Kren Nancy P, Zagon Ian S, McLaughlin Patricia J
Department of Neural & Behavioral Sciences, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, PA, USA.
Department of Neural & Behavioral Sciences, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, PA, USA
Exp Biol Med (Maywood). 2016 Feb;241(3):273-81. doi: 10.1177/1535370215605585. Epub 2015 Sep 30.
Opioid growth factor receptor (OGFr) facilitates growth inhibition in the presence of its specific ligand opioid growth factor (OGF), chemically termed [Met(5)]-enkephalin. The function of the OGF-OGFr axis requires the receptor to translocate to the nucleus. However, the mechanism of nuclear export of OGFr is unknown. In this study, endogenous OGFr, as well as exogenously expressed OGFr-EGFP, demonstrated significant nuclear accumulation in response to leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export, suggesting that OGFr is exported in a CRM1-dependent manner. One consensus sequence for a nuclear export signal (NES) was identified. Mutation of the associated leucines, L217 L220 L223 and L225, to alanine resulted in decreased nuclear accumulation. NES-EGFP responded to LMB, indicating that this sequence is capable of functioning as an export signal in isolation. To determine why the sequence functions differently in isolation than as a full length protein, the localization of subNES was evaluated in the presence and absence of MG132, a potent inhibitor of proteosomal degradation. MG132 had no effect of subNES localization. The role of tandem repeats located at the C-terminus of OGFr was examined for their role in nuclear trafficking. Six of seven tandem repeats were removed to form deltaTR. DeltaTR localized exclusively to the nucleus indicating that the tandem repeats may contribute to the localization of the receptor. Similar to the loss of cellular proliferation activity (i.e. inhibition) recorded with subNES, deltaTR also demonstrated a significant loss of inhibitory activity indicating that the repeats may be integral to receptor function. These experiments reveal that OGFr contains one functional NES, L217 L220 L223 and L225 and can be exported from the nucleus in a CRM1-dependent manner.
阿片样生长因子受体（OGFr）在其特异性配体阿片样生长因子（OGF，化学名称为[Met(5)]-脑啡肽）存在的情况下促进生长抑制。OGF-OGFr轴的功能要求该受体转运至细胞核。然而，OGFr的核输出机制尚不清楚。在本研究中，内源性OGFr以及外源性表达的OGFr-EGFP在受到CRM1依赖性核输出抑制剂雷帕霉素B（LMB）刺激后，均表现出显著的核积累，这表明OGFr以CRM1依赖性方式输出。鉴定出一个核输出信号（NES）的共有序列。将相关的亮氨酸L217、L220、L223和L225突变为丙氨酸会导致核积累减少。NES-EGFP对LMB有反应，表明该序列能够单独作为输出信号发挥作用。为了确定该序列在单独存在时与作为全长蛋白时功能不同的原因，在存在和不存在蛋白酶体降解强效抑制剂MG132的情况下评估了subNES的定位。MG132对subNES的定位没有影响。研究了位于OGFr C末端的串联重复序列在核运输中的作用。去除七个串联重复序列中的六个以形成deltaTR。DeltaTR仅定位于细胞核，表明串联重复序列可能有助于受体的定位。与subNES记录的细胞增殖活性丧失（即抑制）情况类似，deltaTR也表现出显著的抑制活性丧失，表明这些重复序列可能是受体功能所必需的。这些实验表明，OGFr含有一个功能性NES，即L217、L220、L223和L225，并且可以以CRM1依赖性方式从细胞核输出。
