Insulin receptor/IGF-I receptor hybrids are widely distributed in mammalian tissues: quantification of individual receptor species by selective immunoprecipitation and immunoblotting
| Autor: | Stephen R. Orr, Amanda C. Hayward, E M Bailyes, Barbara T. Navé, Maria A. Soos, Kenneth Siddle |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1997 |
| Předmět: |
medicine.drug_class
Receptor expression medicine.medical_treatment Placenta Blotting Western Immunoblotting Monoclonal antibody Biochemistry Radioligand Assay medicine Radioligand Animals Humans Receptor Muscle Skeletal Molecular Biology Insulin-like growth factor 1 receptor biology Insulin Cell Membrane Antibodies Monoclonal Receptors Somatomedin Cell Biology Molecular biology Precipitin Tests Receptor Insulin Insulin receptor Liver biology.protein Rabbits Protein Multimerization Spleen Research Article Protein Binding |
| Popis: | The insulin receptor (IR) and type 1 insulin-like growth factor (IGF-I) receptor (IGFR) are both widely expressed in mammalian tissues, and are known to be capable of heteromeric assembly as insulin/IGF hybrid receptors, in addition to the classically described receptors. By selective immunoadsorption of radioligand/receptor complexes and by immunoblotting we have determined the fraction of insulin receptors and IGF receptors occurring as hybrids in different tissues. Microsomal membranes were isolated from tissue homogenates and solubilized with Triton X-100. Solubilized receptors were incubated with 125I-IGF-I, and radioligand/receptor complexes bound by IR-specific and IGFR-specific monoclonal antibodies were quantified. The fraction of IGF-I binding sites behaving as hybrids (anti-IR-bound/anti-IGFR-bound) was approx. 40% in liver and spleen, 70% in placenta, and 85–90% in skeletal muscle and heart, similar results being obtained in rabbit and human tissues. There was no correlation between the proportion of hybrids and the ratio of 125I-insulin/125I-IGF-I binding in different tissues. The fraction of 125I-insulin bound to hybrids was too low for accurate quantification, because of the relatively low affinity of hybrids for insulin. The fraction of insulin receptors present in hybrids was therefore determined by immunoblotting. Receptors in solubilized human placental microsomal membranes were precipitated with IR-specific or IGFR-specific monoclonal antibodies, and after SDS/PAGE, blots were prepared and probed with IR-specific and IGFR-specific antisera. It was found that 15% of IR and 80% of IGFR were present in hybrids. Consistent with these figures, the overall level of IR was estimated, by blotting with the respective antibodies at concentrations shown to give equal signals with equal amounts of receptor, to be 4-fold greater than IGFR. Overall it was concluded that a significant fraction of both IR and IGFR occurs as hybrids in most mammalian tissues, including those that are recognized targets of insulin and IGF action. The fraction of hybrids in different tissues was not a simple function of the relative levels of IR and IGFR, possibly because of heterogeneity of receptor expression in different cell types. However, in placenta the proportions of IR, IGFR and hybrids were consistent with a process of random assembly reflecting the molar ratio of IR and IGFR half-receptors. |
| Databáze: | OpenAIRE |
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