The rabbit neutrophil N-formyl peptide receptor. cDNA cloning, expression, and structure/function implications

Area of Science:

• Immunology
• Molecular Biology
• Cell Signaling

Background:

• The N-formyl peptide receptor (FPR) in rabbit neutrophils is crucial for immune responses.
• Homologous G protein-coupled receptors exist but lack high-affinity FMLP binding.
• Understanding FPR structure is key to elucidating formyl peptide ligand interactions.

Purpose of the Study:

• To delineate the primary structure of the rabbit FPR.
• To identify structural requirements for formyl peptide ligand binding.
• To characterize FPR-mediated signal transduction in a heterologous system.

Main Methods:

• Screening a rabbit neutrophil cDNA library with a human FPR probe.
• Sequencing the positive cDNA isolate (B6).
• Expressing the rabbit FPR in stably transfected L cell fibroblasts.
• Assessing ligand binding (fMet-Leu-[3H]Phe) and GTPγS effects.
• Measuring FMLP-induced intracellular calcium mobilization and desensitization.

Main Results:

• The cloned rabbit cDNA encodes a 352-amino acid peptide representing a high-affinity FPR.
• Transfected cells exhibited specific binding of fMet-Leu-[3H]Phe with high (Kd = 0.31 nM) and low (Kd = 7.5 nM) affinities.
• GTPγS converted high-affinity sites to low-affinity sites.
• FMLP induced pertussis toxin-sensitive calcium mobilization (EC50 = 0.5 nM) and receptor desensitization.
• Rabbit FPR shares 78% sequence identity with human FPR and 68% with FPR2.
• Sequence analysis suggests extracellular loops 1 and 3, and adjacent transmembrane domains are critical for high-affinity FMLP binding.

Conclusions:

• The cloned rabbit receptor is a functional, high-affinity FPR.
• Early FPR-mediated signal transduction events can be reconstituted in transfected mammalian cells.
• Specific extracellular regions and transmembrane domains are essential for high-affinity formyl peptide binding.