Modulation of B lymphocyte differentiation by calcitonin gene-related peptide (CGRP). I. Characterization of high-affinity CGRP receptors on murine 70Z/3 cells.
Modulation of B lymphocyte differentiation by calcitonin gene-related peptide (CGRP). I. Characterization of high-affinity CGRP receptors on murine 70Z/3 cells.
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DOI：
10.1006/cimm.1993.1207
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发表时间：
1993-09
影响因子：
4.3
通讯作者：
J. Mcgillis;S. Humphreys;Rangnekar;J. Ciallella
中科院分区：
文献类型：
--
作者：
J. Mcgillis;S. Humphreys;Rangnekar;J. Ciallella
降钙素基因相关肽（CGRP）是一种存在于外周和中枢神经元以及某些内分泌组织中的37个氨基酸的神经肽。先前的研究已经鉴定了正常T和B细胞上的特异性高亲和力CGRP受体，并描述了CGRP对新鲜分离的淋巴细胞的调节作用。在这些研究中，筛选了几种淋巴细胞系的125 I-CGRP结合。发现一种细胞系，即鼠70 Z/3前B细胞系，表现出高水平的125 I-CGRP结合，并用于进一步表征淋巴细胞CGRP受体。评价了几种蛋白酶抑制剂稳定125 I-CGRP与70 Z/3细胞结合的能力。杆菌肽，一种相对非特异性的蛋白酶抑制剂，和糜蛋白酶抑制剂，半胱氨酸蛋白酶抑制剂，增强125 I-CGRP结合，表明70 Z/3细胞表达能够灭活CGRP的蛋白酶。125 I-CGRP结合有一个线性依赖于细胞浓度与结合可检测到的少至200，000细胞/ml，是快速的，与平衡结合达到30分钟。饱和结合研究表明，70 Z/3 CGRP受体具有低和高亲和力结合状态，与Kds分别约为0.2和3.3 nM。低亲和力结合位点的密度约为20，000个位点/细胞，并且在LPS处理后没有变化。与此相反，LPS处理48小时诱导的高亲和力位点的密度增加了四倍，从105至401个位点/细胞。在竞争结合研究中，只有CGRP和CGRP拮抗剂CGRP 8 -37抑制125 I-CGRP结合，而无关的神经肽降钙素，SP和CRH没有影响。大鼠CGRP对125 I-CGRP结合的抑制最好用双位点模型描述，Kis为44.1 pM和0.6 nM。人CGRP和CGRP 8 -37对结合的抑制最好通过一个位点模型描述，Kis分别为3.92和6.50 nM。用125 I-CGRP和共价交联剂DSS或BS对70 Z/3 CGRP受体蛋白进行亲和标记，对受体蛋白进行生物化学表征。SDS-PAGE分析显示103，000 MW的主带。此外，还观察到不渗透凝胶的高分子量复合物。70 Z/3细胞上CGRP受体的存在为CGRP的免疫调节作用提供了进一步的证据，并为研究CGRP在调节淋巴细胞分化和功能中的细胞作用机制提供了模型系统。
Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide found in both peripheral and central neurons and in certain endocrine tissues. Previous studies have identified specific high-affinity CGRP receptors on normal T and B cells and have described modulatory effects of CGRP on freshly isolated lymphocytes. In these studies several lymphocyte cell lines were screened for 125I-CGRP binding. One cell line, the murine 70Z/3 pre-B cell line, was found to exhibit a high level of 125I-CGRP binding and was used for further characterization of the lymphocyte CGRP receptor. Several protease inhibitors were evaluated for their ability to stabilize 125I-CGRP binding to 70Z/3 cells. Bacitracin, a relatively nonspecific protease inhibitor, and chymostatin, a cysteine protease inhibitor, enhanced 125I-CGRP binding, suggesting that 70Z/3 cells express a protease capable of inactivating CGRP. 125I-CGRP binding had a linear dependence on cell concentration with binding being detectable with as few as 200,000 cells/ml and was rapid, with equilibrium binding being reached by 30 min. Saturation binding studies demonstrated that the 70Z/3 CGRP receptor has both low- and high-affinity binding states, with Kds of approximately 0.2 and 3.3 nM, respectively. The density of the low-affinity binding site was approximately 20,000 sites/cell and was unchanged following LPS treatment. In contrast, LPS treatment for 48 hr induced a fourfold increase in the density of the high-affinity site, from 105 to 401 sites/cell. In competition binding studies, only CGRP and the CGRP antagonist CGRP8-37 inhibited 125I-CGRP binding, whereas the unrelated neuropeptides calcitonin, SP, and CRH had no effect. Inhibition of 125I-CGRP binding by rat CGRP was best described by a two-site model, with Kis of 44.1 pM and 0.6 nM. Inhibition of binding by human CGRP and CGRP8-37 was best described by a one-site model with Kis of 3.92 and 6.50 nM, respectively. The 70Z/3 CGRP receptor protein was biochemically characterized by affinity labeling the receptor protein with 125I-CGRP and the covalent cross-linking reagents DSS or BS. SDS-PAGE analysis revealed a major band of 103,000 MW. In addition a high-molecular-weight complex which did not penetrate the gel was also observed. The presence of CGRP receptors on 70Z/3 cells provides further evidence for the immunomodulatory role for CGRP and provides a model system for studying the cellular mechanisms of action of CGRP in modulating lymphocyte differentiation and function.