TRAF2-GRK2 Interaction Mediates Non-Canonical Desensitization of Beta-Adrenergic Receptors to TNF-[alpha]

β- Adrenergic receptor (βAR), a prototypical G-protein coupled receptor (GPCR) is a key regulator of cardiac function. Impaired G-protein coupling (desensitization) of βARs is a key hallmark of heart failure. Desensitization occurs by phosphorylation of βARs by GRK2 to sympathetic overdrive leading to β-arrestin binding and endocytosis. Our studies show that TNFα-induces βAR phosphorylation/desensitization via GRK2 recruitment that is independent of the classical Gβγ mechanism suggesting a non-canonical pathway of GRK2 recruitment. Comorbid conditions like obesity/ hypertension have a higher risk of heart failure and is associated with increase in TNFα, a known cardio-depressant. 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 and as TNFα recruits TRAF2 to mediate downstream signaling, we hypothesized that TRAF2 recruits GRK2 to the plasma membrane causing receptor phosphorylation and desensitization. Our study shows TNFα drives βAR dysfunction by recruiting GRK2 to βAR complex through TRAF2. Our studies indicate that TRAF2 and GRK2 form a cytosolic complex and is recruited to the receptor complex post-TNFα treatment. Pharmacologic inhibition of GRK2 results in decreased βAR phosphorylation after TNFα. Furthermore, siRNA knockdown of TRAF2 results in reduction of βAR phosphorylation indicating the key role for TRAF2 in TNFα-mediated desensitization. Given that nothing is known about TRAF2 and GRK2 interaction we performed molecular docking using Haddock software to identify that the N-terminal region of GRK2 may interact with TRAF2. Overexpression of GRK2 N-terminal domain as dominant negative strategy resulted in reduced β2AR phosphorylation in response to TNFα, suggesting the requirement of GRK2 N-terminal in non-canonical β2AR desensitization. We are currently using cardiac specific GRK2 and TRAF2 knockout mice to assess their role in cardiac function in response to TNFα showing the role of GRK2-TRAF2-TNFR axis in mediating agonist-independent βAR desensitization/dysfunction.