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We characterized insulin receptors on a human lymphoblastoid cell line (IM-9) and studied their regulation using anti-receptor antibodies and fluorescence flow cytometry. The fluorescence intensity distribution of insulin receptors on cells was determined by incubating the cells with one of three different anti-receptor antisera (human serum B-9 containing polyclonal autoantibodies, serum from a rabbit with polyclonal antibodies, and a monoclonal antibody to the receptor produced in mouse hybridomas), followed by incubation with an appropriate fluorescein isothiocyanate-labeled second antibody and analysis on an Epics-V flow cytometer. however, the human anti-receptor antisera B-2 and B-9 inhibited the binding of the monoclonal anti-receptor antibody by about 50%, suggesting that these antisera contained autoantibodies directed at the monoclonal antibody binding site. These data indicate that insulin receptors can be regulated by both insulin and anti-receptor antibody and demonstrate the utility of immunofluorescence and flow cytometry as a tool for the study of the insulin receptor.The first step in the action of insulin is binding to a specific glycoprotein receptor on the surface of cells. The primary approach for characterization of the insulin receptor has been through the study of its interaction with 125I-labeled insulin (125I-insulin) (1,2). Antibodies to the insulin receptor also have provided experimental probes of receptor structure and function (3). Anti-insulin-receptor antibodies were initially detected in the sera of patients with a rare form of insulin-resistant diabetes (4), although subsequently it has been possible to raise such antibodies in rabbits and mice by immunization with partially purified receptor (5) and even to produce monoclonal antibodies to the insulin receptor (6, 7). In addition to binding to the receptor, many of these antibodies block insulin binding (8) and mimic many of insulin's biological effects (9).The use of fluorescence probes to study insulin receptors or action has been limited. Murphy et al. studied the binding and internalization of a fluorescein derivative of insulin to Swiss 3T3 cells by flow cytometry (10, 11). Fluorescein has been coupled to insulin directly or via ovalbumin; using these ligands, Schlessinger et al. have shown that insulin receptors undergo patch-and-cap formation at 370C (12). Subsequently, it was shown that fluorescently-labeled anti-receptor antibody cocapped with the rhodamine-coupled insulin (12) and that the rhodamine-insulin-receptor complex was internalized and degraded (13).In the present study, we used fluorescence flow cytometry for identification of insulin receptors and for studying their turnover. Using this technique, we have been able to study (i) the expression of receptors and their regulation by insulin, (ii) compare the effects of acute and chronic exposure of target cells to antibodies directed against insulin receptors, (iii) demonstrate shared antigenic determinants between polyclonal human autoantibodies to th...

Antibodies to the insulin receptor have provided important experimental probes of receptor structure and function. In the present study, we have characterized the insulin receptor on human lymphoblastoid cell lines using polyclonal and monoclonal anti-receptor antibodies and fluorescence flow cytometry. The cell lines were derived by Epstein-Barr virus transformation of peripheral mononuclear leucocytes from normal subjects or patients with disorders that affect the insulin receptor. Fluorescence analysis revealed a high level of specific fluorescence on lymphoid cell lines from normal individuals (mean peak fluorescence 30-50 units above the control) and was similar to the labelling of the spontaneously transformed lymphoblastoid cell line IM-9. Transformed cells from patients with syndromes of insulin resistance, such as the Rabson Mendenhall syndrome, leprechaunism and the type A syndrome of insulin resistance and acanthosis nigricans, exhibited little or no specific fluorescence. In all cases, there was an unimodal distribution of receptors on cells. In addition, there was a good correlation between specific binding of 125I-insulin and percentage peak fluorescence. The data indicate that fluorescence flow cytometry can be used to study the distribution of insulin receptor on different cell lines and to study cells derived from patients with disease states.