doi:10.1016/S0303-7207(97)04050-1 
Copyright © 1997 Elsevier Science Ireland Ltd All rights reserved.
Distribution of insulin/insulin-like growth factor-I hybrid receptors in human tissues
M Federicia, O Porzioa, L Zucaroa, A Fuscoa, P Borbonia, D Lauroa and G Sesti
, a
aLaboratory of Molecular Medicine, Department of Internal Medicine, University of Rome-`Tor Vergata', Via Orazio Raimondo, 00173 Rome, Italy
Received 4 December 1996;
revised 19 February 1997;
accepted 19 February 1997.
Available online 16 September 1997.
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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 IGF-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 
β-heterodimer in an IGF-I responsive form.
Keywords: Insulin receptor; Type 1 IGF receptor; Hybrid receptor
Fig. 1. Inhibition of [125I]insulin or [125I]IGF-I binding to immunoadsorbed 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 -IGF-IR-PA (B). After washing, ligand binding to immunoadsorbed 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.
Fig. 2. Inhibition of [125I]IGF-I binding to immunoadsorbed hybrid receptors. Tissue extracts prepared from skeletal muscle (A) or placenta (B) of normal subjects were added to microwell coated with MA-20 anti-insulin receptor antibody. After washing, ligand binding to immunoadsorbed receptors was assessed by incubating the wells with [125I]IGF-I in the presence or absence of varying concentrations of unlabeled IGF-I (closed symbols) or insulin (open symbols). Data of three experiments carried out in triplicate are shown. Results of IGF-I binding competition expressed as percent of maximal specific binding are presented as mean±S.E.M. Maximal specific [125I]IGF-I binding expressed as percentage of total added counts (B/T) was: 1.5±0.7% for muscle (A), and 2.1±0.5% for placenta, respectively.
Table 1.
Distribution of insulin/IGF-I hybrid receptors in various human tissue and cells
Tissue and cell extracts were added to microwell coated with either -IGF-IR-PA anti-IGF-I receptor or MA-20 anti-insulin receptor antibody. After washing, immoadsorbed receptors were incubated with [125I]IGF-I or [125I]insulin in the presence or absence of increasing concentrations of unlabeled ligand, and radioactivity bound 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 IGF-IR (type 1+hybrids) immobilized with -IGF-IR-PA antibody. Results of three experiments carried out in triplicate are presented as mean±S.E.M.
Table 2.
Distribution of insulin/IGF-I hybrid receptors measured by two antibody sandwich assay
Tissue and cell extracts were added to microwell coated with either -IGF-IR-PA anti-IGF-I receptor or MA-20 anti-insulin receptor antibody. After washing, immoadsorbed receptors were incubated with [125I]IR3 anti-IGF-I receptor or [125I]MA-l0 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 -IGF-IR-PA-adsorbed receptors (column 1). Relative abundance of hybrids vs. total insulin receptors was quantified as the ratio between [125I]IR3 binding and [125I]MA10 binding to MA-20-adsorbed receptors (column 2). Results of three experiments carried out in triplicate are presented as mean±S.E.M.
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