Whitson R H, Wong W L, Itakura K
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Los Angeles, California 91010.
J Cell Biochem. 1991 Sep;47(1):31-42. doi: 10.1002/jcb.240470105.
A low molecular weight inhibitor of TGF-beta 1 binding was detected in partially purified human platelet extracts by using Hep 3B hepatoma cells in the binding assays. The inhibitory protein was purified to homogeneity and was identified as platelet factor 4 on the basis of its amino acid sequence. TGF-beta 1 binding to Hep 3B cells was almost completely inhibited by 100 nM concentrations of platelet factor 4, but TGF-beta 1 binding to NRK 49F fibroblasts was inhibited only slightly. Affinity cross-linking experiments revealed that these differences in the inhibition of TGF-beta 1 binding by platelet factor 4 were due to differences in the complements of TGF-beta 1 binding proteins present on these two cell types. In Hep 3B cells the majority of bound TGF-beta 1 was cross-linked to a complex which had an apparent molecular weight of 70 kDa. TGF-beta 1 binding to this protein was the most sensitive to inhibition by platelet factor 4. Based on its size and TGF-beta 1 binding properties, we believe this protein is the type I TGF-beta 1 receptor. Hep 3B cells also had a high-affinity TGF-beta 1 binding protein which appeared as an 80 kDa complex, and which we believe to be the type II TGF-beta 1 receptor. TGF-beta 1 binding to this protein was not inhibited by platelet factor 4. TGF-beta 1 was also cross-linked to complexes of higher molecular weights in Hep 3B cells, but it was not clear whether any of them represented the type III TGF-beta 1 receptor. In NRK 49F cells, the majority of bound TGF-beta 1 was cross-linked to a high molecular weight complex which probably represented the type III TGF-beta 1 receptor. NRK 49F cells also had type I TGF-beta 1 receptors and platelet factor 4 inhibited binding to these receptors in the NRK cells. Since the type I receptor contributed only a small percentage of total TGF-beta 1 binding, however, the overall effects of platelet factor 4 on TGF-beta 1 binding to NRK 49F cells were negligible. We were unable to demonstrate specific or saturable binding of platelet factor 4 to Hep 3B cells using either direct binding or affinity cross-linking assays. Thus, it is not clear whether platelet factor 4 inhibits TGF-beta 1 binding by competition for binding to the type I receptor. Modest concentrations of TGF-beta 1 reduced the adherence of Hep 3B cells to tissue culture dishes.(ABSTRACT TRUNCATED AT 400 WORDS)
在结合试验中，通过使用Hep 3B肝癌细胞，在部分纯化的人血小板提取物中检测到一种低分子量的TGF-β1结合抑制剂。该抑制蛋白被纯化至同质，并根据其氨基酸序列鉴定为血小板因子4。100 nM浓度的血小板因子4几乎完全抑制了TGF-β1与Hep 3B细胞的结合，但TGF-β1与NRK 49F成纤维细胞的结合仅受到轻微抑制。亲和交联实验表明，血小板因子4对TGF-β1结合抑制的这些差异是由于这两种细胞类型上存在的TGF-β1结合蛋白互补物的差异。在Hep 3B细胞中，大多数结合的TGF-β1与一种表观分子量为70 kDa的复合物交联。TGF-β1与该蛋白的结合对血小板因子4的抑制最为敏感。基于其大小和TGF-β1结合特性，我们认为该蛋白是I型TGF-β1受体。Hep 3B细胞还具有一种高亲和力的TGF-β1结合蛋白，它表现为一种80 kDa的复合物，我们认为它是II型TGF-β1受体。TGF-β1与该蛋白的结合不受血小板因子4的抑制。TGF-β1在Hep 3B细胞中也与更高分子量的复合物交联，但尚不清楚它们中是否有任何一个代表III型TGF-β1受体。在NRK 49F细胞中，大多数结合的TGF-β1与一种高分子量复合物交联，该复合物可能代表III型TGF-β1受体。NRK 49F细胞也具有I型TGF-β1受体，血小板因子4抑制其与NRK细胞中这些受体的结合。然而，由于I型受体仅占总TGF-β1结合的一小部分，因此血小板因子4对TGF-β1与NRK 49F细胞结合的总体影响可以忽略不计。我们无法使用直接结合或亲和交联试验证明血小板因子4与Hep 3B细胞的特异性或饱和结合。因此，尚不清楚血小板因子4是否通过竞争与I型受体结合来抑制TGF-β1结合。适度浓度的TGF-β1降低了Hep 3B细胞对组织培养皿的粘附。（摘要截短至400字）
