Horuk R, Yansura D G, Reilly D, Spencer S, Bourell J, Henzel W, Rice G, Unemori E
Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080.
J Biol Chem. 1993 Jan 5;268(1):541-6.
Human melanoma growth stimulating activity (MGSA) is a mitogenic factor first identified in the conditioned media of human melanoma cells. Structurally, MGSA belongs to a superfamily of proteins that includes interleukin-8 (IL-8) and platelet factor 4. These proteins are involved in inflammatory processes, and an understanding of their mechanism of action should provide insight into their pathophysiology. In this study, we report the high level expression of recombinant human MGSA in Escherichia coli. The structure was confirmed by mass spectrometry and NH2-terminal amino acid sequencing. Receptor binding studies were carried out in a human melanoma cell line, Hs294T, and in U937 cells. Direct binding experiments with 125I-MGSA in Hs294T cells have allowed us to identify a novel MGSA receptor in these cells, with a KD of 3.9-4.25 nM and approximately 52,960-67,758 binding sites/cell. These MGSA-binding sites were specific and could not be displaced by unlabeled IL-8. The MGSA receptor in these cells is biologically active, and the addition of ligand induces cellular proliferation in a dose-dependent manner. In U937 cells, unlabeled IL-8 and MGSA were able to completely displace radiolabeled IL-8. Scatchard analysis of the displacement binding data was consistent with binding to a single class of binding sites, and the calculated KD values were 2.4 +/- 0.6 nM for IL-8 and 3.2 +/- 0.80 nM for MGSA. Treatment of U937 cells with IL-8 or MGSA produced a rapid increase in Ca2+ flux; however, subsequent incubation with either ligand failed to produce any further Ca2+ flux. The IL-8 receptor in U937 cells was covalently labeled with 125I-IL-8 to reveal a protein with a molecular mass of 69 kDa.
人黑素瘤生长刺激活性因子（MGSA）是一种促有丝分裂因子，最初在人黑素瘤细胞的条件培养基中被鉴定出来。在结构上，MGSA属于一个蛋白质超家族，该超家族包括白细胞介素-8（IL-8）和血小板因子4。这些蛋白质参与炎症过程，对其作用机制的了解有助于深入了解它们的病理生理学。在本研究中，我们报道了重组人MGSA在大肠杆菌中的高水平表达。通过质谱分析和N端氨基酸测序对其结构进行了确认。在人黑素瘤细胞系Hs294T和U937细胞中进行了受体结合研究。在Hs294T细胞中用125I-MGSA进行的直接结合实验使我们能够在这些细胞中鉴定出一种新型MGSA受体，其解离常数（KD）为3.9 - 4.25 nM，每个细胞约有52,960 - 67,758个结合位点。这些MGSA结合位点具有特异性，未标记的IL-8不能将其取代。这些细胞中的MGSA受体具有生物活性，添加配体可诱导细胞以剂量依赖方式增殖。在U937细胞中，未标记的IL-8和MGSA能够完全取代放射性标记的IL-8。对置换结合数据的Scatchard分析与结合到单一类别的结合位点一致，计算得出的IL-8的KD值为2.4±0.6 nM，MGSA的KD值为3.2±0.80 nM。用IL-8或MGSA处理U937细胞会使Ca2+通量迅速增加；然而，随后用任何一种配体孵育都未能产生任何进一步的Ca2+通量。用125I-IL-8对U937细胞中的IL-8受体进行共价标记，显示出一种分子量为69 kDa的蛋白质。
