Fig. 5: TM1^MOR interacts with DOR and blocks DM heterodimers, and additionally inhibits MM and DD homodimers, whereas Dpep(20-42)DM only blocks DM heterodimer formation.

a Experimental design for examining the interactions of TM1^MOR, Nterm-TM1^MOR, and TM4^DOR (Fig. 2b) with WT-ORs (top). For the correct orientation of TM4^DOR in the PM, it was linked to the transmembrane domain of LDL receptor (TM^LDLR). Representative (among 20 replicates) single-molecule images of DOR-mGFP (green), MOR-Halo (magenta), and SNAPf-TM1^MOR (cyan) co-expressed at similar levels of 0.5-1.0 fluorescent spots/µm² (bottom). b TM1^MOR and Nterm-TM1^MOR form dimers with WT-DOR as efficiently as WT-MOR does. TM1^DOR and TM4^DOR form dimers with WT-MOR to lesser extents. Expression levels of all molecules were adjusted to 0.5-1.0 fluorescent spots/µm² individually. c Reduction of the DM colocalization index with an increase in the number densities of the TM peptides (TM1^MOR, Nterm-TM1^MOR, and TM4^DOR) relative to that of MOR, expressed in the PM. DOR and MOR were both expressed at levels of 0.5-1.0 fluorescent spots/µm². The effects of TM1^MOR and Nterm-TM1^MOR are stronger than that of TM4^DOR. The rectangular box without the peptides (x = 0) indicates the result of the box plot without datapoints (to avoid excessive complexity in the plot), where the cross indicates the mean value and the box indicates the interquartile range (25–75%). d TM1^MOR and Nterm-TM1^MOR reduce MM and DD homodimers (in addition to DM heterodimers), but 1 µM Dpep(20-42)DM does not. TM1^MOR and Nterm-TM1^MOR expression levels were ≈10x of MOR and DOR, following the results shown in panel c. TM^LDLR is a negative control, representing non-interacting TM domains. In the box plots (b, d), horizontal bars, boxes, and whiskers indicate mean values, interquartile ranges (25–75%), and 10-90% ranges, respectively. * and ns represent significant (p < 0.05) and non-significant (p ≥ 0.05) differences, respectively (Tukey’s multiple comparison test). All of the statistical parameters and analysis results including sample size n and p values are provided in Supplementary Data 2. Source data are provided as a Source Data file.