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Identification of IRAS/Nischarin as an I 1 ‐imidazoline receptor in PC12 rat pheochromocytoma cells
Author(s) -
Sun Zheng,
Chang ChungHo,
Ernsberger Paul
Publication year - 2007
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2006.04413.x
Subject(s) - agonist , imidazoline receptor , microbiology and biotechnology , transfection , receptor , extracellular , biology , western blot , gene expression , signal transduction , prazosin , gene , endocrinology , biochemistry , antagonist
The I 1 ‐imidazoline receptor (I 1 R) is a proposed target for drug action relevant to blood pressure and glucose control. The imidazoline receptor antisera‐selected (IRAS) gene, also known as Nischarin, has several characteristics of an I 1 R. To test the contribution of IRAS to I 1 R binding capacity and cell‐signaling function, an antisense probe targeting the initiating codon of rat IRAS gene was evaluated in PC12 rat pheochromocytoma cells, a well‐established model for I 1 R action. The density of I 1 R was significantly reduced by antisense compared with control transfection ( B max = 400 ± 16 vs. 691 ± 29 fmol/mg protein), without significantly affecting binding affinity ( K d = 0.30 ± 0.04 vs. 0.39 ± 0.05 nmol/L). Thus, IRAS expression is necessary for high‐affinity binding to I 1 R. Western blots with polyclonal anti‐IRAS showed reduced IRAS expression in the major 85‐kDa band relative to an actin reference, paralleling the reduction in binding site density. To determine whether reduced IRAS expression attenuated I 1 R cell signaling, PC12 cells transfected with antisense or sense oligo‐DNA were treated with moxonidine, an I 1 R agonist, then cell lysates were analyzed by western blot. Dose‐dependent activation of extracellular signal‐regulated kinase was attenuated without affecting the potency of the agonist. In contrast, extracellular signal‐regulated kinase activation by insulin was unchanged. The IRAS gene is likely to encode an I 1 R or a functional subunit.