PTP1B-dependent insulin receptor phosphorylation/residency in the endocytic recycling compartment of CHO-IR cells.
Insulin binds to the alpha subunit of the insulin receptor (IR) on the cell surface. The insulin-IR complex is subsequently internalized and trafficked within the cell. Endocytosed receptors, devoid of insulin, recycle back to the plasma membrane through the endocytic recycling compartment (ERC). Using a high content screening system, we investigate the intracellular trafficking of the IR and its phosphorylation state, within the ERC, in response to protein tyrosine phosphatase-1B (PTP1B) inhibition. Insulin stimulates, in a time- and dose-dependent manner, the accumulation of phosphorylated IR (pY(1158,1162,1163 IR) in the ERC of CHO-IR cells. Treatment of CHO-IR cells with PTP1B-specific inhibitors or siRNA leads to dose-dependent increases in IR residency and phosphorylation within the ERC. The results also demonstrate that PTP1B redistributes within CHO-IR cells upon insulin challenge. The established system will allow for efficient screening of candidate inhibitors for the modulation of PTP1B activity.
Cromlish
Wanda A
WA
Department of Biochemistry & Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval, Pointe-Claire-Dorval, Quebec, Canada. wanda_cromlish@merck.com
Tang
Man
M
Kyskan
Robert
R
Tran
Linda
L
Kennedy
Brian P
BP
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D015394:Molecular Structure; D010766:Phosphorylation; D021381:Protein Transport; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011972:Receptor, Insulin / Q000378:metabolism*; D013997:Time Factors
D004791:Enzyme Inhibitors; D007328:Insulin; D011972:Receptor, Insulin; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases