Identification of retinal insulin receptors using site-specific antibodies to a carboxyl-terminal peptide of the human insulin receptor alpha-subunit. Up-regulation of neuronal insulin receptors in diabetes.
Insulin receptor-specific polyclonal antipeptide serum was generated against a synthetic pentadecapeptide (residues 657-670) of the deduced amino acid sequence of human insulin proreceptor cDNA for use in the analysis of insulin receptors in the retina. The affinity-purified antibodies recognized peptide antigen but not keyhole limpet hemocyanin as determined by dot blot analysis and solid phase radioimmunoassay. Addition of either synthetic peptide or the affinity-purified serum had no effect on 125I-insulin binding to placental membranes or to cells in culture. alpha-Subunits of approximately 125 kDa from human placental membranes and liver membranes were labeled by immunoblot analysis with this antiserum. In membranes isolated from human retina and brain, two classes of alpha-subunits of approximately 125 and 115 kDa were detectable. The 115-kDa subunit was neuraminidase resistant whereas the 125-kDa subunit was digested to a band of 115 kDa, indicating that these bands represent peripheral and neuronal receptors, respectively. Analysis of human retinas obtained from type I diabetic donors revealed an increased level of neuronal receptor as compared with normal retinas. These data indicate that human retina expresses neuronal insulin receptor subtypes that are up-regulated in diabetes.
Department of Ophthalmology, Yale University School of Medicine, New Haven, Connecticut 06510.
S A
SA
Rosenzweig
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C
Zetterström
A
A
Benjamin
D000595:Amino Acid Sequence; D000906:Antibodies; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003922:Diabetes Mellitus, Type 1 / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011863:Radioimmunoassay; D011972:Receptor, Insulin / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D012160:Retina / Q000378:metabolism*; D015854:Up-Regulation
D000906:Antibodies; D046911:Macromolecular Substances; D010455:Peptides; D011972:Receptor, Insulin