A truncated form of CKbeta8-1 is a potent agonist for human formyl peptide-receptor-like 1 receptor.
1. Human formyl peptide-receptor-like-1 (FPRL-1) is a promiscuous G protein-coupled receptor (GPCR), and belongs to a chemoattractant receptor family protein. This receptor has been reported to interact with various host-derived peptides and lipids involved in inflammatory responses. We described here, a novel role for FPRL-1 as a high-affinity beta-chemokine receptor for an N-terminally truncated form of the CKbeta8 (CCL23/MPIF-1) splice variant CKbeta8-1 (22-137 aa). 2. RT-PCR analysis of mRNA derived from human tissues and cells revealed a predominant expression of FPRL-1 in inflammatory cells, particularly in neutrophils. 3. Intracellular calcium mobilisation assay, used as screening tool, in recombinant Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293s) cells coexpressing FPRL-1 and Galpha(16), demonstrated FPRL-1 is a functional high-affinity receptor for CKbeta8-1 (46-137 aa, sCKbeta8-1), with pEC(50) values of 9.13 and 8.85, respectively. 4. The FPRL-1 activation in CHO-K1 cells is mediated by Galpha(i)/Galpha(o) proteins, as assessed by pertussis toxin sensitivity and inhibition of forskolin-induced cyclic AMP accumulation. 5. Binding experiments were performed with a radio-iodinated synthetic peptide, [(125-)I]-WKYMVm, a known potent FPRL-1 agonist. CHO-K1 cell membranes expressing FPRL-1 bound [(125-)I]-WKYMVm with a K(d) value of 9.34. Many known FPRL-1 agonists were tested and sCKbeta8-1 was the most effective nonsynthetic ligand in displacing the radiolabelled agonist, with a pIC(50) of 7.97. 6. The functional significance of sCKbeta8-1 interaction with FPRL-1 was further demonstrated by the activation of polymorphonuclear leukocytes (PMNs) calcium mobilisation and chemotaxis. These interactions were shown to be via FPRL-1 by specific blockade of PMNs activation in the presence of an FPRL-1 antibody.
Elagoz
Aram
A
AstraZeneca R&D Montréal, 7171 Frederick-Banting, Ville Saint-Laurent, Québec, Canada, H4S 1Z9. aram.elagoz@astrazeneca.com
Henderson
Duncan
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Babu
Poda Suresh
PS
Salter
Sylvia
S
Grahames
Caroline
C
Bowers
Lorna
L
Roy
Marie-Odile
MO
Laplante
Patricia
P
Grazzini
Eric
E
Ahmad
Sultan
S
Lembo
Paola M C
PM
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D019742:Chemokines, CC / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D002633:Chemotaxis / Q000187:drug effects; D006224:Cricetinae; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D044042:Receptors, Formyl Peptide / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D044067:Receptors, Lipoxin / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
C105504:CCL23 protein, human; D019742:Chemokines, CC; C105101:FPR2 protein, human; D007457:Iodine Radioisotopes; D012333:RNA, Messenger; D044042:Receptors, Formyl Peptide; D043562:Receptors, G-Protein-Coupled; D044067:Receptors, Lipoxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium