Rapid, Facile Detection of Heterodimer Partners for Target Human G-Protein-Coupled Receptors Using a Modified Split-Ubiquitin Membrane Yeast Two-Hybrid System

Potentially immeasurable heterodimer combinations of human G-protein-coupled receptors (GPCRs) result in a great deal of physiological diversity and provide a new opportunity for drug discovery. However, due to the existence of numerous combinations, the sets of GPCR dimers are almost entirely unknown and thus their dominant roles are still poorly understood. Thus, the identification of GPCR dimer pairs has been a major challenge. Here, we established a specialized method to screen potential heterodimer partners of human GPCRs based on the split-ubiquitin membrane yeast two-hybrid system. We demonstrate that the mitogen-activated protein kinase (MAPK) signal-independent method can detect ligand-induced conformational changes and rapidly identify heterodimer partners for target GPCRs. Our data present the abilities to apply for the intermolecular mapping of interactions among GPCRs and to uncover potential GPCR targets for the development of new therapeutic agents.


Document S1. Supplementary Materials and Methods (Plasmid constructions for supporting information)
All plasmids used in this study are summarized in Table S2. All oligonucleotides are listed in Table S1. The bait proteins (X) were fused with a C-terminal ubiquitin moiety linked to an artificial transcription factor (X-Cub) in pBT3-C (Dualsystems Biotech AG, Schlieren, Switzerland). The prey proteins (Y) were fused with an N-terminal moiety of split-ubiquitin with I13G mutation (Y-NubG) in pPR3-C (Dualsystems Biotech AG).

Bait vectors:
For bait vectors, several promoters exhibiting distinctive expression strength were substituted for the original weak CYC1 promoter of pBT3-C as follows.
PHO5 promoter (stronger than CYC1 promoter) was PCR-amplified with oligonucleotides o1 and o2. The SacII-XbaI PHO5 promoter was inserted at the SacII-XbaI site on pBT3-C, resulting in the plasmid pBPH3-C.
TPI1 promoter (stronger than PHO5 promoter) was PCR-amplified with oligonucleotides o3 and o4. The SacII-XbaI TPI1 promoter was inserted at the SacII-XbaI site on pBT3-C, resulting in the plasmid pBTP3-C.
TDH3 promoter (stronger than TPI1 promoter) was PCR-amplified with oligonucleotides o5 and o6. The SacII-XbaI TDH3 promoter was inserted at the SacII-XbaI site on pBT3-C, resulting in the plasmid pBTD3-C.

GPCR expression plasmids:
The bait and prey plasmids used for the expression of GPCRs were constructed as follows. Full length STE2 genes encoding yeast pheromone receptor were PCR-amplified with oligonucleotide pairs: o7 and o8; o9 and o10. The XbaI-HindIII STE2 gene fragments were inserted at the XbaI-HindIII site on pBT3-C, pBPH3-C, pBTP3-C and pBTD3-C, resulting in the plasmids pBT3-STE2, pBPH3-STE2, pBTP3-STE2 and pBTD3-STE2, respectively. The SpeI-EcoRI STE2 gene fragment was inserted at the SpeI-EcoRI site on pPR3-C, resulting in the plasmid pPR3-STE2.
Truncated STE2 genes that lack the C-terminal domain (Ste2ΔC) were PCR-amplified

Supporting Information:
Rapid, facile detection of heterodimer partners for target human G-protein-coupled receptors using a modified split-ubiquitin membrane yeast two-hybrid system 2 with oligonucleotide pairs: o7 and o11; o9 and o12. The XbaI-HindIII STE2ΔC gene fragments were inserted at the XbaI-HindIII site on pBT3-C and pBTP3-C, resulting in the plasmid pBT3-STE2ΔC and pBTP3-STE2ΔC, respectively. The SpeI-EcoRI STE2ΔC gene fragment was inserted at the SpeI-EcoRI site on pPR3-C, resulting in the plasmid pPR3-STE2ΔC.
The HXT1 gene encoding glucose transporter was PCR-amplified with oligonucleotides o17 and o18. The SpeI-EcoRI HXT1 gene fragment was inserted at the SpeI-EcoRI site on pPR3-C, resulting in the plasmid pPR3-HXT1.
GABBR1a genes encoding GABAB1a receptor were PCR-amplified with oligonucleotide pairs: o19 and o20; o21 and o22. The XbaI-HindIII GABBR1a gene fragment was inserted at the XbaI-HindIII site on pBTP3-C, resulting in the plasmid pBTP3-GABBR1a. The SpeI-EcoRI GABBR1a gene fragment was inserted at the SpeI-EcoRI site on pPR3-C, resulting in the plasmid pPR3-GABBR1a.
GABBR2 genes encoding GABAB2 receptor were PCR-amplified with oligonucleotide pairs: o23 and o24; o25 and o26. The XbaI-HindIII GABBR2 gene fragments were inserted at the XbaI-HindIII site on pBTP3-C and pBTD3-C, resulting in the plasmid pBTP3-GABBR2 and pBTD3-GABBR2, respectively. The SpeI-EcoRI GABBR2 gene fragment was inserted at the SpeI-EcoRI site on pPR3-C, resulting in the plasmid pPR3-GABBR2.

Supporting Information:
Rapid, facile detection of heterodimer partners for target human G-protein-coupled receptors using a modified split-ubiquitin membrane yeast two-hybrid system 3 AGTR1 genes encoding AT1 (angiotensin type 1) receptor were PCR-amplified with oligonucleotide pairs: o27 and o28; o29 and o30. The XbaI-HindIII AGTR1 gene fragments were inserted at the XbaI-HindIII site on pBT3-C and pBTP3-C, resulting in the plasmid pBT3-AGTR1 and pBTP3-AGTR1, respectively. The SpeI-EcoRI AGTR1 gene fragment was inserted at the SpeI-EcoRI site on pPR3-C, resulting in the plasmid pPR3-AGTR1.
SSTR5 genes encoding somatostatin receptor 5 were PCR-amplified with oligonucleotide pairs: o43 and o44; o45 and o46. The XbaI-HindIII SSTR5 gene fragment was inserted at the XbaI-HindIII site on pBTD3-C, resulting in the plasmid pBTD3-SSTR5. The SpeI-EcoRI SSTR5 gene fragment was inserted at the SpeI-EcoRI site on pPR3-C, resulting in the plasmid pPR3-SSTR5.
ADRB2 genes encoding β2-adrenergic receptor were PCR-amplified with