Abstract

Recycling of G protein-coupled receptors determines the functional resensitization of receptors and is implicated in switching β₂ adrenoceptor (β₂AR) G protein specificity in cardiomyocytes. The human β₂AR carboxyl end binds to the N-ethylmaleimide-sensitive factor (NSF), an ATPase integral to membrane trafficking machinery. It is interesting that the human β₂AR (hβ₂AR) carboxyl end pulled down NSF from mouse heart lysates, whereas the murine one did not. Despite this difference, both β₂ARs exhibited substantial agonist-induced internalization, recycling, and G_i coupling in cardiomyocytes. The hβ₂AR, however, displayed faster rates of agonist-induced internalization and recycling compared with the murine β₂AR (mβ₂AR) and a more profound G_i component in its contraction response. Replacing the mβ₂AR proline (-1) with a leucine generated a gain-of-function mutation, mβ₂AR-P417L, with a rescued ability to bind NSF, faster internalization and recycling than the mβ₂AR, and a significant enhancement in G_i signaling, which mimics the hβ₂AR. Selective disruption of the mβ₂AR-P417L binding to NSF inhibited the receptor coupling to G_i. Mean-while, inhibiting NSF with N-ethylmaleimide blocked the mβ₂AR recycling after agonist-induced endocytosis. Expressing the NSF-E329Q mutant lacking ATPase activity inhibited the mβ₂AR coupling to G_i in cardiomyocytes. Our results revealed a dual regulation on hβ₂AR trafficking and signaling by NSF through direct binding to cargo receptor and its ATPase activity and uncovered an unprecedented role for the receptor binding to NSF in regulating G protein specificity that has diverged between mouse and human β₂ARs.