Co-expression 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 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)
Maegawa
H
H
Third Department of Medicine, Shiga University of Medical Science, Japan.
Kashiwagi
A
A
Haruta
T
T
Egawa
K
K
Ugi
S
S
Tachikawa-Ide
R
R
Hasegawa
M
M
Kobayashi
M
M
Shigeta
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D010766:Phosphorylation; D017354:Point Mutation; D011498:Protein Precursors / Q000096:biosynthesis / Q000378:metabolism*; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012694:Serine; D014162:Transfection
D007328:Insulin; D046911:Macromolecular Substances; D011498:Protein Precursors; C051512:insulin proreceptor; D012694:Serine; D011972:Receptor, Insulin