Analysis of Tyr to Phe and fa/fa leptin receptor mutations in the PC12 cell line.
Weight regulation through body-fat content and energy homeostasis, is regulated mainly through the actions of leptin. Herein, we analyse the effect of mutations in the mouse leptin receptor using the PC12 pheochromocytoma cell line as a model system. Both the induction of pancreatitis associated protein 1 and metallothionein-II, two leptin regulated genes in PC12, was evaluated. Tyr to Phe mutations in the cytoplasmic tail of the mouse leptin receptor confirmed the critical role of Tyr1138 (a YxxQ motif) and STAT-3 activation for induction of leptin-induced genes in PC12. In addition, the Tyr985Phe mutation showed enhanced responsiveness to leptin, which was even more pronounced in combination with Tyr1077Phe. The short isoform of the leptin receptor showed complete loss of stimulation of both genes. In contrast, a leptin receptor devoid of all Tyr residues in its cytoplasmic tail was still capable of a limited induction of the PAP 1 gene. A mutant mouse leptin receptor containing the fa/fa mutation showed constitutive signalling and impaired responsiveness to leptin. Treatment with the adenylate cyclase activator forskolin alone, in the absence of leptin was sufficient to obtain full induction of both genes.
Eyckerman
S
S
Flanders Interuniversity Institute for Biotechnology, Dept VIB09, Medical Protein Research, Faculty of Medicine, University of Ghent, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
Waelput
W
W
Verhee
A
A
Broekaert
D
D
Vandekerckhove
J
J
Tavernier
J
J
D000209:Acute-Phase Proteins / Q000235:genetics; D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000502:physiology; D017781:Genes, Immediate-Early / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D037181:Lectins, C-Type; D020738:Leptin / Q000378:metabolism / Q000494:pharmacology; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D009154:Mutation / Q000235:genetics*; D020932:Nerve Growth Factor / Q000494:pharmacology; D016716:PC12 Cells; D000073718:Pancreatitis-Associated Proteins; D010649:Phenylalanine / Q000235:genetics*; D011506:Proteins; D051381:Rats; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D050796:STAT3 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects; D015534:Trans-Activators / Q000502:physiology; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014443:Tyrosine / Q000235:genetics*
D000209:Acute-Phase Proteins; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D037181:Lectins, C-Type; D020738:Leptin; D000073718:Pancreatitis-Associated Proteins; D011506:Proteins; C000612141:REG3A protein, human; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; C490253:Reg3b protein, mouse; C000612144:Reg3b protein, rat; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; C494088:Stat3 protein, rat; D015534:Trans-Activators; C515447:leptin receptor, mouse; D005576:Colforsin; D014443:Tyrosine; D010649:Phenylalanine; D008668:Metallothionein; D020932:Nerve Growth Factor