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N-Ethylmaleimide-Sensitive Factor Regulates β₂ Adrenoceptor Trafficking and Signaling in Cardiomyocytes

Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois (Y.W., Y.X.); Program in Pharmaceutical Sciences and Pharmacogenomics and Department of Pharmaceutical Chemistry (B.L.), Department of Psychiatry and Department of Cellular and Molecular Pharmacology (M.V.Z.), University of California at San Francisco, San Francisco, California; and Department of Molecular and Cellular Physiology, Stanford Medical Center, Palo Alto, California (B.K.K.)

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.

Address correspondence to: Dr. Yang Xiang, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, B523 Burrill Hall, MC-114, 407 S. Goodwin Avenue, Urbana, IL 61801. E-mail: kevinyx{at}uiuc.edu

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				Y. Wang, V. De Arcangelis, X. Gao, B. Ramani, Y.-s. Jung, and Y. Xiang Norepinephrine- and Epinephrine-induced Distinct 2-Adrenoceptor Signaling Is Dictated by GRK2 Phosphorylation in Cardiomyocytes J. Biol. Chem., January 25, 2008; 283(4): 1799 - 1807. [Abstract] [Full Text] [PDF]