IDENTIFICATION AND CHARACTERIZATION OF INSULIN RECEPTOR IN R3230AC MAMMARY ADENOCARCINOMA OF RAT

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  • 1 January 1976
    • journal article
    • research article
    • Vol. 36  (11) , 3993-4000
Abstract
The binding of labeled insulin to dissociated R3230AC mammary adenocarcinoma cells from diabetic and intact rats was investigated in vitro. At 20.degree. C specific binding (total binding minus binding in the presence of 1000-fold excess or 10-6 M unlabeled insulin) reached a plateau at 45-60 min and was directly related to the number of cells used. Degradation of labeled insulin, as measured by trichloroacetic acid precipitation, was related to the number of cells used, was not prevented by trasylol or phenylmethylsulfonyl fluoride (general proteolytic enzyme inhibitors), but was prevented by addition of 1-2% bovine serum albumin to the incubation medium. Specificity of insulin binding was examined by competition with insulin analogs. Proinsulin, guinea pig insulin and desoctapeptide insulin were capable of competing for insulin binding in an order of potency related to their relative biological activity; prolactin and glucagon were unable to compete for insulin binding. Scatchard analysis of the binding data demonstrated a curvilinear plot; specific binding (over the concentration range of 10-11-10-10 M insulin) showed a high affinity (Kd .apprx. 1 to 3 .times. 10-10 M), and the estimated number of sites was greater in tumors from diabetic animals than in tumors from intact animals or intact animals given insulin prior to sacrifice. Reversibility of insulin binding was studied by dissociation experiments; dissociation was enhanced in the presence of added unlabeled insulin compared to dissociation examined under conditions of infinite dilutions only. Maximum dissociation of bound insulin was observed in the presence of 10-7 M unlabeled insulin, with less of an effect at lower or higher concentrations of added insulin (no effect seen at 10-10 M insulin). Two techniques were investigated for separating cells from unbound labeled insulin; the procedure using centrifugation was more efficient. In the R3230AC mammary adenocarcinoma, data obtained on saturability, reversibility and specificity of insulin binding indicated the existence of an insulin receptor with properties similar to those found in normal cells.