Formyl peptide receptor expression in birds.
N-formyl-methionyl-leucyl-phenylalanine (fMLP) is a major chemotactic factor produced by Escherichia coli and other Gram-negative bacteria. The prototypal human fMLP receptor 1 (FPR1) was cloned in 1990 from a differentiated HL-60 myeloid leukemia cell cDNA library. In transfected cells, FPR1 binds fMLP with high affinity and is activated by picomolar to low nanomolar concentrations of fMLP in chemotaxis and calcium ion mobilization assays. Two additional human genes, designated FPR-like 1 (FPRL1) and FPR-like 2 (FPRL2), were later isolated by low-stringency hybridization using FPR1 cDNA as a probe, and these were shown to cluster with FPR1 on chromosome 19q13.3. In avian models the fMLP effects and the possible expression of FPRs have been poorly investigated. In this study we demonstrated that stimulation with fMLP of cultured cells isolated from the 10-day chick embryo brain causes superoxide anion and nitric oxide release and protein phosphorylation at serine, threonine, and tyrosine residues. These effects were abrogated by pretreatment with pertussis toxin, suggesting the involvement of a G-protein-coupled receptor (GPCR). Although specific N-formyl peptide receptors have so far been demonstrated only in mammals, a specific polyclonal antihuman-FPR1 antibody proved to bind to the membrane of both neurons and glial cells isolated from the chick brain. Immunoblot analysis revealed a single band corresponding to 60 kDa ca. A BLAST search and aa sequence alignments demonstrated that a number of avian 7-transmembrane (7TM) GPCRs share some homologies with the human FPR1. Furthermore, the CXCR4 ligand, SDF-1alpha, seems to compete with the antihuman-FPR1 polyclonal antibody used in our experiments. We thus advance the hypothesis that in birds one (or more) of the expressed 7TM GPCRs, most probably chemokine receptors belonging to the CXCR4 subfamily, also may act as fMLP receptors.
Panaro
M A
MA
Department of Human Anatomy and Histology, University of Bari, Bari, Italy. ma.panaro@anatomia.uniba.it
Cianciulli
A
A
Lisi
S
S
Sisto
M
M
Acquafredda
A
A
Mitolo
V
V
D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002633:Chemotaxis / Q000187:drug effects / Q000502:physiology; D002642:Chick Embryo; D002645:Chickens / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D018922:HL-60 Cells; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism; D044042:Receptors, Formyl Peptide / Q000096:biosynthesis* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D019718:Receptors, CXCR4; D044042:Receptors, Formyl Peptide; D009240:N-Formylmethionine Leucyl-Phenylalanine