2026/06/25 by Claudia Garrigós, Joan Serrano-Marín, Joan Serrano‐Marín +8
Biochemistry, Genetics and Molecular Biology · Neuroscience · Nursing · #Biochemical Analysis and Sensing Techniques #Cysteine #Extracellular #G protein-coupled receptor #Intracellular #Ligand (biochemistry) #Olfactory and Sensory Function Studies #Protein–protein interaction #Receptor #Receptor Mechanisms and Signaling #Signal transduction
paper · doi:10.1016/j.bcp.2026.118191
published in Biochemical Pharmacology 251(Pt 2), 118191 (Elsevier BV)
openalex publication_date 2026/06/25 · openalex created_date 2026/06/26 · openalex updated_date 2026/07/25
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.Ser174Val183del , 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.