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Title: A G Protein-Coupled Receptor with a Lipid Kinase Domain Is Involved in Cell-Density Sensing.      
keywords:
Transcriptome or Gene expression
ID:
PRJNA100019
description:
One mechanism multicellular structures use for controlling cell number [1, 2] involves the secretion and sensing of a factor, such as leptin [3] or myostatin [4], in mammals. Dictyostelium cells secrete autocrine factors for sensing cell density prior to aggregation and multicellular development [5, 6] such as CMF (conditioned-medium factor), which enables starving cells to respond to cAMP pulses [7, 8, 9]. Its actions are mediated by two receptors. CMFR1 activates a G protein-independent signaling pathway regulating gene expression [10]. An unknown Gα1-dependent receptor activates phospholipase C (PLC), which regulates the lifetime of Gα2-GTP [11, 12, 13]. Here, we describe RpkA, an unusual seven-transmembrane receptor that is fused to a C-terminal PIP5 kinase domain and that localizes in membranes of a late endosomal compartment. Loss of RpkA resulted in formation of persistent loose aggregates and altered expression of cAMP-regulated genes. The developmental defect can be rescued by full-length RpkA and the transmembrane domain only. The PIP5 kinase domain is dispensable for the developmental role of RpkA. rpkA− cells secrete and bind CMF but are unable to induce downstream responses. Inactivation of Gα1, a negative regulator of CMF signaling, rescued the developmental defect of the rpkA− cells, suggesting that RpkA actions are mediated by Gα1. Keywords: Comparison of wild type AX2 cells with rpkA minus cells Overall design: Microarray analysis for the rpkA- mutant. Developmental stage Microarray Wild type Mutant Total RNA dye Total RNA dye T0 12874460 Culture A Cy3 Culture A Cy5 12945001 Culture B Cy3 Culture B Cy5 12874461 Culture A Cy5 Culture A Cy3 12945002 Culture B Cy5 Culture B Cy3 T8 12882232 Culture A Cy3 Culture A Cy5 12953218 Culture B Cy3 Culture B Cy5 12882233 Culture A Cy5 Culture A Cy3 12953220 Culture B Cy5 Culture B Cy3 AX2 cells and rpkA- cells were starved (5 × 10E6 cells/cm2) on phosphate agar plates and total RNA was prepared at 0 and 8 hrs of development. cDNA was prepared from total RNA (10 ug) using the Fairplay Kit (Stratagene Europe, Amsterdam, The Netherlands) as described in the manufacturers protocol. For each microarray experiment, an experimental (mutant, rpkA-) sample was labeled with Cy5 or Cy3 and the reference (wild type, AX2) sample with Cy3 or Cy5, respectively. Fluorescently labeled cDNA of the rpkA- mutant was generated and compared to labeled cDNA from AX2. Microarray production, expression profiling and data analysis were carried out as described in [Farbrother et al., Cell. Microbiology 2006, 3:438-456].
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landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA100019
authentication:
none
authorization:
none
ID:
pmid:17481898
name:
Dictyostelium discoideum
ncbiID:
ncbitax:44689
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject