Evidence that inhibition of insulin receptor signaling activity by PC-1/ENPP1 is dependent on its enzyme activity.
Plasma cell membrane glycoprotein-1 or ectonucleotide pyrophosphatase/phosphodiesterase (PC-1/ENPP1) has been shown to inhibit insulin signaling, and its genetic polymorphism or increased expression is associated with type 2 diabetes in humans. Therefore, PC-1 inhibition represents a potential strategy in treating diabetes. Since patients with phosphodiesterase/pyrophosphatase deficient PC-1 manifest abnormal calcification, enhancing insulin signaling by inhibiting PC-1 for the treatment of diabetes will be feasible only if PC-1 phosphodiesterase/pyrophosphatase activity needs not be significantly diminished. However, whether inhibition of insulin receptor signaling by PC-1 is dependent upon its phosphodiesterase/pyrophosphatase activity remains controversial. In this study, the extracellular domain of the human PC-1 in its native form or with a T256A or T256S mutation was overexpressed and purified. Enzymatic assays showed that both mutants have less than 10% of the activity of the wild-type protein. In HEK293 cells stably expressing recombinant insulin receptor or insulin-like growth factor 1 (IGF1) receptor, transient expression of wild-type full length PC-1 (PC-1.FL.WT) but not the T256A or T256S mutants inhibits insulin signaling without affecting IGF1 signaling. Western blot and FACS analysis showed that the wild-type and mutant full length PC-1 proteins are expressed at similar levels in the cells, and were localized to the similar levels on the cell surface. Overexpression of PC-1.FL.WT did not affect insulin receptor mRNA level, total protein and cell surface levels. Together, these results suggest that the inhibition of insulin signaling by PC-1 is somewhat specific and is dependent upon the enzymatic activity of the phosphodiesterase/pyrophosphatase.
Biologics Research, Merck & Co, Inc, West Point, PA 19486, United States.
Chen-Ni
CN
Chin
Qing
Q
Dallas-Yang
Franklin
F
Liu
Thu
T
Ho
Kenneth
K
Ellsworth
Paul
P
Fischer
Tajneen
T
Natasha
Cheryl
C
Ireland
Ping
P
Lu
Cai
C
Li
I-Ming
IM
Wang
William
W
Strohl
Joel P
JP
Berger
Zhiqiang
Z
An
Bei B
BB
Zhang
Guoqiang
G
Jiang
D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D009154:Mutation; D010727:Phosphoric Diester Hydrolases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011755:Pyrophosphatases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011972:Receptor, Insulin / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection
D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases