MOR1K expression and binding pattern. (A) The schematic diagram illustrates the exonic composition and relative positions of PCR primers designed to amplify the major MOR1 isoform and the newly identified alternative MOR1K isoform. The relative positions of translation initiation start and stop codons are designated by ATG and TGA, respectively. The predicted protein structure of MOR1 and MOR1K isoforms is schematically presented. Translation of the MOR1K variant results in a 6TM receptor, truncated at the N-terminus. (B) Real-time PCR was performed on total RNA samples from the human brain regions known to express MOR1. Primers specific for exons 1 and 2 were used to measure MOR1 and primers specific for exons 13 and exon 2 were used to measure MOR1K. GAP3DH was used as a control for cDNA loading and PCR efficiency. (C) Confocal images of C-terminally MYC-tagged MOR1 or FLAG-tagged MOR1K overexpressed in HEK293 cells and stained with either Anti-MYC-Tag Antibody (Alexa Fluor 647 Conjugate) or Anti-DYKDDDDK Tag Antibody (Alexa Fluor 555 conjugate). Cells transfected with MOR1 showed membrane expression of receptor, while cell transfected with MOR1K express receptor only intracellular. (D) Confocal images of C-terminally FLAG-tagged MOR1K overexpressed in Be2C cells and stained with either Anti-FLAG M2 Antibody Alexa Fluor Conjugate or fluorescent-labeled naloxone (FNAL). Cells transfected with MOR1K showed intracellular retention of FNAL that co-localized with antibody-labeled receptor. (E) The binding of naloxone to MOR1K was assessed using flow cytometry to measure FNAL retention. Be2C cells transfected with either MOR1 or MOR1K isoforms showed increased retention of FNAL at concentrations of 0.1 and 1 μM. FNAL retention was abolished in the presence of 10 μM unlabelled naloxone (Nal). In panel E, data are presented as mean + SEM. *P < 0.05 different from controls).