Photoaffinity labeling of the erythropoietin receptor and its identification in a ligand-free form.
Photoaffinity labeling of the erythropoietin receptor and its identification in a ligand-free form.
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促红细胞生成素受体的光亲和标记及其无配体形式的鉴定。
DOI：
10.1021/bi00216a004
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Krantz,SB
中科院分区：
文献类型：
--
作者：
Hosoi,T;Sawyer,ST;Krantz,SB
纯的人重组促红细胞生成素（EP）通过伯氨基残基用交联剂7V-[[3-[[4-[（p-叠氮基-m-[125 I]碘苯基）偶氮]苯甲酰基]氨基]丙酰基]氧基]-琥珀酰亚胺（Denny-Jaffe试剂）酰化，所述交联剂是光反应性的并且在偶氮残基处可裂解。通过反相高压液相色谱法从未修饰的EP中纯化得到的缀合激素（DJ-EP），并保持其与红系祖细胞上的EP受体结合的能力。EP的受体先前通过与125 I-EP化学交联而被鉴定为100和85 kDa分子量的两种相关蛋白质。最近，D ′ Andrea和同事[（1989）Cell 57，277-285]克隆了编码55-66 kDa蛋白质的cDNA，该蛋白质被认为是EP受体。在本报告中，通过单功能DJ-EP与受体交联，标记了与125 I-EP和辛二酸二琥珀酰亚胺酯交联的相同140和125 kDa分子量条带（100和85 kDa蛋白质）。此外，DJ-EP受体复合物的偶氮键被连二亚硫酸钠裂解（80 ℃，5分钟）证明105和90 kDa的蛋白质被DJ-EP标记为无配体形式。红细胞生成素是一种糖蛋白激素，它对于未成熟红细胞完全成熟为红细胞是必需的（Graber和Krantz，1989;索耶，1990）。在最依赖于该激素的细胞中，EP 1的受体在未成熟的红系细胞上数量最多，这些细胞被称为红系集落形成单位。当这些细胞分化成成熟的成红细胞时，EP受体的数量已显示消失（索耶，1990; Landschulz等，1989; Sawada等人，1987年）。EP受体首先在从Friend病毒贫血株感染的小鼠脾脏中纯化的未成熟红系细胞（FVA细胞）上鉴定。这些FVA细胞对EP具有较高和较低的亲和力受体，125 I-EP在这些细胞的溶酶体中被迅速内吞和降解（索耶等人，1987年a）。与来自FVA细胞的膜结合的125 I-EP的交联鉴定出两个放射性条带，当从交联条带中减去EP的分子量时，其具有100和85 kDa的表观分子量（索耶等人，1987 b，1989;索耶，1989）。两种交联受体蛋白的肽图谱表明氨基酸序列相似（如果不完全相同），表明存在单一基因，以及共同前体的差异加工或低分子量蛋白质的蛋白水解生成（索耶，1989）。
Pure human recombinanterythropoietin (EP) was acylated through a primary aminoresidue with a cross-linking reagent, 7V-[[3-[[4-[(p-azido-m-[125I] iodophenyl) azo] benzoyl] amino] propanoyl] oxy]-succinimide (Denny-Jaffe reagent), which is photoreactive and cleavable at the azo residue. The resulting conjugated hormone (DJ-EP) was purified from unmodified EP by reverse-phase high-pressure liquid chromatography and maintained its capacity to bind to receptors for EP on erythroid progenitor cells. The receptor for EP was previously identified as two related proteins of 100 and 85 kDa molecular mass by chemical cross-linking to 125I-EP. Recently, D’Andrea and co-workers [(1989) Cell 57, 277-285] cloned a cDNA that codes for a protein of 55-66 kDa, which is thought to be the EP receptor. In this report, cross-linking to the receptor through the monofunctional DJ-EP labeled the same 140-and 125-kDa molecular mass bands (100-and 85-kDa proteins) cross-linked with 125I-EP and disuccinimidyl suberate. Furthermore, cleavage of the azo bond of the DJ-EP receptor complex by sodium dithionite (80 C, 5 min) demonstrated that proteins of 105 and 90 kDa were labeled in ligand-free form by DJ-EP. This result demonstrates that artifactual cross-linking of multiple proteins or other artifacts of cross-linking do not explain the difference in molecular mass of the EP receptor identified by cross-linking and the receptor identified by expression cloning.Ijrythropoietin is the glycoprotein hormone that is essential for the complete maturation of immature erythroid cells into red blood cells (Graber & Krantz, 1989; Sawyer, 1990). Receptors for EP1 are found on immature erythroid cells in greatest numbers in cells that are most dependent on the hormone and these cells are known as colony-forming units-erythroid. As these cells differentiate into mature erythro-blasts, the EP receptor number has been shown to disappear (Sawyer, 1990; Landschulz et al., 1989; Sawada et al., 1987). Receptors for EP were first identified on immature erythroid cells purified from the spleens of mice infected with theanemia strain of Friend virus (FVA cells). These FVA cells have higher and lower affinity receptors for EP, and 125I-EP is rapidly endocytosed and degraded in the lysosomes of these cells (Sawyer et al., 1987a). Cross-linking of 125I-EP bound to membranes from FVA cells identified two bands of radioactivity, which had apparent molecular masses of 100 and 85 kDa when the molecular mass of EP was subtracted from the cross-linked bands (Sawyer et al., 1987b, 1989; Sawyer, 1989). Peptide mapping from the two cross-linked receptor proteins indicated similar if not identical sequences of amino acids, suggesting a single gene and either differential processing of a common precursor or proteolytic generation of the lower molecular mass protein (Sawyer, 1989).
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大规模采购促红细胞生成素反应性红系细胞：促红细胞生成素生物活性测定。
影响因子：
--
作者：
Sawyer,ST;Koury,MJ;Bondurant,MC
通讯作者：
Bondurant,MC
促红细胞生成素与源自小鼠胎肝细胞的 CFU-E 的结合。
影响因子：
2.6
作者：
H. Fukamachi;T. Saito;A. Tojo;T. Kitamura;A. Urabe;F. Takaku
通讯作者：
F. Takaku
影响因子：
20.3
作者：
S. Jones;Alan D. D’Andrea;Lora L. Haines;Gordon G. Wong
通讯作者：
S. Jones;Alan D. D’Andrea;Lora L. Haines;Gordon G. Wong
Friend脾病灶形成病毒糖蛋白gp55与内质网中的促红细胞生成素受体相互作用并影响受体代谢。
DOI：
10.1073/pnas.87.11.4139
发表时间：
1990
影响因子：
11.1
作者：
Yoshimura,A;D'Andrea,AD;Lodish,HF
通讯作者：
Lodish,HF
使用生物素化促红细胞生成素检测和分离促红细胞生成素受体。
影响因子：
20.3
作者：
A. W. Wognum;Peter M. Lansdorp;R. Humphries;Gerald Krystal
通讯作者：
Gerald Krystal