EXPRESSION CLONING OF A RECEPTOR FOR C5A ANAPHYLATOXIN ON DIFFERENTIATED HL-60 CELLS
EXPRESSION CLONING OF A RECEPTOR FOR C5A ANAPHYLATOXIN ON DIFFERENTIATED HL-60 CELLS
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DOI：
10.1021/bi00226a002
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发表时间：
1991-03-26
期刊：
影响因子：
2.9
通讯作者：
VIGNAIS, P
中科院分区：
文献类型：
--
作者：
BOULAY, F;MERY, L;VIGNAIS, P
编码人C5 a过敏毒素受体的cDNA克隆已通过表达克隆从CDM 8表达文库中分离，所述CDM 8表达文库由用二丁酰腺苷环磷酸分化为粒细胞表型的人髓系HL-60细胞的mRNA制备。该cDNA克隆能够将特异性结合碘化人重组C5 a的能力转移到COS-7细胞。 该cDNA长2.3 kb，开放阅读框编码350个氨基酸残基的多肽。 碘化C5 a与转染COS细胞质膜的交联揭示了具有52-55 kDa的表观分子量的复合物，类似于在分化的HL-60细胞或人嗜中性粒细胞中观察到的组成型表达受体。 尽管分化的HL-60细胞显示出一类结合位点，解离常数约为800-900 pM，但COS细胞中表达的C5 a-R cDNA产生高亲和力（1.7 nM）和低亲和力（20-25 nM）受体。 序列比较确定C5 a受体和N-甲酰肽受体之间的序列同一性程度为34%。
A cDNA clone encoding the human C5a anaphylatoxin receptor has been isolated by expression cloning from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with dibutyryladenosine cyclic monophosphate. The cDNA clone was able to transfer to COS-7 cells the capacity to specifically bind iodinated human recombinant C5a. The cDNA was 2.3 kb long, with an open reading frame encoding a 350-residue polypeptide. Cross-linking of iodinated C5a to the plasma membrane of transfected COS cells revealed a complex with an apparent molecular mass of 52-55 kDa, similar to that observed for the constitutively expressed receptor in differentiated HL-60 cells or human neutrophils. Although differentiated HL-60 cells display a single class of binding sites, with a dissociation constant of approximately 800-900 pM, the C5a-R cDNA, expressed in COS cells, generates both high-affinity (1.7 nM) and low-affinity (20-25 nM) receptors. Sequence comparison established that the degree of sequence identity between the C5a receptor and the N-formylpeptide receptor is 34%.