Mechanism of GPR84 allosteric modulation at a helix 8-proximate site.

Allosteric modulators offer opportunities for pathway-selective G protein-coupled receptor (GPCR) signaling, but the mechanisms enabling biased allosteric modulation remain poorly understood. We identify a helix 8-proximate allosteric site in the immune-metabolic receptor GPR84 and define how it achieves Gi-biased signaling. Cryo-electron microscopy structures of the GPR84-Gi complexes bound to the orthosteric agonist OX04539 alone or with the positive allosteric modulator PSB-16671 reveal a helix 8-proximate allosteric site for PSB-16671 formed between transmembrane helices 1 (TM1) and TM7. Molecular dynamics simulations and mutagenesis uncover a polar interaction network linking orthosteric and allosteric sites through conserved residues including Asp662.50, Asn1043.36, and Asn3627.45, and disrupting this network enhances allosteric cooperativity. PSB-16671 stabilizes a receptor conformation with pronounced TM6 displacement that favors Gi coupling while disfavoring β-arrestin recruitment, sustaining macrophage phagocytosis of cancer cells without the desensitization induced by balanced agonists. Sequence analysis suggests that receptor-specific helix 8-proximate allosteric sites may be broadly targetable across class A GPCRs. These findings establish mechanistic principles for biased allosteric modulation applicable beyond GPR84.