CXCR1 and CXCR2

22 Pages·0.759 MB·English

by  Roth I., Hebert C.

CXCR1 and CXCR2
Iris Roth* and Caroline Hebert
Department of Molecular Oncology, Genentech, 1 DNA Way South San Francisco,
San Francisco, CA 94080, USA
*corresponding author tel: [phone], fax: [phone], e-mail: [email protected]
DOI: 10.1006/rwcy.2000.21001.
SUMMARY inflammation by transducing the signal for one
ligand, IL-8. These receptors are attractive targets
forthedevelopmentoftherapeuticsforinflammatory
Chemokines induce cell migration and activation by
disease,asGPCRshaveproventobegoodtargetsfor
binding to specific seven transmembrane (7TM) G
small molecule antagonists. However, the relative
protein-coupled cell surface receptors (GPCRs) on
contributionsofeachreceptortodiseaseprocesshave
targetcells(Murphy,1994;PremackandSchall,1996).
not yet been established, thus it is still not known
Chemokine receptors, like all members of the GPCR
which of the two must be targeted for drug develop-
superfamily, mediate signal transduction through G
ment. Selective antagonists for CXCR1 and CXCR2
proteins. They have a single polypeptide chain and
have begun to emerge, and these will be useful tools
have25–80%aminoacidsequenceidentityamongall
forfurtherelucidatingtheroleofeachreceptorinthe
family members. CXCR1 and CXCR2 were the first
pathophysiology of inflammatory diseases. Because
members of the chemokine receptor family to be
thetworeceptorsshareamultitudeofcharacteristics,
cloned, and share a high degree of homology with
including expression patterns and functional proper-
receptors for C5a and formyl peptide. There is also a
ties, they will be discussed together in this review.
highlevelofhomologybetweenreceptorsfromhuman,
This chapter will include brief descriptions of their
rabbit, rat, and mouse. Like the other chemokine
genetics, structure–function analysis, ligand-binding
receptor family members, CXCR1 and CXCR2 are
requirements,andsignaltransductionmechanisms,as
expressed on a variety of cells and have a charac-
wellasexpressionpatternsandrolesinnormaltissues
teristically narrow ligand-binding profile, including a
as well as in disease states.
subgroup of human CXC chemokines defined by the
conserved sequence motif glutamic acid-leucine-
arginine (ELR). The human ELR-containing CXC
BACKGROUND
chemokines IL-8, growth-related oncogene (GRO)(cid:11),
GRO(cid:12), GRO(cid:13), neutrophil-activating peptide-2
Discovery
(NAP-2), epithelial cell-derived neutrophil activating
peptide 78 (ENA-78), and granulocyte chemoattrac-
tantprotein2(GCP-2)are40–90%identicalinamino The discovery of IL-8 marked the emergence of the
acidsequence.CXCR1andCXCR2aredistinguished chemokine field. Previously described chemotactic
by their different selectivities for these chemokine factors, including the formyl peptide N-formyl
ligands, with CXCR1 displaying a relatively narrow methionyl-leucyl-phenylalanine (fMLP), complement
selectivity and preference for IL-8. factor C5a, platelet-activating factor (PAF), and
Both receptors are expressed on a wide variety of leukotrieneB (LTB ),werepromiscuous,actingona
4 4
cell types, including neutrophils, monocytes, CD8(cid:135) variety of leukocyte subsets (Locati and Murphy,
T cells, mast cells, basophils, natural killer cells, 1999). IL-8, produced by lipopolysaccharide (LPS)-
keratinocytes, fibroblasts, neurons, endothelial cells, stimulated monocytes, was active on neutrophils but
and melanocytes. They are distinguished by their dif- not monocytes/macrophages. This chemoattractant
ferential affinities for a group of chemokine ligands. wasdescribedbyindependentworkinseverallabora-
CXCR1 and CXCR2 play an important role in acute tories and named neutrophil-activating factor (NAF)
1982 Iris Roth and Caroline Hebert
(Walz et al., 1987), monocyte-derived neutrophil- acid protein with seven hydrophobic domains. The
activating peptide (MONAP) (Schroder et al., 1987), IL-8 receptor described in this study is 79% identical
and monocyte-derived neutrophil chemotactic factor to F3R at the amino acid level. Because F3R shares
(MDNC) (Yoshimura et al., 1987). It was subse- only a 26% identity with the human fMLP receptor,
quently shown that this factor is produced by vari- the authors suggested that this sequence might
ous cell types and has multiple targets, thus it was actually encode an IL-8 receptor.
