Wang Yongyu, Lauffer Benjamin, Von Zastrow Mark, Kobilka Brian K, Xiang Yang
Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, B523 Burrill Hall, MC-114, 407 S. Goodwin Avenue, Urbana, IL 61801, USA.
Mol Pharmacol. 2007 Aug;72(2):429-39. doi: 10.1124/mol.107.037747. Epub 2007 May 17.
Recycling of G protein-coupled receptors determines the functional resensitization of receptors and is implicated in switching beta2 adrenoceptor (beta2AR) G protein specificity in cardiomyocytes. The human beta2AR carboxyl end binds to the N-ethylmaleimide-sensitive factor (NSF), an ATPase integral to membrane trafficking machinery. It is interesting that the human beta2AR (hbeta2AR) carboxyl end pulled down NSF from mouse heart lysates, whereas the murine one did not. Despite this difference, both beta2ARs exhibited substantial agonist-induced internalization, recycling, and Gi coupling in cardiomyocytes. The hbeta2AR, however, displayed faster rates of agonist-induced internalization and recycling compared with the murine beta2AR (mbeta2AR) and a more profound Gi component in its contraction response. Replacing the mbeta2AR proline (-1) with a leucine generated a gain-of-function mutation, mbeta2AR-P417L, with a rescued ability to bind NSF, faster internalization and recycling than the mbeta2AR, and a significant enhancement in Gi signaling, which mimics the hbeta2AR. Selective disruption of the mbeta2AR-P417L binding to NSF inhibited the receptor coupling to Gi. Mean-while, inhibiting NSF with N-ethylmaleimide blocked the mbeta2AR recycling after agonist-induced endocytosis. Expressing the NSF-E329Q mutant lacking ATPase activity inhibited the mbeta2AR coupling to Gi in cardiomyocytes. Our results revealed a dual regulation on hbeta2AR 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 beta2ARs.
G蛋白偶联受体的再循环决定了受体的功能再敏化，并与心肌细胞中β2肾上腺素能受体（β2AR）G蛋白特异性的转换有关。人β2AR的羧基末端与N-乙基马来酰亚胺敏感因子（NSF）结合，NSF是膜运输机制中不可或缺的一种ATP酶。有趣的是，人β2AR（hβ2AR）的羧基末端能从小鼠心脏裂解物中拉下NSF，而小鼠β2AR的羧基末端则不能。尽管存在这种差异，但两种β2AR在心肌细胞中均表现出显著的激动剂诱导的内化、再循环和Gi偶联。然而，与小鼠β2AR（mβ2AR）相比，hβ2AR表现出更快的激动剂诱导的内化和再循环速率，并且在其收缩反应中具有更显著的Gi成分。将mβ2AR的脯氨酸（-1）替换为亮氨酸产生了一种功能获得性突变体mβ2AR-P417L，其具有恢复的结合NSF的能力，比mβ2AR更快的内化和再循环，以及Gi信号的显著增强，这模拟了hβ2AR。选择性破坏mβ2AR-P417L与NSF的结合会抑制受体与Gi的偶联。同时，用N-乙基马来酰亚胺抑制NSF会阻断激动剂诱导的内吞作用后mβ2AR的再循环。表达缺乏ATP酶活性的NSF-E329Q突变体可抑制心肌细胞中mβ2AR与Gi的偶联。我们的结果揭示了NSF通过直接结合货物受体及其ATP酶活性对hβ2AR运输和信号传导的双重调节，并揭示了受体与NSF结合在调节小鼠和人β2AR之间已发生分歧的G蛋白特异性方面前所未有的作用。
