The TM3-ECL2 disulfide bridge is a structural checkpoint for productive G_olf 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: OR51E2^C96S,C178S, designed to disrupt the conserved TM3-ECL2 disulfide bridge, and OR51E2^{p.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-G_αolf 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 A_2A receptor, although the interaction profile was altered. Together, these results reveal how extracellular constraints differentially regulate OR51E2 signaling and GPCR heteromerization.