Landsman Tanya, Waxman David J
Division of Cell and Molecular Biology, Department of Biology, Boston University, MA 02215, USA.
J Biol Chem. 2005 Nov 11;280(45):37471-80. doi: 10.1074/jbc.M504125200. Epub 2005 Sep 8.
The cytokine-inducible SH2 domain-containing protein CIS inhibits signaling from the growth hormone (GH) receptor (GHR) to STAT5b by a proteasome-dependent mechanism. Here, we used the GH-responsive rat liver cell line CWSV-1 to investigate the role of CIS and the proteasome in GH-induced GHR internalization. Cell-surface GHR localization and internalization were monitored in GH-stimulated cells by confocal immunofluorescence microscopy using an antibody directed against the GHR extracellular domain. In GH naïve cells, GHR was detected in small, randomly distributed granules on the cell surface and in the cytoplasm, with accumulation in the perinuclear area. GH treatment induced a rapid (within 5 min) internalization of GH.GHR complexes, which coincided with the onset of GHR tyrosine phosphorylation and the appearance in the cytosol of distinct granular structures containing internalized GH. GHR signaling to STAT5b continued for approximately 30-40 min, however, indicating that GHR signaling and deactivation of the GH.GHR complex both proceed from an intracellular compartment. The internalization of GH and GHR was inhibited by CIS-R107K, a dominant-negative SH2 domain mutant of CIS, and by the proteasome inhibitors MG132 and epoxomicin, which prolong GHR signaling to STAT5b. GH pulse-chase studies established that the internalized GH.GHR complexes did not recycle back to the cell surface in significant amounts under these conditions. Given the established specificity of CIS-R107K for blocking the GHR signaling inhibitory actions of CIS, but not those of other SOCS/CIS family members, these findings implicate CIS and the proteasome in the control of GHR internalization following receptor activation and suggest that CIS-dependent receptor internalization is a prerequisite for efficient termination of GHR signaling.
细胞因子诱导含SH2结构域蛋白CIS通过蛋白酶体依赖机制抑制生长激素（GH）受体（GHR）向信号转导子和转录激活子5b（STAT5b）的信号传导。在此，我们使用对GH有反应的大鼠肝细胞系CWSV-1来研究CIS和蛋白酶体在GH诱导的GHR内化中的作用。通过共聚焦免疫荧光显微镜，使用针对GHR细胞外结构域的抗体，监测GH刺激细胞中细胞表面GHR的定位和内化。在未接触GH的细胞中，GHR在细胞表面和细胞质中呈小的、随机分布的颗粒状被检测到，且在核周区域积聚。GH处理诱导GH.GHR复合物迅速（5分钟内）内化，这与GHR酪氨酸磷酸化的开始以及含有内化GH的独特颗粒结构在胞质溶胶中的出现同时发生。然而，GHR向STAT5b的信号传导持续约30 - 40分钟，这表明GHR信号传导以及GH.GHR复合物的失活均从细胞内区室进行。GH和GHR的内化受到CIS-R107K（CIS的显性负性SH2结构域突变体）以及蛋白酶体抑制剂MG132和环氧霉素的抑制，这些抑制剂会延长GHR向STAT5b的信号传导。GH脉冲追踪研究表明，在这些条件下，内化的GH.GHR复合物不会大量循环回到细胞表面。鉴于已确定CIS-R107K对阻断CIS的GHR信号抑制作用具有特异性，但对其他细胞因子信号抑制蛋白（SOCS）/CIS家族成员的抑制作用无特异性，这些发现表明CIS和蛋白酶体参与受体激活后对GHR内化的控制，并提示CIS依赖的受体内化是有效终止GHR信号传导的前提条件。
