COEXPRESSION OF MUTANT AND NORMAL HUMAN INSULIN-RECEPTORS IN COS-7 CELLS

In order to assess the interference of the mutant insulin proreceptor on normal receptor function and formation of proreceptor-receptor hete rotrimers (alpha beta-proreceptor), COS 7 cells were transfected with the same amount of expression plasmid (pGEM3SV) containing wild-type, a mutant proreceptor cDNA and both, using the DEAE-dextran method. Sca tchard analysis of insulin binding data revealed that there was an app rox. 50-fold higher receptor concentration in the transfected cells th an in untransfected cells. After 0.025% trypsin treatment, insulin bin ding to the cells expressed with wild-type, proreceptor and both incre ased by 1-fold, 2.9-fold and 1.5-fold of the untreated cells, respecti vely. In the presence of 167 nM insulin, the amounts of phosphate inco rporated into the 95 kDa protein beta-subunits and 210 kDa proreceptor s from co-transfected cells, were identical to those of an in vitro mi xture of the wild-type and the mutant receptors. At 10 nM insulin, the proreceptors from co-transfected cells normally autophosphorylated by insulin stimulation, whereas those mixed in vitro did not (73.3 +/- 9 .3 vs. 29.6 +/- 2.6% of the maximal effect, n = 4, P < 0.01). However, at a similar concentration of insulin, the phosphate incorporation in to Glu-80/ Tyr-20 polymers by receptors from co-transfected cells was decreased when compared with a in vitro mixture (9.0 +/- 2.6 vs. 22.5 +/- 6.7% of the maximal effect at 4 nM, n = 6, P < 0.01), although the basal and maximally stimulated phosphate incorporation were comparabl e among these groups. These results suggested that similar amounts of both receptor types were expressed and heterotrimers might be formed r esulting that a partially activated proreceptor could interfere with n ormal receptor kinase activity in the co-transfected cells.