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Functional high efficiency expression of cloned leucocyte chemoattractant receptor cDNAs
Author(s) -
Didsbury Jolin R.,
Uhing Ronald J.,
Tomhave Eric,
Gerard Craig,
Gerard Norma,
Snyderman Ralph
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)80555-u
Subject(s) - receptor , chemotaxis , c5a receptor , pertussis toxin , microbiology and biotechnology , biology , intracellular , transfection , calcium in biology , g protein coupled receptor , g protein , biochemistry , gene , immunology , complement system , antibody
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 ( K d s of ca. 0.43 nM and 39 nM), which in the presence of 100μM GTPγS 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 ED 50 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.