Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells.
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%.
Boulay
F
F
Département de Recherche Fondamentale, Centre d'Etudes Nucléaires, Grenoble, France.
Mery
L
L
Tardif
M
M
Brouchon
L
L
Vignais
P
P
D000595:Amino Acid Sequence; D000706:Anaphylatoxins / Q000235:genetics*; D000818:Animals; D015152:Blotting, Northern; D002454:Cell Differentiation; D002460:Cell Line; D003001:Cloning, Molecular; D015936:Complement C5a / Q000235:genetics*; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics; D004285:Dogs; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011869:Radioligand Assay; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D000706:Anaphylatoxins; D003432:Cross-Linking Reagents; D012333:RNA, Messenger; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a; D004247:DNA