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.
Footnotes
-
ABBREVIATIONS: GPCR, G-protein-coupled receptor; PCR, polymerase chain reaction; TM, transmembrane; CXCR1-2-1, CXCR1 receptor with amino acids 168 to 218 (CXCR2 numbering system, equivalent to 159 to 209 in the CXCR1 sequence) substituted from CXCR2; CXCR1-2long, CXCR1 receptor with C-terminal 60 amino acids substituted from CXCR2; CXCR2-1long, CXCR2 receptor with C-terminal 59 amino acids substituted from CXCR1; CXCR1-2short, CXCR1 receptor with C-terminal 34 amino acids substituted from CXCR2; CXCR2-1short, CXCR2 receptor with C-terminal 33 amino acids substituted from CXCR1; HEK, human embryonic kidney; KT5720, hexyl (5R,6S,8S)-6-hydroxy-5-methyl-13-oxo-5,6,7,8,14,15-hexahydro-13H-5,8-epoxy-4b,8a,14-triazadibenzo[b,h]cycloocta[1,2,3,4-jkl]cyclopenta[e]-as-indacene-6-carboxylate.
-
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
-
↵1 Current affiliation: GlaxoSmithKline, Clinical Immunology, Biopharm CEDD, Stevenage, United Kingdom.
- Received December 21, 2007.
- Accepted August 1, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|