Mapping MOR Interacting Proteins Using Y2H and MYTH Screens

The traditional Y2H screening method involves the reconstitution of the GAL4 transcription factor through the interaction of a bait protein fused to the GAL4 DNA binding domain and a prey protein (from a fetal brain cDNA library) fused to the transcriptional activating domain of GAL4 [21] (link). This method is biased for cytosolic and nuclear proteins, as the protein complex must be imported into the nucleus to activate transcription. Therefore, only the cytosolic portion of integral membrane proteins is usually employed in this type of screen. Traditional Y2H screens in this study were performed as previously described [22] , [23] using the second intracellular loop (IL2; amino acids 166–187) of the MOR as bait to screen a fetal human brain cDNA library. The MOR-IL2 was cloned into the yeast GAL4 DNA binding domain expression vector pAS2-1 (Clontech, Palo Alto, CA), while the human fetal brain cDNA library was provided in the GAL4 activation domain vector pACT2 (Clonetech). Bait and prey plasmids were successively transformed into yeast strain MaV103 [22] . Transformation of yeast with the fetal human brain library produced ∼2×10⁶ transformants/µg of DNA on quadruple dropout plates (-Leu/−Trp/−His/−Ura; Clonetech) containing 3-amino-1,2,4-triazol (3AT). Interactions were assayed for β-galactosidase (β-gal) activity via the nitrocellulose lift method [22] . cDNAs were extracted from yeast colonies, sequenced, and subjected to Basic Local Alignment Search Tool (BLAST) analysis to determine their identities.
To identify additional MOR interacting proteins (MORIPs), a modified split-ubiquitin membrane yeast two-hybrid (MYTH) screen was performed as previously described [24] (link). The MYTH system uses the split-ubiquitin method, in which the reconstitution of ubiquitin is mediated by a specific protein-protein interaction. Ubiquitin-specific proteases cleave at the C-terminus of ubiquitin, which releases a transcription factor that can translocate to the nucleus and activate transcription of a reporter gene [25] (link). The unique advantage of MYTH is that full-length integral membrane proteins can be used as bait and are amenable to protein-protein interaction analyses in their natural membrane environment [26] (link), [27] (link). For this study, full-length human MOR cDNA in the bait vector pCCW-STE (Dualsystems Biotech AG, Switzerland) and a human fetal brain library in the prey vector pPR3-N (Dualsystems) were sequentially transformed into S. cerevisiae reporter strain THY.AP4. Transformation of yeast with the human brain library yielded 6×10⁶ transformants/µg DNA on quadruple drop out plates (−Trp/−Leu/−His/−Ade; Clonetech) containing 3AT. Fifty transformants were positive for β-gal activity. These colonies were picked and their cDNAs extracted, sequenced and subjected to BLAST analysis. From this screen we identified four novel MORIPs (Table 1) that were subjected to further biochemical analysis.
To map sites of interaction between the MOR and the newly identified MORIPs, each MOR intracellular loop (IL) was tested for interaction with individual MORIPs using the traditional Y2H method. MOR-IL domains (IL1, amino acids 97–102; IL2, amino acids 166–187; IL3, amino acids 259–282; and C-tail residues 361–420) were separately ligated into pAS2-1 and assayed for interaction with candidate MORIP cDNA clones in pACT2. Bait and prey plasmids were simultaneously co-transformed into S. cerevisiae strain MaV103 and interactions assayed for β-gal activity as described above.