ABSTRACT
The chemokine receptors CXCR1 and CXCR2 are G-protein-coupled receptors (GPCRs) implicated in mediating cellular functions associated with the inflammatory response. Potent CXCR2 receptor antagonists have been discovered, some of which have recently entered clinical development. The aim of this study was to identify key amino acid residue differences between CXCR1 and CXCR2 that influence the relative antagonism by two compounds that have markedly different chemical structures. By investigating the effects of domain switching and point mutations, we found that the second extracellular loop, which contained significant amino acid sequence diversity, was not important for compound antagonism. We were surprised to find that switching the intracellular C-terminal 60 amino acid domains of CXCR1 and CXCR2 caused an apparent reversal of antagonism at these two receptors. Further investigation showed that a single amino acid residue, lysine 320 in CXCR2 and asparagine 311 in CXCR1, plays a predominant role in describing the relative antagonism of the two compounds. Homology modeling studies based on the structure of bovine rhodopsin indicated a potential intracellular antagonist binding pocket involving lysine 320. We conclude that residue 320 in CXCR2 forms part of a potential allosteric binding pocket on the intracellular side of the receptor, a site that is distal to the orthosteric site commonly assumed to be the location of antagonist binding to GPCRs. The existence of a common intracellular allosteric binding site at GPCRs related to CXCR2 may be of value in the design of novel antagonists for therapeutic intervention.
Subject(s)
Receptors, Interleukin-8A/metabolism , Receptors, Interleukin-8B/metabolism , Allosteric Site , Amino Acid Sequence , Cell Line , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Radioligand Assay , Receptors, Interleukin-8A/chemistry , Receptors, Interleukin-8A/drug effects , Receptors, Interleukin-8A/genetics , Receptors, Interleukin-8B/chemistry , Receptors, Interleukin-8B/drug effects , Receptors, Interleukin-8B/genetics , Sequence Homology, Amino AcidABSTRACT
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.
