Identification of IRAS/Nischarin as an I1-imidazoline receptor in PC12 rat pheochromocytoma cells.
The I1-imidazoline receptor (I1R) 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 I1R. To test the contribution of IRAS to I1R 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 I1R action. The density of I1R was significantly reduced by antisense compared with control transfection (Bmax = 400 +/- 16 vs. 691 +/- 29 fmol/mg protein), without significantly affecting binding affinity (Kd = 0.30 +/- 0.04 vs. 0.39 +/- 0.05 nmol/L). Thus, IRAS expression is necessary for high-affinity binding to I1R. 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 I1R cell signaling, PC12 cells transfected with antisense or sense oligo-DNA were treated with moxonidine, an I1R 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 I1R or a functional subunit.
Sun
Zheng
Z
Department of Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4906, USA.
Chang
Chung-Ho
CH
Ernsberger
Paul
P
D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D001794:Blood Pressure / Q000187:drug effects / Q000502:physiology; D001921:Brain / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D054755:Imidazoline Receptors; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D016716:PC12 Cells; D051381:Rats; D011955:Receptors, Drug / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000959:Antihypertensive Agents; D007093:Imidazoles; D054755:Imidazoline Receptors; D047908:Intracellular Signaling Peptides and Proteins; C458505:NISCH protein, human; C519767:Nisch protein, rat; D016376:Oligonucleotides, Antisense; D011955:Receptors, Drug; C410887:imidazoline I1 receptors; C043482:moxonidine; D048049:Extracellular Signal-Regulated MAP Kinases