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 Gi 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 Gi 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 Gi signaling, which mimics the hβ2AR. Selective disruption of the mβ2AR-P417L binding to NSF inhibited the receptor coupling to Gi. Meanwhile, 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 Gi 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.
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