Summary
Intracellular insulin-binding sites were directly traced in fixed monolayer cultures of a variety of cell types with the use of two fluorescent derivatives of insulin, viz. fluorescein isothiocyanate (FITC)-labelled and tetramethyl rhodamine isothiocyanate (TMRITC)-labelled insulin. Both derivatives retained the property of stimulating DNA synthesis in fibroblasts. Insulin-binding sites were found in the nuclear envelope, nucleoplasm, nucleoli, and in mitochondria and rough endoplasmic reticulum. The identity of these structures was established by concomitant studies on the same cell by means of phase contrast optics and immunocytochemical tracing with specific antibodies to nuclei, mitochondria, or ribosomes. Binding of insulin to the nuclear and cytoplasmic structures was rapid, reversible and saturable, temperature and pH-dependent, and inhibited by an excess of native, but not other, hormones. The staining reactions were sensitive to treatment by the nonionic detergents, NP-40 and TX-100, and to trypsin and pronase, but not to DNase and RNase, suggesting that the binding sites are protein in nature.
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References
Carpentier J-L, Gorden P, Barazzone P, Freychet P, Le Cam A, Orci L (1979) Intracellular localization of 125I-labelled insulin in hepatocytes from intact rat liver. Proc Natl Acad Sci 76:2803–2807
Doniach D, Roitt OM, Walker JG, Sherlock S (1966) Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other liver diseases and their clinical implications. Clin Exp Immunol 1:237–262
Goldfine I, Jones AL, Hradek GT, Wong KY, Mooney JS (1978) Entry of insulin into cultured lymphocytes: electron microscope autoradiographic analysis. Science 202:760–763
Hollenberg MD, Cuatrecasas P (1975) Insulin and epidermal growth factor: human fibroblast receptors related to deoxyribonucleic acid synthesis and amino acid uptake. J Biol Chem 250:3845–3853
Kahn CR (1976) Membrane receptors for hormone and neurotransmitters. J Cell Biol 70:261–286
Lee ND, Williams RH (1954) The intracellular localization of labelled thyroxine and labelled insulin in mammalian liver. Endocrinology 54:5–19
Mathy JP, Baum R, Toh BH (l980) Antibody to ribosomes and systemic lupus erythematosus. Clin Exp Immunol (in press)
Nairn RC (1976) Fluorescent protein tracing. 4th Ed Edinburgh, Livingston
Shields R (1977) Growth hormones and serum factors. Nature 267:308–310
Sótéló J, Toh BH, Lolait SJ, Yildiz A, Osung O, Holborow EJ (1980) Cytoplasmic intermediate filaments in cultured glial cells. Neuropathol Appl Neurobiol (in press)
Toh BH, Hard GC (1977) Actin co-caps with concanavalin A receptors. Nature 269:695–697
Toh BH, Muller HK, Elrick WL (1976) Smooth muscle associated antigen in astrocytes and astrocytoma. Brit J Cancer 33:195–202
Toh BH, Yildiz A, Sótéló J, Osung O, Holborow EJ, Fairfax A (1979) Distribution of actin andmyosin in muscle and non-muscle cells. Cell Tissue Res 199:117–126
Vigneri, R, Goldfine ID, Wong KY, Smith GJ, Pezzino V (1978) The nuclear envelope: The major site of insulin-binding in rat liver nuclei. J Biol Chem 253:2098–2103
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Supported by a grant from the Anti-Cancer Council of Victoria. We thank Mrs. I. Burns for technical assistance, Dr. H.A. Ward and staff for preparation of the conjugated insulins, and Prof. R.C. Nairn for advice
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Lolait, S.J., Toh, B.H. Binding sites of fluorescent derivatives of insulin in nuclei, rough endoplasmic reticulum, and mitochondria. Cell Tissue Res. 210, 145–153 (1980). https://doi.org/10.1007/BF00232150
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DOI: https://doi.org/10.1007/BF00232150