding and [125I]~IR3 binding to c~-IGF-IRPA-adsorbed receptors (column 1). Relative abundance of hybrids vs. total insulin receptors was quantified as the ratio between [125I]eIR3 binding and [~25I]MAI0 binding to MA-20-adsorbed receptors (column 2). Results of three experiments carried out in triplicate are presented as mean _+ S.E.M.

However, the dose-response curve for IGF-I negative co-operativity is not bell-shaped, and resembles the monophasic curve observed with insulin analogues modified at the hexamer-forming surface which possess an enhanced mitogenic potency [19]. Therefore, the selectivity for metabolic versus mitogenic signaling for insulin and IGF-I, respectively, may result from differences in their binding kinetic properties. It is not known whether hybrid receptors have kinetic binding properties similar to those of the insulin receptor, the type 1 IGF receptor or some intermediate version of these receptors. However, the findings that hybrid receptors bind IGF-I, but not insulin, with high affinity raise the possibility that in hybrid receptors insulin binds to its e-subunit through the dimerforming surface, but does not cross-link the type 1 IGF receptor ~-subunit through the hexamer-forming surface. On the contrary, IGF-I binding to hybrid receptors is not subject to the same constraints since combination of a type 1 IGF receptor e-subunit with an insulin receptor e-subunit in hybrid receptors does not appear to affect high affinity IGF-I binding. This would be expected to have some biological relevance since IGF-I binding is associated with enhanced mitogenic activity. Such a scenario would be consistent with the hypothesis that in human tissues hybrid receptors may be primarily responsive to mitogenic effects of IGF-I rather than metabolic effects of insulin under physiological conditions. However, it is still unclear whether mitogenic and metabolic effects of both insulin and IGF-I can be equally signaled via hybrid receptors. Since trans-phosphorylation is the major mechanism of intramolecular autophosphorylation, IGF-I binding to hybrid receptors may activate cellular substrates specifically associated with the insulin receptor fl-subunit or common signaling different kinetics of activation thus exerting more pronounced

126

M. Federici et al./ Molecular and Cellular Endocrinology 129 (1997) 121-126

m e t a b o l i c effects in a d d i t i o n to t y p i c a l g r o w t h - p r o m o t i n g effects. Overall, these d a t a raise the p o s s i b i l i t y t h a t tissue-specific differences in e x p r e s s i o n o f h y b r i d receptors m a y c o n t r i b u t e to r e g u l a t e tissue sensitivity to insulin and IGFI.

Acknowledgements W e are g r a t e f u l to P r o f e s s o r R e n a t o L a u r o ( R o m e , Italy) for his advice, a n d h e l p f u l discussions. T h i s w o r k was s u p p o r t e d i n p a r t b y g r a n t s f r o m B I O M E D 2 E C - P r o g r a m m e n ° E R B B M H 4 C T 9 6 - 0 7 5 1 (G. Sesti), C o n s i g l i o N a z i o n a l e delle R i c e r c h e n. 9 5 . 0 0 9 0 8 . P F 4 1 a n d 9 6 . 0 3 7 2 4 . C T 1 4 (G. Sesti).

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[8] Soos, M.A., Field, C.E., and Siddle, K (1993) Purified hybrid insulin/insulin-like growth factor-I receptors bind insulin-like growth factor-I, but not insulin, with high affinity. Biochem. J. 290, 419-426. [9] Langlois, W.J., Sasaoka, T., Yip, C.C., and Olefsky, J.M. (1995) Functional characterization of hybrid receptors composed of a truncated insulin receptor and wild type Insulin-like growth factor 1 or insulin receptor. Endocrinology 136, 1978 1986. [10] Frattali, A.L., and Pessin, J.E. (1993) Relationship between a subunit ligand occupancy and fl subunit autophosphorylation in insulin/insulin-like growth factor-I hybrid receptors. J. Biol. Chem. 268, 7393-7400. [11] Seely, B.L., Reichart, D.R., Takata, Y., Yip, C.C., and Olefsky J.M. (1995) A functional assessment of insulin/insulin-like growth factor I hybrid receptors. Endocrinology 136, 1635 1641. [12] Sesti, G., D'Alfonso, R., Vargas Punti, M.D., Frittitta, L., Trischitta, V., Liu, Y.Y., Borboni, P., Longhi, R., Montemurro, A., and Lauro, R. (1995) Peptide-based radioimmunoassay for the two isoforms of the human insulin receptor. Diabetologia 38, 445 453. [13] Forsayeth, J.R., Montemurro, A., Maddux, B.A., De Pirro, R., and Goldfine, I.D. (1987) Effect of monoclonal antibodies on human insulin receptor autophosphorylation, negative cooperativity, and down-regulation. J. Biol. Chem. 262, 4134-4140. [14] Sesti, G., Marini, M.A., Montemurro, A., Condorelli, L., Borboni, P., Haring, H.U., Ullrich, A., Goldfine, I.D., De Pirro, R., Lauro, R., (1992) Evidence that two naturally occurring human insulin receptor c~-subunit variants are immunologicatly distinct. Diabetes 4t, 6 11. [15] Grigorescu, F., White, M.F., and Kahn, C.R. (1983) Insulin binding and insulindependent phosphorylation of the insulin receptor solubilized from human erythrocytes. J. Biol. Chem. 258, 13708 13716. [16] Bradford, M.M. (1978) 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. [17] Federici, M., Zucaro, L., Porzio, O., Massoud, R., Borboni, P., Lauro, D., and Sesti, G.(1996) Increased expression of insulin/insulin-like growth factor-I hybrid receptors in skeletal muscle of non-insulin-dependentdiabetes mellitus subjects. J. Clin. Invest. 98, 2887 2893. [18] Valensise, H., Liu, Y.Y., Federici, M., Lauro, D., Dell'anna, D., Romanini, C., Sesti, G. (1996) Increased expression of lowaffinity insulin receptor isoform and insulin/insulin-likegrowth factor I hybrid receptors in term placenta from insulin resistant women with gestational hypertension. Diabetologia 39, 952-960. [19] De Meyts, P. (1994) The structural basis of insulin and insulinlike growth factor-I receptor binding and negative cooperativity, and its relevance to mitogenic versus metabolic signalling. Diabetologia 37, [Suppl 2] 135-148.