Control of signalling properties of human somatostatin receptor subtype-5 by additional signal sequences on its amino-terminus in yeast.
The yeast Saccharomyces cerevisiae is known as an available host for human G-protein-coupled receptor (GPCR) ligand screening. Although several types of yeast signal sequences (SS) attached with the GPCRs could improve their productivities and facilitate transportation of the GPCRs to the yeast plasma membrane, the effects of additional SS on ligand-specific signalling functions of GPCRs are not reported. Here, we demonstrated the controlling signalling properties by addition of SS using engineered yeast as a host. Prepro and pre regions of alpha-factor and amino-terminal sequence of Ste2 (Ste2N) were used as SS, and somatostatin (SST) receptor subtype-5 (SSTR5) was used as a model GPCR. We also constructed a yeast-based fluorescent assay system for monitoring the activation levels of SSTR5 signalling by a green fluorescent protein (GFP) reporter gene. The production levels and localisation patterns of the SS-attached SSTR5 were more significantly improved than those of wild-type SSTR5. In addition, we successfully controlled the pharmacological efficacy and potency by introducing SS. Among four types of SSTR5 receptors, Ste2N-SSTR5 responded at the lowest ligand concentration. This finding will be informative for constructing optimal yeast-based ligand screening systems to discriminate the cells on the basis of signalling levels.
Iguchi
Yusuke
Y
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe 657-8501, Japan.
Ishii
Jun
J
Nakayama
Hideki
H
Ishikura
Atsushi
A
Izawa
Keiko
K
Tanaka
Tsutomu
T
Ogino
Chiaki
C
Kondo
Akihiko
A
D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D008856:Microscopy, Fluorescence; D021382:Protein Sorting Signals / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism*; D017481:Receptors, Somatostatin / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction; D013004:Somatostatin / Q000378:metabolism; D014162:Transfection
D021382:Protein Sorting Signals; D043562:Receptors, G-Protein-Coupled; D017481:Receptors, Somatostatin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013004:Somatostatin; C094573:somatostatin receptor 5