renamed interleukin 8 (IL-8) (Balkwill and Burke, In an accompanying paper, Murphy and Tiffany
1989; Larsen et al., 1989). Lack of cross-desensiti- screened a differentiated HL-60 cDNA library to
zation suggested that this chemokine acts through identify the human homolog of F3R (Murphy and
selective receptors on neutrophils distinct from those Tiffany, 1991). One clone shared a 69% amino acid
for fMLP, C5a, PAF, and LTB (Peveri et al., 1988). identity to F3R, yet demonstrated a pattern of
4
Samanta et al. first demonstrated specific binding expression more like that of IL-8-binding sites than
of [125I]IL-8 to the surface of human neutrophils N-formyl peptide-binding sites. When expressed in
(Samanta et al., 1989). Scatchard analysis yielded a Xenopus oocytes, this clone was found to respond to
curvilinear plot, which the authors interpreted as re- IL-8, but not to fMLP. Because specific binding
presentingasinglebindingaffinityreceptor.Chemical was not saturated at the highest concentration of
crosslinking of ligand–receptor complexes identified [125I]IL-8 tested, a dissociation constant could not be
two IL-8-binding proteins, thought to be noncova- determined. Thus, the receptor encoded by this
lentlyassociatedsubunitsofthisreceptor.Subsequent cDNA appeared to be a low-affinity IL-8 receptor
binding studies using different preparations of IL-8 (CXCR2). The structurally related ligands NAP-2
resulted in conflicting interpretations. Besemer et al. and melanoma growth-stimulatory activity (MGSA)/
also observed nonlinear Scatchard plots, but con- growth-related oncogene (GRO(cid:11)) induced calcium
cludedthattheirdatawereconsistentwithtwopopu- flux in transfected oocytes, though with decreased
lations of receptors (Besemer et al., 1989). Others potency compared to IL-8. These results correlated
demonstrated a linear Scatchard plot and only one withtheeffectivenessofthesechemokinestocompete
protein chemically crosslinked to IL-8 – results con- withIL-8forbindingtoneutrophils,asdemonstrated
sistentwiththepresenceofonlyoneclassofreceptors by Moser et al. (1991).
on neutrophils (Grob et al., 1990). The binding Meanwhile, the original rabbit F3R clone was
affinities ranged from 11pM to 2nM, with estimated expressed in COS cells and found to confer satura-
receptor numbers ranging from 20,000 to 75,000 table specific binding of IL-8, not fMLP (Thomas
per cell. et al., 1991). IL-8 bound neutrophils and F3R-
The search for chemokine receptors began when transfected cells with apparent K values of 1.2 and
d
severalgroupsutilizedexpressioncloningstrategiesto 1.4nM, respectively. F3R antibodies specifically
identify the human formyl peptide receptor (FPR) immunoprecipitated [125I]IL-8 bound to the mem-
(Boulayetal.,1990;CoatsandNavarro,1990;Murphy branes of F3R-transfected COS cells as well as to
et al., 1990). One such cDNA clone was capable of neutrophils. With these results, Navarro’s laboratory
conferring specific fMLP binding to transfected and others then correctly identified the F3R protein
mammalianCOScells(Boulayetal.,1990).Aputative as the rabbit homolog of CXCR1 (Thomas et al.,
rabbit homolog, F3R, was later cloned from a 1991; Cerretti et al., 1993a).
neutrophil cDNA library and shown to bind fMLP
when expressed in Xenopus oocytes (Thomas et al., Alternative names
1990). However, F3R was only 26% homologous to
the clone previously identified as the human fMLP
CXCR1: IL-8RA, IL-8R1, IL-8R(cid:11)
receptor.
CXCR2: IL-8RB, IL-8R2, IL-8R(cid:12)
Unique clones for two distinct human IL-8
The official names CXCR1 and CXCR2 were
receptorswerelaterindependentlydescribedbyHolmes
assigned by a consensus nomenclature agreement at
and Murphy in the same issue of Science (Holmes
the second Gordon Conference on Chemotactic
et al., 1991; Murphy and Tiffany, 1991). Using an
Cytokines in Plymouth, NH, USA in 1996.
expression cloning strategy in COS-7 cells, Holmes
et al. isolated the clone for CXCR1 from a human
Structure
neutrophil cDNA library. COS cells transfected with
this cDNA specifically bound IL-8 with a K of
d
3.6nM, within the range of 0.8–4nM reported for Human CXCR1 and CXCR2 have a single polypep-
binding to neutrophils. The cDNA contains a single tide chain 350, and 355 or 360 amino acids in length,
long open reading frame, which encodes a 350 amino respectively. The receptors share 76% amino acid
CXCR1 and CXCR2 1983
identitytooneanother(Figure3)(Holmesetal.,1991; various animal models (Koch et al., 1992; Strieter
Murphy, 1994). As members of the GPCR super- et al., 1995).
family, CXCR1 and CXCR2 are 7TM receptors that
signal via G proteins (Murphy, 1994; Premack and
Schall, 1996). Their structure can be inferred from GENE
homology to another family member, rhodopsin,
which signals light sensitivity in retinal cones (Luo Accession numbers
et al., 1997). The seven hydrophobic regions of the
receptors are embedded in the plasma membrane,
Human CXCR1: L19592
giving it a serpentine appearance (Figure 1). The free
Rat CXCR1-like: U71089
N-terminal tail is extracellular, and the C-terminus
Gorilla CXCR1: X91110
is in the cytoplasm.
