Prabakaran D, Kim P, Kim K R, Arvan P
Division of Endocrinology, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts.
J Biol Chem. 1993 Apr 25;268(12):9041-8.
In addition to thyroglobulin, primary thyrocytes secrete into the culture medium significant quantities of p500, a protein so named because of its M(r) > or = 500,000. Epithelial monolayers cultured on porous filters serve as a useful model system in which to study protein secretion. From these monolayers, thyroglobulin, the precursor in thyroid hormonogenesis, was released with apical predominance, while p500 was found mostly in the basolateral medium. Thyrocyte exposure to thyrotropin augmented selectively thyroglobulin but not p500 production. By contrast, exposure to cycloheximide actually augmented p500 production, a response observed for immediate-early proto-oncogenes. Using thyrocyte conditioned medium, the p500 protein was purified to homogeneity. Peptide sequencing of tryptic fragments of purified p500 showed identity to thrombospondin. Immunoprecipitation of thrombospondin from media bathing primary thyrocytes and the FRTL5 cell line quantitatively recovered p500, confirming its identity and indicating an epithelial origin. Gel filtration of secreted thrombospondin eluted at a high molecular weight, suggesting complexation with components of the extracellular matrix. Further, immunofluorescence showed cellular codistribution of thrombospondin and thyroglobulin, although thrombospondin exhibited predominantly an extracellular, basolateral deposition. It seems likely that thrombospondin production by thyrocytes plays a role in the growth or development of the thyroid epithelium.
除甲状腺球蛋白外，原代甲状腺细胞还向培养基中分泌大量的p500，这种蛋白质因其相对分子质量（Mr）≥500,000而得名。在多孔滤膜上培养的上皮单层细胞是研究蛋白质分泌的有用模型系统。在这些单层细胞中，甲状腺激素生成的前体甲状腺球蛋白以顶端优势释放，而p500大多存在于基底外侧培养基中。甲状腺细胞暴露于促甲状腺激素可选择性增加甲状腺球蛋白的生成，但不增加p500的生成。相比之下，暴露于环己酰亚胺实际上会增加p500的生成，这是即刻早期原癌基因所观察到的反应。利用甲状腺细胞条件培养基，将p500蛋白纯化至同质。对纯化的p500胰蛋白酶片段进行肽测序，结果显示其与血小板反应蛋白相同。从培养原代甲状腺细胞和FRTL5细胞系的培养基中免疫沉淀血小板反应蛋白，定量回收了p500，证实了其身份并表明其来源于上皮细胞。分泌的血小板反应蛋白经凝胶过滤后在高分子量处洗脱，提示其与细胞外基质成分形成复合物。此外，免疫荧光显示血小板反应蛋白和甲状腺球蛋白在细胞中共分布，尽管血小板反应蛋白主要表现为细胞外基底外侧沉积。甲状腺细胞产生的血小板反应蛋白似乎在甲状腺上皮的生长或发育中起作用。
