Functional reconstitution of fMet-Leu-Phe receptor in Xenopus laevis oocytes

fMet-Leu-Phe (fMLP) receptors were functionally reconstituted into Xenopus laevis oocytes by microinjection with RNA isolated from promyelocytic leukemia cells (HL-60) differentiated with 750 μM N⁶, O₂-dibutyryl cyclic adenosine 3′,5′-monophosphate. fMLP-induced Ca²⁺ mobilization was monitored by measurement of photon emission elicited by aequorin coinjected with RNA into albino X. laevis oocytes. Maximal expression of the fMLP receptor was achieved 48 h after microinjection of RNA. Dose-response experiments revealed a K_0.5 of 9.5 nM fMLP which is in good agreement with the dissociation constants of the fMLP receptor complex in human neutrophils. Furthermore, the fMLP-induced Ca²⁺ mobilization in Xenopus oocytes was blocked by the fMLP receptor inhibitor t-butoxycarbonyl-Met-Leu-Phe. Size fractionation of the RNA and microinjection of the individual fractions indicated that messenger RNA for the fMLP receptor is between 1.5 and 2.0 kilobases. Reconstitution of the fMLP receptor into Xenopus oocytes can be employed to isolate the cDNA encoding the fMLP receptor as well as to study the regulation of the fMLP receptor in a functional system.