D (Table 1 and Fig. S3). Since clear evidence for the functional

D (Table 1 and Fig. S3). Since clear evidence for the functional role of GPCR homodimer and heterodimer pairs was first obtained for class C receptors, such as GABAB buy 58-49-1 receptors [17], we 10781694 tested the dimerization of GABAB2 receptor (GABBR2) with GABAB1a receptor (GABBR1a). The b-galactosidase assay clearly showed the specific activities both for GABAB2/GABAB2 and GABAB2/GABAB1a couples (Fig. 5A and Fig. S4A). This result was coincident with the fact that GABAB2 receptor could form not only heterodimer with GABAB1a receptor but also homodimer [18,19], indicating that the split-ubiquitin-based approach could detect the homodimerization and heterodimerization of GABABScreening of Human GPCR HeterodimerFigure 5. Dimerization assays of human GPCRs. Quantitative bgalactosidase assay. (A) Detection of GABAB1a/GABAB2 (GABBR1a/ GABBR2) heterodimers. (B) Detection of AT1/AT2 (AGTR1/AGTR2) heterodimers. Error bars represent the standard deviations (n = 3). The control prey plasmid was pPR3-C mock vector. doi:10.1371/journal.pone.0066793.g005 Figure 4. Detection for dimerization of yeast Ste2p deletion mutants (TM1? and TM6?) in NMY62 strain. (A) Schematic of Ste2DC and the deletion mutants. Transmembrane (TM) domains are indicated with pillar-type boxes, and the Cub (Cub-LexA-VP16) or NubG (Nub with I13G mutation) is depicted as a rounded rectangle. (B) Growth assay without a-factor (left panels) and with 5 mM a-factor (right panels). Each cell was spotted in serial 10-fold dilutions on SD eu, Trp, Ade and His dropout plate. (C) Quantitative b-galactosidase activity in yeast cells containing various combinations of plasmids. Error bars represent the standard deviations (n = 3). The control prey plasmids were pPR3-C mock vector (empty vector) and pPR3-HXT1. doi:10.1371/journal.pone.0066793.greceptors. Additionally, the b-galactosidase assay for dimerization of GABAB1a receptor also showed similar results (Fig. S5). In contrast to the widely accepted concept of class C GPCR dimerization, the significance of in vivo dimerization of class A GPCRs remains controversial [20]. Since a growing amount of evidence indicates that class A GPCRs are able to form dimers or higher-ordered oligomers in vivo [21,22], we next evaluated class A GPCR heterodimer pairs. As class A GPCRs, AT1 and AT2 angiotensin receptors (AGTR1 and AGTR2) were selected. Consistent with previous reports [23,24], the b-galactosidase assay also illustrated the formation of heterodimers between AGTR1 and AGTR2 (Fig. 5B and Fig. S4B). Next, we aimed to apply our system to screen new candidate heterodimer FD&C Yellow 5 partners of AGTR1 receptor within the class A GPCRs, except for AGTR2. The CYC1 promoter was selected forexpressing AGTR1 as a bait protein (Fig. S4B). As a model candidate library, we constructed the prey vectors to express AGTR1 (as a positive control), b2-adrenergic receptor (ADRB2), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1A), somatostatin receptor 2 (SSTR2), somatostatin receptor 5 (SSTR5), endothelin receptor type B (EDNRB), neurotensin receptor 1 (NTSR1) and neurotensin receptor 2 (NTSR2) (Table S2 and S3) and then mixed equal amounts of these 9 prey vectors (containing pPR3-C mock vector). After introduction of the constructed library into the NMY63 yeast strains harboring AGTR1 bait vector, the selection with ADE2/HIS3 growth reporter genes was performed (Fig. 6A). A total of 30 colonies was generated on the adenine/histidine-deficient selection media (Fig. 6A). Following iso.D (Table 1 and Fig. S3). Since clear evidence for the functional role of GPCR homodimer and heterodimer pairs was first obtained for class C receptors, such as GABAB receptors [17], we 10781694 tested the dimerization of GABAB2 receptor (GABBR2) with GABAB1a receptor (GABBR1a). The b-galactosidase assay clearly showed the specific activities both for GABAB2/GABAB2 and GABAB2/GABAB1a couples (Fig. 5A and Fig. S4A). This result was coincident with the fact that GABAB2 receptor could form not only heterodimer with GABAB1a receptor but also homodimer [18,19], indicating that the split-ubiquitin-based approach could detect the homodimerization and heterodimerization of GABABScreening of Human GPCR HeterodimerFigure 5. Dimerization assays of human GPCRs. Quantitative bgalactosidase assay. (A) Detection of GABAB1a/GABAB2 (GABBR1a/ GABBR2) heterodimers. (B) Detection of AT1/AT2 (AGTR1/AGTR2) heterodimers. Error bars represent the standard deviations (n = 3). The control prey plasmid was pPR3-C mock vector. doi:10.1371/journal.pone.0066793.g005 Figure 4. Detection for dimerization of yeast Ste2p deletion mutants (TM1? and TM6?) in NMY62 strain. (A) Schematic of Ste2DC and the deletion mutants. Transmembrane (TM) domains are indicated with pillar-type boxes, and the Cub (Cub-LexA-VP16) or NubG (Nub with I13G mutation) is depicted as a rounded rectangle. (B) Growth assay without a-factor (left panels) and with 5 mM a-factor (right panels). Each cell was spotted in serial 10-fold dilutions on SD eu, Trp, Ade and His dropout plate. (C) Quantitative b-galactosidase activity in yeast cells containing various combinations of plasmids. Error bars represent the standard deviations (n = 3). The control prey plasmids were pPR3-C mock vector (empty vector) and pPR3-HXT1. doi:10.1371/journal.pone.0066793.greceptors. Additionally, the b-galactosidase assay for dimerization of GABAB1a receptor also showed similar results (Fig. S5). In contrast to the widely accepted concept of class C GPCR dimerization, the significance of in vivo dimerization of class A GPCRs remains controversial [20]. Since a growing amount of evidence indicates that class A GPCRs are able to form dimers or higher-ordered oligomers in vivo [21,22], we next evaluated class A GPCR heterodimer pairs. As class A GPCRs, AT1 and AT2 angiotensin receptors (AGTR1 and AGTR2) were selected. Consistent with previous reports [23,24], the b-galactosidase assay also illustrated the formation of heterodimers between AGTR1 and AGTR2 (Fig. 5B and Fig. S4B). Next, we aimed to apply our system to screen new candidate heterodimer partners of AGTR1 receptor within the class A GPCRs, except for AGTR2. The CYC1 promoter was selected forexpressing AGTR1 as a bait protein (Fig. S4B). As a model candidate library, we constructed the prey vectors to express AGTR1 (as a positive control), b2-adrenergic receptor (ADRB2), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1A), somatostatin receptor 2 (SSTR2), somatostatin receptor 5 (SSTR5), endothelin receptor type B (EDNRB), neurotensin receptor 1 (NTSR1) and neurotensin receptor 2 (NTSR2) (Table S2 and S3) and then mixed equal amounts of these 9 prey vectors (containing pPR3-C mock vector). After introduction of the constructed library into the NMY63 yeast strains harboring AGTR1 bait vector, the selection with ADE2/HIS3 growth reporter genes was performed (Fig. 6A). A total of 30 colonies was generated on the adenine/histidine-deficient selection media (Fig. 6A). Following iso.