RRC ID 22732
Author Togawa S, Ishii J, Ishikura A, Tanaka T, Ogino C, Kondo A.
Title Importance of asparagine residues at positions 13 and 26 on the amino-terminal domain of human somatostatin receptor subtype-5 in signalling.
Journal J Biochem
Abstract N-linked oligosaccharides or asparagine residues are often involved in G protein-coupled receptor functions. Focusing on Asn13 and Asn26 positioned on N-linked glycosylation motifs in the amino-terminal domain of human somatostatin receptor subtype-5 (hSSTR5), we performed site-directed mutagenesis and evaluated the mutants by using yeast cells as the host strain. This is because analysing the complicated signalling in mammalian cell lines is simplified by the utilization of the monopolistic pheromone signalling pathway in yeast. Western blot analysis and confocal laser scanning microscope observation showed that Asn13 and/or Asn26 mutations had no effects on cell-surface expression of hSSTR5 in yeast. By using an engineered yeast strain of Saccharomyces cerevisiae, which induces the expression of the green fluorescent protein (GFP) reporter gene in response to the agonist-specific signal transduction, it was demonstrated that a single mutation of two asparagine residues attenuated the somatostatin-specific signalling levels, and the double mutant significantly lost the signalling ability. These results clearly show the importance of these asparagine residues in the agonist-specific signalling of hSSTR5, although it was not enough to identify the consequence of oligosaccharides.
Volume 147(6)
Pages 867-73
Published 2010-6-1
DOI 10.1093/jb/mvq022
PII mvq022
PMID 20207824
MeSH Amino Acid Substitution Asparagine Blotting, Western Glycosylation Green Fluorescent Proteins Humans Membrane Glycoproteins / chemistry* Membrane Glycoproteins / metabolism Microscopy, Confocal Mutagenesis, Site-Directed Receptors, G-Protein-Coupled / chemistry* Receptors, G-Protein-Coupled / metabolism Receptors, Somatostatin* / chemistry Receptors, Somatostatin* / genetics Receptors, Somatostatin* / metabolism Saccharomyces cerevisiae Signal Transduction / physiology* Somatostatin / metabolism Transfection
IF 2.476
Times Cited 20
Yeast NA