Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3-F442A fibroblasts and adipocytes.
Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3-F442A fibroblasts and adipocytes.
复制标题
生长激素促进小鼠 3T3-F442A 成纤维细胞和脂肪细胞中生长激素受体的酪氨酰磷酸化。
DOI：
10.1021/bi00401a049
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Carter-Su,C
中科院分区：
文献类型：
--
作者：
Foster,CM;Shafer,JA;Rozsa,FW;Wang,XY;Lewis,SD;Renken,DA;Natale,JE;Schwartz,J;Carter-Su,C
密歇根大学医学院生理学和生物化学系，安阿伯，密歇根州48109，1987年2月13日接收; 1987年9月11日接收的修订版Mandarin pt摘要：由于许多生长因子受体是配体激活的酪氨酸蛋白激酶，生长激素（GH），一种与人类生长有关的激素，研究了促进其受体的酪氨酰磷酸化。用~（125）I标记的人生长激素（GH）与完整的3 T3-F442 A成纤维细胞（一种对GH有反应而分化为脂肪细胞的细胞系）的受体共价交联。将交联的细胞溶解并通过固定在蛋白A-琼脂糖上的磷酸酪氨酰结合抗体柱。用半抗原（磷酸对硝基苯酯）洗脱免疫吸附蛋白，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和放射自显影进行分析。来自抗体柱的洗脱液含有Mr 134000的125 I-GH-受体复合物。当使用脂肪细胞形式的3 T3-F442 A细胞代替成纤维细胞形式时，获得了类似的结果。O-磷酸酪氨酸阻止125 I-GH-受体复合物与抗体柱结合，而O-磷酸丝氨酸和O-磷酸苏氨酸没有。在用[32 P] Pj代谢标记的3 T3-F442 A成纤维细胞中，GH促进磷酸化的研究显示，GH刺激形成与固定化磷酸酪氨酸结合抗体结合的32 P标记蛋白。该蛋白质的分子量为114000，与非交联GH受体的预期分子量相似。Mr 114000磷酸化蛋白可与抗GH抗体发生免疫沉淀，表明GH在吸附和洗脱过程中与该蛋白保持非共价结合。在有限的酸水解和广泛的碱水解的Mr 114 000磷蛋白后的Phos-phoamino酸分析证实了磷酸酪氨酸残基的存在。这些观察结果提供了强有力的证据，GH与其受体的结合刺激GH受体中酪氨酰残基的磷酸化。生长激素（GH）1在60多年前被鉴定为生长因子，但其作用机制知之甚少（Greep，1974）。最近，许多生长促进肽的受体已显示经历配体活化的酪氨酰磷酸化[Cohen等人，1980; Kasuga等人，1982 a; Roth & Cassell，1983; Pang等人，1985 a; Nishimura等人，1982; Frackelton等人，1984; Jacobs等人，1983; Rubin等人，1983; Petruzzeli等人，1984; Huang & Huang，1986;综述参见Carter-Su and Pratt（1984）]。作为确定GH受体是否是配体激活的酪氨酸激酶的第一步，我们研究了GH受体是否经历酪氨酰磷酸化以及GH是否刺激3 T3-F442 A细胞中其受体的磷酸化。该细胞系经历GH促进的从成纤维细胞分化为脂肪细胞形式（Mor-ikawa et al.，1982年;尼克松和绿色，1984年）。此外，GH改变该细胞系脂肪细胞形式中的碳水化合物和脂质代谢（Schwartz，1984; Schwartz等人，1985年）。在本研究中，我们结合使用交联剂。这项工作得到了赠款DK 34171（授予CC-S.和JS）和DK 35249（授予JAS）。CMF是美国国立卫生研究院博士后奖学金（AM 07245）和临床副医师奖（5 M01 RR 2）的获得者。JEN是密歇根大学的摄政王。
Departments of Physiology and Biochemistry, The University of Michigan Medical School, Ann Arbor, Michigan 48109 Received February 13, 1987; Revised Manuscript Received September 11, 1987 abstract: Because many growth factor receptors are ligand-activated tyrosine protein kinases, thepossibility that growth hormone (GH), a hormone implicated in human growth, promotes tyrosyl phosphorylation of its receptor was investigated. 125I-Labeled human GH was covalently cross-linkedto receptors in intact 3T3-F442A fibroblasts, a cell line which differentiates into adipocytes in response to GH. Thecross-linked cells were solubilized and passed over a column of phosphotyrosyl binding antibody immobilized on protein A-Sepharose. Immunoadsorbed proteins were eluted with a hapten (p-nitrophenyl phosphate) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The eluate from the antibody column contained an Mr 134000 125I-GH-receptor complex. A similar result was obtained when the adipocyte form of 3T3-F442A cells was used in place of the fibroblast form. O-Phosphotyrosine prevented 125I-GH-receptor complexes from binding to the antibody column, whereas O-phosphoserine and O-phosphothreonine did not. In studies of GH-promotedphosphorylation in 3T3-F442A fibroblasts labeled metabolically with [32P] Pj, GH was shown to stimulate formation of a 32P-labeled protein which boundto immobilized phosphotyrosyl binding antibodies. The molecular weight of 114000 obtained for this protein is similar to that expected for non-cross-linked GH receptor. The Mr 114 000 phosphorylated protein could be immunoprecipitated with anti-GH antibody, indicating that GH remained noncovalently boundto this protein during absorption to and elution from the immobilized phosphotyrosyl binding antibody. Phos-phoamino acid analysis after bothlimited acid hydrolysis and extensive base hydrolysis of the Mr 114 000 phosphoprotein confirmed the presence of phosphotyrosyl residues. These observations provide strong evidence that binding of GH to its receptor stimulates phosphorylation of tyrosyl residues in the GH receptor.(jrowth hormone (GH) 1 was identified as a growth factor more than 60 years ago, yet its mechanism of action is poorly understood (Greep, 1974). Recently, the receptors for a number of growth-promoting peptides have been shown to undergo ligand-activated tyrosyl phosphorylation [Cohen et al., 1980; Kasuga et al., 1982a; Roth & Cassell, 1983; Pang et al., 1985a; Nishimura et al., 1982; Frackelton et al., 1984; Jacobs et al., 1983; Rubin et al., 1983; Petruzzeli et al., 1984; Huang & Huang, 1986; for a review, see Carter-Su and Pratt (1984)]. As a first step toward determining whether the GH receptor is a ligand-activated tyrosine kinase, we investigated whether the GH receptor undergoes tyrosyl phosphorylation and whether GH stimulates the phosphorylation of its receptor in 3T3-F442A cells. This cell line undergoes GH-promoted differentiation from a fibroblast to an adipocyte form (Mor-ikawa et al., 1982; Nixon & Green, 1984). Additionally, GH alters carbohydrate and lipid metabolism in the adipocyte form of this cell line (Schwartz, 1984; Schwartz et al., 1985). In the present study, we used cross-linking agents in combination^ This work was supported by research funds provided by Grants DK34171 (awarded to CC-S. and JS) and DK35249 (awarded to JAS) from the National Institutes of Health. CMF is a recipient of a postdoctoral fellowship (AM07245) and a clinical associate physician award (5M01RR2) from the National Institutes of Health. JEN is a University of Michigan Regent …