G protein-coupled receptors (GPCRs) represent the largest family of cellular receptors and are involved in a wide range of physiological activities. Traditionally, they were described as on/off signal transducers, located on the plasma membrane to convey signals from the outside to the inside of the cell, and composed of seven transmembrane (TM) domains as the minimum structural unit to achieve stability and function. All these concepts have gradually been broadened and made more complex with research progress.
The classical view of the on/off activation of GPCRs was substituted with a more complex scenario, with a continuum of conformations where the thermal energy drives the receptor into preferred energy wells stabilized upon ligand binding, transducer coupling and other factors influencing their dynamic fluctuations. While plasma membrane localization remains predominant, GPCRs have also been found in various intracellular compartments, including the endoplasmic reticulum, Golgi apparatus, and mitochondria, with unique roles determined by their subcellular localization. The necessity for seven TM domains to function is no longer absolute, as some truncated or short forms of receptors were shown to have a role in cell physiology (such as a dominant negative effect on their wild-type counterpart) or retain their functionality. Notably, truncated GPCRs typically include the early TM domains but terminate around TM5. To date, no tail isoforms (missing initial TM domains but retaining the final ones and the C-terminus) have been described.
In this study, we identified and characterized an internal ribosome entry site (IRES) within the third intracellular loop (IL3) of the M2 muscarinic receptor, enabling cap-independent translation of a highly truncated C-terminal fragment comprising TM6 and 7, termed M2tail. The M2tail localizes unexpectedly to the inner mitochondrial membrane, where it interacts with the F0F1 ATP-synthase complex, thereby reducing basal mitochondrial oxygen consumption and reactive oxygen species levels. This localization suggests a protective role for M2tail under oxidative stress conditions.
Extending our investigation to the dopamine D2 receptor, we discovered a significant sequence overlap with the M2 IRES in the IL3 region upstream of methionine 346 (Met346). To replicate the steps that led to the M2 IRES discovery, we split the receptor into trunk and
tail segments and verified that the activity could be reconstituted. Functional assays using a stop codon mutant revealed that, unlike the M2 stopped mutant, the D2 mutant (D2stop283) was unable to produce a tail fragment and reconstitute receptor activity. However, using antibodies targeted to the C-terminal portion of the receptor we could detect in western blot analysis some light bands similar to those observed for the M2 receptor. By mutating methionine residues in IL3 that could serve as alternative initiation points for cap-independent translation, we identified that the 15kDa band was absent in the D2M346A mutant, suggesting Met346 as the likely initiation site for D2tail synthesis.
The mitochondrial localization and function of D2tail, if confirmed, could provide valuable insights into dopaminergic neuron vulnerability to oxidative and bioenergetic stressors, which contribute to neurodegenerative processes, including those implicated in Parkinson’s disease. These findings suggest that certain GPCR fragments play crucial roles beyond traditional cell-surface signaling, directly influencing mitochondrial bioenergetics and cellular stress responses. Future research employing animal models with mutations in the start codons of these GPCR fragments could elucidate their physiological and pathological roles.