Background <p>CCR5 is a critical receptor for anti-infectious immunity. It acts by binding chemokines, which activate Gαβγ protein-dependent signaling and ultimately regulate leukocyte recruitment. However, CCR5 is also implicated in numerous pathological conditions, raising interest in its potential as a therapeutic target. Notably, CCR5 serves as a coreceptor for HIV-1 envelope glycoproteins (Envs), allowing viral entry into host cells and initiating infection. Whether and how Envs trigger CCR5 signaling remains debated, with conflicting evidence regarding their ability to mimic chemokine-induced activation of the receptor. This study aims to clarify the nature of Env-induced CCR5 signaling.</p> Methods <p>We used a real-time bioluminescence resonance energy transfer (BRET)-based assay in living cells to monitor the activation of ten Gα subunits from all four major G protein families in response to a prototypical CCR5 chemokine or HIV-1 Envs. Functional consequences of receptor activation were assessed via Ca²⁺ flux and cAMP inhibition assays. Molecular dynamics simulations were performed to investigate differences in ligand-induced conformational changes in CCR5. The role of membrane cholesterol in ligand engagement was studied using methyl-β-cyclodextrin treatments and binding assays.</p> Results <p>HIV Envs act as biased agonists at CCR5, selectively activating distinct G protein families compared to chemokines. Notably, Env-induced signaling, but not chemokine signaling, was strictly dependent on specific Gα subunit abundance and CCR5/G protein stoichiometry. This dependency likely explains the conflicting data in the literature regarding Env agonism. Molecular modeling revealed that Envs and chemokines stabilize distinct active conformations of CCR5, particularly at the G protein interaction site, likely accounting for their differential signaling behaviors. Additionally, cholesterol depletion impaired chemokine, but not HIV-1 Env, binding to CCR5, suggesting that the two ligand types engage the receptor within distinct membrane microenvironments.</p> Conclusions <p>These findings reveal that CCR5 exists in a mosaic of distinct membrane-associated functional states, differentially accessed by viral and endogenous ligands. This spatial and signaling compartmentalization provides a mechanistic framework to understand CCR5’s functional diversity in health and disease and highlights the potential for developing context-specific CCR5-targeted therapeutic strategies.</p>