N-homocysteinylation of β-arrestins biases GPCR signaling and promotes platelet activation

• GPCRs downstream of β-arrestins are the direct pathogenic targets of hcy.

• HHcy acts as a modulator of GPCR-biased signaling, suggesting that G-biased agonists are better choices for disease treatment under HHcy.

Hyperhomocysteinemia (HHcy) is strongly associated with cardiovascular diseases (CVDs), and HHcy has been identified as a risk factor for thrombotic diseases. Most patients with HHcy die from various complications closely related to thrombotic diseases. However, the underlying mechanisms have not been fully elucidated. G-protein coupled receptors (GPCRs), the central regulators of the cardiovascular system, primarily control platelet activation. By examining the effects of HHcy on a panel of GPCRs involved in platelet aggregation, we found that HHcy systematically modulated biased GPCR signaling through the inhibition of desensitization by β-arrestins and the amplification of G protein signals. We further revealed that the N-homocysteinylation of β-arrestin1/2 at lysine (K) residues (K294/K296) disrupted the interaction between β-arrestins and GPCRs. The above phenomenon may be universal because HHcy was found to modulate the signaling bias of nine other randomly selected GPCRs. Moreover, we found that the proinflammatory effects of Hcy and HTL were weakened in Arrb2-/- mice and that the reintroduction of WT but not K296R β-arrestin2 (in mice) mutants into primary peritoneal macrophages reversed these effects. Notably, Arrb2K296R mice reversed HHcy-induced thrombus formation and platelet aggregation. Our results suggest that a G-biased agonist could be a better choice for disease therapy under HHcy conditions. Collectively, our findings demonstrate that the N-homocysteinylation of β-arrestin1/β-arrestin2 actively modulates the biased property of GPCR signaling, which contributes to the pathophysiology of HHcy-related CVDs and provides insight into the selection of agonists for the treatment of diseases under HHcy conditions.