Importance of asparagine residues at positions 13 and 26 on the amino-terminal domain of human somatostatin receptor subtype-5 in signalling.
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.
Togawa
Shota
S
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.
Ishii
Jun
J
Ishikura
Atsushi
A
Tanaka
Tsutomu
T
Ogino
Chiaki
C
Kondo
Akihiko
A
D019943:Amino Acid Substitution; D001216:Asparagine; D015153:Blotting, Western; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism; D017481:Receptors, Somatostatin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction / Q000502:physiology*; D013004:Somatostatin / Q000378:metabolism; D014162:Transfection
D008562:Membrane Glycoproteins; D043562:Receptors, G-Protein-Coupled; D017481:Receptors, Somatostatin; D049452:Green Fluorescent Proteins; D013004:Somatostatin; D001216:Asparagine; C094573:somatostatin receptor 5