Tumor Necrosis Factor-α Prevents Desensitization of Gαs-Coupled Receptors by Regulating GRK2 Association with the Plasma Membrane

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Abstract
We have reported previously that interleukin-1 and tumor necrosis factor (TNF)-α increase expression and function of adenosine A2A receptors (A2ARs), although the increased function is disproportionate to the increment in expression. We therefore studied the effect of TNF-α on A2A R function and desensitization in human monocytoid THP-1 cells. We observed that TNF-α regulates activity of A2A Rs and other G protein-coupled receptors (GPCRs) by altering their ligand-mediated desensitization. Pretreatment of resting cells with the A2AR agonist 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680) or the pan-adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine quickly desensitized cAMP responses to CGS 21680 restimulation, but TNF-α treatment prevented A2AR desensitization. As expected, A2A R occupancy induced translocation of GPCR kinase-2 (GRK2) to the plasma membrane (PM). We were surprised to find that after TNF-α treatment, A2AR occupancy not only failed to induce GRK2 translocation to PM but also decreased GRK2 association with PM. TNF-α altered GRK2 translocation in response to the β-adrenergic receptor agonist isoproterenol in a similar manner. Similar to GRK2, β-arrestin associated with PM after A2A R stimulation in control cells but not in TNF-α-treated cells. C2 -ceramide, a downstream mediator in the sphingomyelinase (SMase)-dependent pathway, mimicked the effect of TNF-α on GRK2 translocation. Moreover, inhibitors of the SMases and an inhibitor of c-Jun NH2-terminal kinase, also a downstream effector in the SMase pathway, reversed TNF-α-mediated effects on GRK2 translocation and A2A R desensitization. These results suggest a novel form of cross-talk between TNF-α receptors and GPCRs; TNF-α enhances GPCR function by preventing agonist-induced desensitization of GPCRs by diminishing agonist-dependent recruitment of GRK2 and β-arrestin to PM by a SMase pathway-mediated mechanism.