Abstract 403: TNFα-receptor Associated Factor 2 (TRAF2) Interaction With GRK2 Mediates βAR Desensitization to TNFα

Abstract

β- Adrenergic receptor (βAR), a prototypical G-protein coupled receptor (GPCR) is a key regulator of cardiac function. Loss of surface receptors (downregulation) and impaired G-protein coupling (desensitization) of βARs are hallmarks of heart failure. Desensitization occurs by phosphorylation of βAR by GRK2 in response to sympathetic overdrive leading to β-arrestin binding and its endocytosis into endosomes. Our studies indicate that TNFα induces βAR phosphorylation/desensitization in HEK 293, neonatal and adult cardiomyocyte cells via GRK2 recruitment. The TNFα induced GRK2 increase is independent of agonist or Gβγ pathway, suggesting a non-canonical mechanism of GRK2 recruitment to the plasma membrane. In conditions of obesity and type 2 diabetes which are clear risk factors for heart failure are consistently associated with significant increase in TNFα which is a known cardio-depressant. Correspondingly, β-blockers are known to be contra-indicative in obesity which we believe is due to βAR desensitization through non-canonical GRK2 recruitment to the βAR in response to elevated TNFα. However the underlying mechanisms for βAR desensitization are not known. We hypothesized that TRAF2 (TNFα-receptor associated factor 2) recruits GRK2 to the plasma membrane causing receptor phosphorylation. Using co-immunoprecipitation studies, we identified that scaffolding protein TRAF2 (responsible for TNFR signaling) interacts with GRK2 in untreated whole cell lysate. Furthermore, co-immunoprecipitation and immunoblotting show that TRAF2-GRK2 interaction is increased in the plasma membrane after 30 minutes of TNFα treatment. Immunoblotting also shows that TNF receptor 2 (TNFR2) engages more robustly with GRK2-TRAF2 complex than TNFR1, suggesting GRK2-TRAF2 dependent phosphorylation of βARs with TNFα is TNFR2 driven. Furthermore, TNFα induced βAR phosphorylation/desensitization is GRK2 dependent as significant loss in βAR phosphorylation is observed with GRK2 inhibitor paroxetine despite presence of TNFα. These observations suggest that cardiac dysfunction mediated by TNFα in conditions of obesity could occur through a novel GRK2-TRAF2-TNFR2 axis that could mediate agonist-independent βAR phosphorylation.