1. Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells
- Author
-
Pierre V. Vignais, Laurence Brouchon, Laurence Mery, Marianne Tardif, and François Boulay
- Subjects
Anaphylatoxins ,Cellular differentiation ,Molecular Sequence Data ,Complement C5a ,Biology ,Molecular cloning ,Transfection ,Biochemistry ,C5a receptor ,Cell Line ,Radioligand Assay ,Dogs ,Complementary DNA ,Sequence Homology, Nucleic Acid ,Animals ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,Receptor ,Receptor, Anaphylatoxin C5a ,COS cells ,Anaphylatoxin receptors ,Cell Differentiation ,DNA ,Blotting, Northern ,Molecular biology ,Receptors, Complement ,Cross-Linking Reagents ,Gene Expression Regulation ,Expression cloning ,Sequence Alignment - Abstract
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-formlypeptide receptor is 34%.
- Published
- 1991