Functional high efficiency expression of cloned leucocyte chemoattractant receptor cDNAs
- 10 February 1992
- journal article
- Published by Wiley in FEBS Letters
- Vol. 297 (3) , 275-279
- https://doi.org/10.1016/0014-5793(92)80555-u
Abstract
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 ds 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 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.Keywords
This publication has 13 references indexed in Scilit:
- The chemotactic receptor for human C5a anaphylatoxinNature, 1991
- Synthesis and use of a novel N-formyl peptide derivative to isolate a human N-formyl peptide receptor cDNABiochemical and Biophysical Research Communications, 1990
- Molecular cloning of a new human G protein Evidence for two Giα‐like protein familiesFEBS Letters, 1987
- Construction and characterization of an active factor VIII variant lacking the central one-third of the moleculeBiochemistry, 1986
- A pertussis/choleratoxin-sensitive N protein may mediate chemoattractant receptor signal transductionBiochemical and Biophysical Research Communications, 1986
- Potential role for a guanine nucleotide regulatory protein in chemoattractant receptor mediated polyphosphoinositide metabolism, Ca++ mobilization and cellular responses by leukocytesBiochemical and Biophysical Research Communications, 1985
- A chemoattractant receptor on macrophages exists in two affinity states regulated by guanine nucleotides.The Journal of cell biology, 1984
- Guanine nucleotides modulate the binding affinity of the oligopeptide chemoattractant receptor on human polymorphonuclear leukocytes.Journal of Clinical Investigation, 1983
- The oligopeptide chemotactic factor receptor on human polymorphonuclear leukocyte membranes exists in two affinity statesBiochemical and Biophysical Research Communications, 1982
- THE ATTRACTIONS OF PROTEINS FOR SMALL MOLECULES AND IONSAnnals of the New York Academy of Sciences, 1949