Activation of transfected M1 or M3 muscarinic acetylcholine receptors induces cell-cell adhesion of Chinese hamster ovary cells expressing endogenous cadherins.
Activation of transfected M1 or M3 muscarinic acetylcholine receptors induces cell-cell adhesion of Chinese hamster ovary cells expressing endogenous cadherins.
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转染的 M1 或 M3 毒蕈碱乙酰胆碱受体的激活诱导表达内源钙粘蛋白的中国仓鼠卵巢细胞的细胞粘附。
DOI：
10.1006/excr.1998.4385
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Williams,CL
中科院分区：
文献类型：
--
作者：
Shafer,SH;Puhl,HL;Phelps,SH;Williams,CL
中国仓鼠卵巢（CHO）细胞表达内源性钙粘蛋白以前没有报道。然而，我们观察到，CHO细胞粘附到另一个转染的毒蕈碱乙酰胆碱受体（mAChR）激活后，表明细胞表达内源性钙粘蛋白。从CHO细胞中扩增了一个160碱基对的RT-PCR产物，与人E-钙粘蛋白的胞质结构域具有100%的同源性。从这些细胞扩增的第二RT-PCR产物与人钙粘蛋白-9的胞质结构域具有92%的同源性，与人钙粘蛋白-6的胞质结构域具有86%的同源性。蛋白质印迹法表明CHO细胞表达由E-钙粘蛋白抗体识别的165-kDa蛋白和由钙粘蛋白C-末端序列的抗体识别的120-kDa蛋白。转染的mAChR亚型调节钙粘蛋白介导的CHO细胞粘附的能力通过测量辣根过氧化物酶穿过汇合的CHO细胞单层的渗透、通过细胞的显微镜检查和通过聚集测定来测试。在激活与蛋白激酶C（PKC）功能性偶联的转染的M1或M3 mAChR的15分钟内诱导细胞-细胞粘附。相反，CHO细胞粘附不受激活转染的M2mAChR的影响，M2mAChR在功能上与其他效应物偶联。用佛波醇酯活化PKC也诱导所有测试的CHO亚系的细胞-细胞粘附。免疫荧光分析表明，内源性钙粘蛋白重新分布在CHO细胞的质膜上的mAChR或PKC激活。通过去除细胞外Ca 2+使钙粘蛋白失活，可以消除mAChR或PKC激活诱导的粘附。我们的研究表明，只有奇数mAChR亚型的激活诱导钙粘蛋白介导的粘附，表明细胞对M1或M3 mAChR刺激的独特反应可能涉及钙粘蛋白激活。
Expression of endogenous cadherins by Chinese hamster ovary (CHO) cells has not been previously reported. However, we observed that CHO cells adhere to one another upon activation of transfected muscarinic acetylcholine receptors (mAChR), suggesting that the cells express endogenous cadherins. A 160-base pair RT-PCR product with 100% homology to the cytoplasmic domain of human E-cadherin was amplified from CHO cells. A second RT-PCR product amplified from these cells has 92% homology to the cytoplasmic domain of human cadherin-9 and 86% homology to the cytoplasmic domain of human cadherin-6. Western blotting indicates that CHO cells express a 165-kDa protein recognized by E-cadherin antibodies and a 120-kDa protein recognized by an antibody to the cadherin C-terminus sequence. The ability of transfected mAChR subtypes to regulate cadherin-mediated adhesion of CHO cells was tested by measuring the permeation of horseradish peroxidase across confluent CHO cell monolayers, by microscopic examination of the cells, and by aggregation assays. Cell–cell adhesion is induced within 15 min of activating transfected M1or M3mAChR which functionally couple to protein kinase C (PKC). In contrast, CHO cell adhesion is not affected by activating transfected M2mAChR which functionally couple to other effectors. Activation of PKC with phorbol esters also induces cell–cell adhesion of all CHO sublines tested. Immunofluorescence assays reveal that endogenous cadherins redistribute on the plasma membrane of CHO cells following mAChR or PKC activation. Inactivation of cadherins by removal of extracellular Ca2+abrogates adhesion induced by mAChR or PKC activation. Our demonstration that activation of only odd-numbered mAChR subtypes induces cadherin-mediated adhesion suggests that the unique responses of cells to M1or M3mAChR stimulation may involve cadherin activation.