Insulin-independent and wortmannin-resistant targeting of IRS-3 to the plasma membrane via its pleckstrin homology domain mediates a different interaction with the insulin receptor from that of IRS-1.
OBJECTIVE
In primary adipocytes, although IRS-1 and IRS-3 are expressed in comparable amounts, these proteins manifest distinct distribution and significance in insulin signalling. We investigated the molecular basis of the difference between these two proteins.


METHODS
In Cos-1 cells transiently expressing rat IRS-1, IRS-3, or chimeric proteins of these two proteins we examined the tyrosine phosphorylation via the wild-type or mutant insulin receptors and evaluated their targeting to the plasma membrane by immunostaining the membrane ghost.


RESULTS
In contrast to IRS-1, IRS-3 was tyrosine-phosphorylated by the insulin receptor altering Tyr960 to Phe (Y960F), which disrupts the binding site of the PTB domain of IRSs, to an extent comparable to the wild-type receptor. The tyrosine phosphorylation of IRS-3 with the PH domain replacement via the Y960F insulin receptor markedly decreased, whereas that of IRS-3 with the PTB domain alteration was mildly impaired. Insulin-stimulated translocation of IRS-1 to the plasma membrane, as well as that of IRS-3 with the PH domain replacement, was wortmannin-sensitive, although that of IRS-3 was insulin-independent and wortmannin-resistant.


CONCLUSIONS
The affinity of the PH domain for the phospholipids in the plasma membrane seems to influence the receptor-substrate interaction required for IRS tyrosine phosphorylation, indicating that the PH domain and the PTB domain of IRSs cooperatively function in insulin-stimulated tyrosine phosphorylation of these proteins.
Kaburagi
Y
Y
The Department of Metabolic Disease, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Satoh
S
S
Yamamoto-Honda
R
R
Ito
T
T
Ueki
K
K
Akanuma
Y
Y
Sekihara
H
H
Kimura
S
S
Kadowaki
T
T
D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics*; D019556:COS Cells / Q000378:metabolism; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011817:Rabbits; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D017385:Sequence Homology; D013329:Structure-Activity Relationship; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D001798:Blood Proteins; D004791:Enzyme Inhibitors; C526292:IRS1 protein, human; C491585:IRS3P protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; C108872:Irs3 protein, mouse; C491584:Irs3 protein, rat; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; C039077:platelet protein P47; D019000:Phosphotyrosine; D011972:Receptor, Insulin; D000077191:Wortmannin