Functional high efficiency expression of cloned leukocyte chemoattractant receptor cDNAs.
Human kidney 293 TSA cells were transfected by a calcium phosphate method with human formylpeptide and C5a receptor cDNAs with high efficiency. Formylpeptide receptor positive transfectants expressed a total of 968,000 +/- 34,000 receptors per cell with two affinity states (Kds of ca. 0.43 nM and 39 nM), which in the presence of 100 microM GTP gamma S decreased by ca. 4-fold the number of high-affinity sites. The ligand binding pharmacology of cloned and expressed formylpeptide receptors were indistinguishable from endogenous receptors on human neutrophils. Expressed formylpeptide and C5a receptors were functionally active in mobilizing intracellular calcium via a pertussis toxin sensitive mechanism with an ED50 for formylpeptide of ca. 0.5-1.0 nM. This expression system, in which receptor expression can be monitored by flow cytometric methods and in which intracellular calcium responses are measurable, unlike in the more popular COS-7 cell expression system, will provide a useful basis for the analysis of chemoattractant receptor structure-function relationships.
Didsbury
J R
JR
Department of Medicine, Duke University Medical Center, Durham, NC 27710.
Uhing
R J
RJ
Tomhave
E
E
Gerard
C
C
Gerard
N
N
Snyderman
R
R
D001483:Base Sequence; D002628:Chemoreceptor Cells / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA; D005434:Flow Cytometry; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D009240:N-Formylmethionine Leucyl-Phenylalanine; D004247:DNA; D019204:GTP-Binding Proteins