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Co-expression of mutant and normal human insulin receptors in COS 7 cells
H Maegawa1, A Kashiwagi, T Haruta
1Third Department of Medicine, Shiga University of Medical Science, Japan.
Abstract:
In order to assess the interference of the mutant insulin proreceptor on normal receptor function and formation of proreceptor-receptor heterotrimers (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. Scatchard analysis of insulin binding data revealed that there was an approx. 50-fold higher receptor concentration in the transfected cells than in untransfected cells. After 0.025% trypsin treatment, insulin binding to the cells expressed with wild-type, proreceptor and both increased by 1-fold, 2.9-fold and 1.5-fold of the untreated cells, respectively. In the presence of 167 nM insulin, the amounts of phosphate incorporated into the 95 kDa protein beta-subunits and 210 kDa proreceptors from co-transfected cells, were identical to those of an in vitro mixture 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 into 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 comparable among these groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
A mutant insulin proreceptor interferes with normal receptor function, affecting insulin binding and signaling pathways. This study investigates how the mutant proreceptor impacts insulin receptor heterotrimer formation and autophosphorylation.
Area of Science:
- Molecular Endocrinology
- Cell Biology
- Signal Transduction
Background:
- The insulin receptor plays a crucial role in glucose metabolism and cellular signaling.
- Understanding the function of insulin proreceptors and their potential interference is vital for metabolic research.
Purpose of the Study:
- To investigate the interference of a mutant insulin proreceptor on normal receptor function.
- To examine the formation of proreceptor-receptor heterotrimers and their impact on insulin signaling.
Main Methods:
- COS 7 cells were transfected with wild-type and mutant insulin proreceptor cDNAs.
- Scatchard analysis was used to assess insulin binding.
- Trypsin treatment and insulin stimulation were employed to evaluate receptor activity and phosphorylation.
Main Results:
- Transfected cells showed a 50-fold increase in receptor concentration.
- Insulin binding increased after trypsin treatment, with variations between wild-type, proreceptor, and co-transfected cells.
- Co-transfected proreceptors exhibited altered autophosphorylation and decreased phosphate incorporation into specific polymers compared to in vitro mixtures.
Conclusions:
- The mutant insulin proreceptor interferes with normal insulin receptor function and heterotrimer formation.
- The mutant proreceptor affects insulin-stimulated signaling pathways, including autophosphorylation and substrate phosphorylation.