The TM3-ECL2 disulfide bridge is a structural checkpoint for productive Golf coupling of the odorant receptor OR51E2.

Olfactory receptors (ORs) constitute the largest subfamily of G protein-coupled receptors (GPCRs), yet their activation mechanisms remain poorly understood, particularly outside canonical olfaction. The ectopically expressed receptor OR51E2 responds to microbiota-derived short-chain fatty acids (SCFAs) and has been linked to cancer, metabolism, and neuroprotection. However, the structural determinants governing OR51E2 activation, G protein coupling, and receptor-receptor interactions remain incompletely defined. Here, we combined targeted mutagenesis, functional signaling assays, BRET-based interaction analyses, and molecular dynamics simulations to examine key extracellular structural elements of OR51E2. Two variants were generated: OR51E2C96S,C178S, designed to disrupt the conserved TM3-ECL2 disulfide bridge, and OR51E2p.Ser174_Val183del, lacking part of ECL2. Both mutants trafficked efficiently to the plasma membrane and were defective in SCFA-induced cAMP signaling. In the double cysteine mutant, NFAT-based assays further confirmed a severe loss of ligand-induced signaling. BRET analyses revealed that disruption of the disulfide bridge markedly impairs productive OR51E2-Gαolf engagement and abolishes ligand-induced changes in receptor-G protein proximity. Consistently, molecular dynamics simulations predicted preserved ligand binding but preferential TM6/TM7 displacement, suggesting a signaling-incompetent intracellular architecture not properly organized for Gαolf coupling. These findings support a model in which the TM3-ECL2 disulfide bridge acts as an extracellular structural checkpoint that converts ligand occupancy into productive G protein engagement. Notably, its disruption did not prevent heteromerization with the adenosine A2A receptor, although the interaction profile was altered. Together, these results reveal how extracellular constraints differentially regulate OR51E2 signaling and GPCR heteromerization.