Schroering A, Carey D J
Weis Center for Research, Penn State College of Medicine, Danville, Pennsylvania 17822-2613, USA.
J Biol Chem. 1998 Nov 13;273(46):30643-50. doi: 10.1074/jbc.273.46.30643.
The beta-heregulin sensory and motor neuron-derived factor (SMDF) has been suggested to be an important regulator of Schwann cell development and proliferation. In the present study, human SMDF was expressed in cultured cell lines. The cells and the recombinant protein were used to examine the membrane association and biological activity of the growth factor. Transfection of cells with SMDF cDNA constructs bearing FLAG epitope tags at either the amino- or carboxyl-terminal ends of the polypeptide resulted in expression of anti-FLAG immunoreactive polypeptides of approximately 44 and 83 kDa. The 83-kDa polypeptide was the major form expressed on the cell surface, as demonstrated by sensitivity to proteolysis in intact cells and surface biotinylation. SMDF was tightly associated with membranes isolated from transfected cells but was solubilized by Triton X-100. Immunofluorescent staining and immunoprecipitation experiments using cells expressing amino- or carboxyl-terminal tagged SMDF revealed that only the carboxyl-terminal end of the protein is exposed on the cell surface. Membranes from SMDF-transfected cells stimulated tyrosine phosphorylation of the beta-heregulin receptor ErbB3 in Schwann cells. Conditioned medium from transfected cells contained a similar activity, suggesting that SMDF is subject to proteolytic release from the plasma membrane. In contrast with other beta-heregulin isoforms, SMDF failed to bind heparin. Stimulation of Schwann cell ErbB3 receptor phosphorylation by SMDF was not affected by inhibition of Schwann cell heparan sulfate proteoglycan synthesis. These results demonstrate that SMDF is a type II transmembrane protein. This orientation places the active epidermal growth factor homology domain, which is located near the carboxyl-terminal end of the polypeptide, on the cell surface, where it can function as a membrane-anchored growth factor.
β-调理性素感觉和运动神经元衍生因子（SMDF）被认为是施万细胞发育和增殖的重要调节因子。在本研究中，人SMDF在培养的细胞系中表达。利用这些细胞和重组蛋白来检测生长因子的膜结合及生物学活性。用在多肽的氨基或羧基末端带有FLAG表位标签的SMDF cDNA构建体转染细胞，导致表达出大小约为44 kDa和83 kDa的抗FLAG免疫反应性多肽。83 kDa的多肽是细胞表面表达的主要形式，完整细胞中的蛋白水解敏感性和表面生物素化证明了这一点。SMDF与从转染细胞分离的膜紧密结合，但可被 Triton X-100溶解。使用表达氨基或羧基末端标记的SMDF的细胞进行免疫荧光染色和免疫沉淀实验表明，只有该蛋白的羧基末端暴露在细胞表面。来自SMDF转染细胞的膜刺激了施万细胞中β-调理性素受体ErbB3的酪氨酸磷酸化。转染细胞的条件培养基含有类似的活性，表明SMDF可从质膜进行蛋白水解释放。与其他β-调理性素异构体不同，SMDF不能结合肝素。SMDF对施万细胞ErbB3受体磷酸化的刺激不受施万细胞硫酸乙酰肝素蛋白聚糖合成抑制的影响。这些结果表明，SMDF是一种II型跨膜蛋白。这种取向将位于多肽羧基末端附近的活性表皮生长因子同源结构域置于细胞表面，在那里它可以作为一种膜锚定生长因子发挥作用。
