Control of signalling properties of human somatostatin receptor subtype-5 by additional signal sequences on its amino-terminus in yeast

Abstract

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.

Oligomerisation of C. elegans Olfactory Receptors, ODR-10 and STR-112, in Yeast

“…Although some of ODR-10 was localised to the yeast plasma membrane, much of it was localised intracellularly (Figure 1), which has also previously been observed for mammalian chemoreceptors [34], [43], [44]. However, it has also been shown that mammalian olfactory receptors are functional whether they are localised to yeast ER, Golgi or plasma membrane when tested in vitro [34], [43], [44]. Based on these previous reports, we believe it is reasonable to infer that ODR-10 is correctly folded whether it is located in the plasma or intracellular membranes [35].…”

“…In yeast cells co-expressing ODR-10-GFP 2 and ODR-10-Rluc, ODR-10-GFP 2 was localised to the plasma membrane in ≈32% of cells after a 4 hour induction (Figure 1). Although some of ODR-10 was localised to the yeast plasma membrane, much of it was localised intracellularly (Figure 1), which has also previously been observed for mammalian chemoreceptors [34], [43], [44]. However, it has also been shown that mammalian olfactory receptors are functional whether they are localised to yeast ER, Golgi or plasma membrane when tested in vitro [34], [43], [44].…”

“…To investigate whether ODR-10 can form hetero-oligomers with other GPCRs, we probed the interaction of ODR-10 with a second C. elegans chemoreceptor, STR-112 and two mammalian receptors, the rat I7 chemoreceptor [33], [60] and the human somatostatin receptor subtype 5 (SSTR5) [34], [61]. STR-112 is the closest homologue of ODR-10, having 79.6% amino acid identity with it.…”

“…Rat I7 receptor was amplified by PCR from the plasmid pCA4-I7-IRES-GFP, kindly provided by Prof S. Firestein [33]. SSTR5 receptor was amplified by PCR from the plasmid pGK-SSTR5-HA, kindly provided by Dr Jun Ishii [34]. The BRET tags Rluc and GFP 2 were sourced from the plasmid pGFP 2 -MCS-Rluc(h) (Perkin Elmer) and introduced at the C-termini of odr-10 , I7 and sstr5 , by PCR amplification as described previously [35].…”

Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface

“…We constructed several negative controls by eliminating the S-14 peptide or by replacing it with agonistic peptides for other GPCRs (Figure 2B and Table 1). We expressed hemagglutinin (HA)-tagged human SSTR5 on the yeast cell surface using previously reported plasmids [5], [6] (Table 1). We used these expression and mock plasmids to investigate the ability of the S-14–Flag–Flo42 autocrine peptide to activate GPCR signaling (Figure 4).…”