Leucine-rich repeat (LRR) proteins feature tandem leucine-rich motifs that form a protein-protein interaction domain. Plants contain diverse classes of LRR proteins, many of which take part in signal transduction. We have identified a novel family of nine Arabidopsis LRR proteins that, based on predicted intracellular location and LRR motif consensus sequence, are related to Ras-binding LRR proteins found in signaling complexes in animals and yeast. This new class has been named plant intracellular Ras group-related LRR proteins (PIRLs). We have characterized PIRL cDNAs, rigorously defined gene and protein annotations, investigated gene family evolution and surveyed mRNA expression. While LRR regions suggested a relationship to Ras group LRR proteins, outside of their LRR domains PIRLs differed from Ras group proteins, exhibiting N- and C-terminal regions containing low complexity stretches and clusters of charged amino acids. PIRL genes grouped into three subfamilies based on sequence relationships and gene structures. Related gene pairs and dispersed chromosomal locations suggested family expansion by ancestral genomic or segmental duplications. Expression surveys revealed that all PIRL mRNAs are actively transcribed, with three expressed differentially in leaves, roots or flowers. These results define PIRLs as a distinct, plant-specific class of intracellular LRR proteins that probably mediate protein interactions, possibly in the context of signal transduction. T-DNA knock-out mutants have been isolated as a starting point for systematic functional analysis of this intriguing family.

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are cytokines which signal through receptor complexes that include the receptor subunits glycoprotein 130 (gp130) and the LIF receptor (LIFR), but CNTF also requires the non-signal transducing CNTF receptor (CNTFR) for binding. We show here that in IMR-32 neuronal cells endogenously expressing the receptor subunits for LIF and CNTF, CNTFR, but not gp130 or LIFR, is found in detergent-resistant lipid rafts. In addition, stimulation of these cells with CNTF resulted in a rapid translocation of a portion of gp130 and LIFR into detergent-resistant lipid rafts while an equivalent stimulation with LIF did not. Disruption of lipid rafts by cholesterol depletion of cell membranes blocked the CNTF-induced translocation of LIFR and gp130. Interestingly, while cholesterol-depletion did not inhibit signal transducer and activator of transcription 3 phosphorylation by either CNTF or LIF stimulation, it strongly inhibited both CNTF-and LIF-mediated phosphorylation of extracellular signal-regulated kinases 1 and 2 and Akt. LIF and CNTF generally appear to have redundant effects in cells responsive to both cytokines. Intriguingly, the data presented here suggest a possible mechanism whereby CNTF or other cytokines that signal through CNTFR could generate signals distinct from those elicited by cytokines such as LIF which utilize a LIFR/gp130 heterodimer, via association with or exclusion from lipid rafts.

The cytokines that signal through the leukemia inhibitory factor (LIF) receptor are members of the neuropoietic cytokine family and have varied and numerous roles in the nervous system. In this report we have determined the effects of growth factor stimulation on LIF receptor (LIFR) expression and signal transduction in the human neuroblastoma cell line NBFL. We show here that stimulation of NBFL cells with either epidermal growth factor or fibroblast growth factor decreases the level of LIFR in an extracellular signal-regulated kinase (Erk)1/2-dependent manner and that this downregulation is due to an increase in the apparent rate of lysosomal LIFR degradation. Growth factor-induced decreases in LIFR level inhibit both LIF-stimulated phosphorylation of signal transducers and activators of transcription 3 (STAT3) and LIFRmediated gene induction. We also show that Ser1044 of LIFR, which we have previously shown to be phosphorylated by Erk1/2, is required for the inhibitory effects of growth factors. Neurons are exposed to varying combinations and concentrations of growth factors and cytokines that influence their growth, development, differentiation and repair in vivo. These findings demonstrate that LIFR expression and signaling in neuroblastoma cells can be regulated by growth factors that are potent activators of the mitogen activated protein kinase pathway, and thus illustrate a fundamental mechanism that underlies cross-talk between receptor tyrosine kinase and neuropoietic cytokine signaling pathways. Keywords leukemia inhibitory factor; gp130; growth factor; MAP Kinase; cytokine The leukemia inhibitory factor receptor (LIFR), together with glycoprotein 130 (gp130), acts as a signal transducing receptor for a group of related cytokines that include leukemia Address correspondence to: Neil M. Nathanson, Department of Pharmacology, University of Washington, Box 357750, 1959 NE Pacific, Seattle, WA, 98195-7750, Tel. 206-543-9457; Fax. 206-616-4230; E-mail: nathanso@u.washington 1 The abbreviations used in the text are: CaMK, Ca 2+ /calmodulin-dependent kinase; CLC, cardiotrophin-like cytokine; CNTF, ciliary neurotrophic factor; CNTFR, ciliary neurotrophic factor receptor; CT-1, cardiotrophin-1; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; DTT, dithriotreithol; EGF, epithelial growth factor; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; FGF, fibroblast growth factor; G-CSF, granulocyte colony stimulating factor; G-CSFR, G-SCF receptor; gp130, glycoprotein 130; IL, interleukin; JAK, Janus kinase; LIF, leukemia inhibitory factor; LIFR, leukemia inhibitory factor receptor; MAPK, mitogen activated protein kinase; MEK, MAPK kinase; NGF, nerve growth factor; OsM, Oncostatin M; PERK, phosphorylated ERK; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PIAS, protein induced by activated STATs; PMA, phorbol 12-myristate 13-acetate; PY-STAT3, Tyr705-phosphorylated STAT3; SDS, sodium dodecyl sulphate; SHP, src homology 2 domain-containing phos...