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ted extra sets of chimeric receptors by replacing ACTHR TM domains with corresponding regions of MC4R in various combinations (Ch3 h15 in Table S1 in Supplementary Material). Subsequent evaluation with the membrane transportation efficiency revealed that retention occurred only when each TM3 and TM4 from ACTHR had been present simultaneously, suggesting this area, or components of this region, is accountable for intracellular retention. As well as membrane transportation, we also carried out binding and cAMP response analyses. Although, on account of their sensitivity, the initial objective of those assays was to allow the detection of low-level membrane trafficking, the outcomes also identified regions that establish ACTHRligand-binding specificity. MC4R-based chimeric receptors were in a position to bind ligands when TM4 and TM6 were replaced with the corresponding components of ACTHR, but binding was not observed when each regions have been replaced simultaneously. As a result, we speculated that there has to be some form of interaction between these two components, which disrupts the conformation essential for formation in the MC consensus pharmacophore (M ) binding pocket (199). A year right after publication of our report, Hinkle and colleagues reported on employment of remarkably related strategy (201). Indeed, quite a few from the chimeric receptors have been identical in spite of becoming independently designed. Having said that, they employed distinct procedures for characterization with the chimeric receptor properties (i.e., measurement of surface transport and activation efficiency), and additionally they evaluated the effects of co-expression with MRAP1. As in our study, the chimeric receptors have been generated in sets. The initial set included ACTHR-based chimeric receptors using a variety of TM domains replaced to corresponding regions from MC4R (2C1C6 in Table S1 in Supplementary Material), whilst the second set mirrored these so that the identical TM domains inside MC4R have been replaced with the corresponding regions from ACTHR (4C1C6 in Table S1 in Supplementary Material). Functional analysis confirmed that co-expression with MRAP1 promoted the cell-surface trafficking of ACTHR-based chimeric receptors, but this was decreased with MC4R-based receptors. Nonetheless, the ACTHR-based receptor with only the N-terminus, TM1 domain, and IL1 replaced for the corresponding regions of MC4R (2C1 in Table S1 in Supplementary Material) was an exception, considering that it was located on the cell surface even in the absence of MRAP1, but despite its profitable transportation it was functionally inactive. To investigate this additional, Hinkle and colleagues developed an additional set of chimeric receptors (C2C1a, C2C1b, and C2C1c in Table S1 in Supplementary Material), and subsequent evaluation of their properties narrowed the area accountable for intracellular retention to TM1 alone, since the corresponding receptor variant (C2C1b in Table S1 in Supplementary Material) was both proficiently transported towards the cell plasma membrane, and functionally active inside the presence of MRAP1. Functional analysis of receptors in the major set also revealed a further surprising discovery; when co-expressed with MRAP1, ACTHR-based chimeric receptor with TM2, EL1, and TM3 replaced for the corresponding regions of MC4R (2C2 in Table S1 in Supplementary Material) was able to induce intracellular response upon stimulation with each NDP-MSH and ACTH and also displayed substantial constitutive activity. Considering the fact that both of these traits are known to become characterist