Janus kinase 2 enhances the stability of the mature growth hormone receptor
Janus kinase 2 enhances the stability of the mature growth hormone receptor
复制标题
DOI：
10.1210/en.2005-0514
复制
发表时间：
2005-11-01
期刊：
影响因子：
4.8
通讯作者：
Frank, SJ
中科院分区：
文献类型：
--
作者：
He, K;Loesch, K;Frank, SJ
表面GH受体（GHR）的丰度是细胞GH敏感性的重要决定因素，并在转录和转录后水平上受到调节。在先前对表达GHR的Janus激酶2（JAK 2）缺陷型人纤维肉瘤细胞（γ 2A-GHR）的研究中，我们证明了用JAK 2稳定转染导致成熟GHR（endoH抗性;相对分子量，115 - 140 kDa）相对于前体GHR（endoH敏感;相对分子量，100 kDa）的稳态水平增加.我们现在通过比较γ 2A-GHR和用JAK 2稳定重建的γ 2A-GHR细胞（C14细胞）进一步检查JAK 2对GHR运输的影响。在JAK 2的存在下，GHR表面表达增加，如通过表面生物素化、I-125标记的人GH细胞表面结合和免疫荧光显微镜测定所评估的。虽然JAK 2的缺乏排除了GH刺激的信号传导，但GH诱导的GHR二硫键（GH诱导的GHR二聚体构象变化的代表）独立于JAK 2表达进行，表明GH诱导的GHR触发的最早步骤不受JAK 2缺乏的阻止. RNA干扰介导的C14细胞中JAK 2的敲低导致成熟与前体比率降低，支持JAK 2在增强GHR生物发生或抑制成熟GHR降解中的主要作用。为了解决这些潜在的机制，进行了代谢脉冲追踪标记实验和其中在放线菌酮处理后通过抗GHR免疫印迹跟踪先前合成的GHR的命运的实验（放线菌酮追踪实验）。这些表明JAK 2的存在赋予GHR成熟的适度增强（1.3- 1.5倍），但显著延长成熟GHR的t（1/2），表明对成熟GHR稳定性的主要影响。用金属蛋白酶、蛋白酶体和溶酶体抑制剂进行的环己酰亚胺追踪实验表明，JAK 2赋予的成熟GHR的增强的稳定性与对组成型受体金属蛋白酶解的影响无关，而是成熟GHR的组成型内体/溶酶体降解减少的结果。这些结果的背景下，JAK家族成员如何调节其他细胞因子受体的表面表达的新兴信息进行了讨论。
The abundance of surface GH receptor ( GHR) is an important determinant of cellular GH sensitivity and is regulated at both transcriptional and posttranscriptional levels. In previous studies of GHR- expressing Janus kinase 2 ( JAK2)- deficient human fibrosarcoma cells (gamma 2A- GHR), we demonstrated that stable transfection with JAK2 resulted in increased steadystate levels of mature GHR ( endoH- resistant; relative molecular mass, 115 - 140 kDa) relative to precursor GHR ( endoHsensitive; relative molecular mass, 100 kDa). We now examine further the effects of JAK2 on GHR trafficking by comparing gamma 2A- GHR to gamma 2A- GHR cells stably reconstituted with JAK2 ( C14 cells). In the presence of JAK2, GHR surface expression was increased, as assessed by surface biotinylation, I-125- labeled human GH cell surface binding, and immunofluorescence microscopy assays. Although the absence of JAK2 precluded GH- stimulated signaling, GH- induced GHR disulfide linkage ( a proxy for the GH- induced conformational changes in the GHR dimer) proceeded independent of JAK2 expression, indicating that the earliest steps in GH- induced GHR triggering are not prevented by the absence of JAK2. RNA interference- mediated knockdown of JAK2 in C14 cells resulted in a decreased mature to precursor ratio, supporting a primary role for JAK2 either in enhancing GHR biogenesis or dampening mature GHR degradation. To address these potential mechanisms, metabolic pulse- chase labeling experiments and experiments in which the fate of previously synthesized GHR was followed by anti- GHR immunoblotting after cycloheximide treatment ( cycloheximide chase experiments) were performed. These indicated that the presence of JAK2 conferred modest enhancement ( 1.3- to 1.5- fold) in GHR maturation but substantially prolonged the t(1/2) of the mature GHR, suggesting a predominant effect on mature GHR stability. Cycloheximide chase experiments with metalloprotease, proteasome, and lysosome inhibitors indicated that the enhanced stability of mature GHR conferred by JAK2 is not related to effects on constitutive receptor metalloproteolysis but rather is a result of reduced constitutive endosomal/ lysosomal degradation of the mature GHR. These results are discussed in the context of emerging information on how JAKfamily members modulate surface expression of other cytokine receptors.