Chimpanzee CXCR1: X91109
Human CXCR2: M99412
Main activities and Mouse CXCR2: L23637
Rat CXCR2: U70988
pathophysiological roles
Gorilla CXCR2: X91114
Rhesus CXCR2: X91116
CXCR1 and CXCR2 mediate a diversity of chemo- Orangutan CXCR2: X91115
kine functions in a variety of cell types. In leukocytes Chimpanzee CXCR2: X91113
they are important in mediating antimicrobial host
defenses.Bothreceptorsacttoinducechemotaxisand
Chromosome location and linkages
calcium fluxin differentleukocyte subsets. In neutro-
phils,receptoractivationalsostimulatesthereleaseof
granule enzymes as well as the generation of super- ThehumangenesforCXCR1andCXCR2(il8raand
oxide in respiratory burst (Loetscher et al., 1994; il8rb,respectively),alongwithonepseudogene(il8rp),
Hammondetal.,1995;Jonesetal.,1996).Inaddition have been mapped to chromosome 2q34–35, 20kb
to their effects on immune cells, CXCR1 and/or apart (Ahuja et al., 1992; Morris et al., 1992; Lloyd
CXCR2 may be important in regulating vasculogen- etal.,1993).Thecloseclusteringofthethreegenes,as
esisandconsequenttumorgrowth(Kochet al.,1992; well as their high degree of homology, suggests that
Strieter et al., 1995). ELR-containing CXC chemo- they may have arisen by duplication of a common
kines signal via these receptors to induce endothelial ancestral gene. The CXCR2 gene is approximately
cell chemotaxis in vitro as well as angiogenesis in 12kbinlengthwhileil8raspansjust4kb(Kelvinetal.,
Figure1 PutativemodeloftheproposedtertiarystructureofCXCR1and
CXCR2.Thetransmembrane(cid:11)helicaldomainsareindicatedbythetubular
structures, numbered 1–7. Three extracellular loops are designated EL1-3.
Twopotentialdisulfidebridges,linkingtheextracellularN-terminaldomain
with the third extracellular loop, and the first and second extracellular
loops, are shown in red. (Full colour figure can be viewed online.)
1984 Iris Roth and Caroline Hebert
1993; Ahuja et al., 1994). In both cases, the open constitutive activity when cloned upstream from a
reading frame (ORF) and 30 untranslated regions are chloramphenicol acetyltransferase (CAT) reporter
contained in a single exon, while the 50 UTR is more gene. A granulocyte colony-stimulating factor (G-
complex. CSF) responsive element was mapped within the first
Thereisonepseudogene,namedil8rp,containedin 118 nucleotides upstream of the transcription start
the CXCR1/CXCR2 gene cluster. This locus has site of il8rb (Sprenger et al., 1995). Expression ana-
an 87% identity to CXCR2, but contains multiple lyses of additional regulatory regions suggested that
frameshifts and point mutations introducing stop both promoters are negatively controlled by silencer
codons (Ahuja et al., 1992; Mollereau et al., 1993). elements. Recent work has identified promoter ele-
The50 UTRofil8raresidesinthefirsttwoofthree ments of il8ra and demonstrated that the Ets family
exons (Ahuja et al., 1994). Neutrophils contain two transcription factor PU.1 is a major regulator for
equallyabundantmRNAsforCXCR1,2.0and2.4kb activation of the CXCR1 promoter (Wilkinson and
in length, the result of usage of two alternative poly- Navarro, 1999).
adenylation signals (Figure 2). For il8rb, alternative
splicingof11exonsformssevendistinctmRNAswith
one predominant splice variant, designated IL8RB3.
PROTEIN
Primer extension analysis identified two major tran-
scriptional start sites for il8ra and 11 for il8rb. The
Accession numbers
promoters of both genes appeared to be very similar:
a nonclassical TATA box and a GC-rich 50 flanking
region was identified immediately upstream of the Human CXCR1: P25024
transcription start site (Sprenger et al., 1995). These Rabbit CXCR1: P21109
minimal promoters were sufficient to induce strong Rat CXCR1-like protein: P70612
Figure 2 Genomic organization of CXCR1 (a) and CXCR2 (b). Boxes, exons; lines connecting exons,
introns;dottedlines,gapsinthesequence;H,sitesforHindIIIcleavage.Exonsarenumberedintheupperleftof
thecorrespondingbox;intronsarenumberedinblue(CXCR1)orred(CXCR2)boxes,andtheirlengthsare
indicatedinbasepairsbelowtheintronnumber.Splicingpatternsareshownbeloweachgenemap,andthe
nameforeachmRNAformisgivenattherightofthesplicepatterndiagram.CXCR2mRNAnamesarebased
onthenumberofthe50-mostexon.HorizontallinesforeachmRNAindicatethecorrespondingexoninthe
gene,andinternaltickmarksindicateexonboundaries;dashedlinesindicatesplicingpatterns.(Fullcolour
figurecanbeviewedonline.)
CXCR1 and CXCR2 1985
Gorilla CXCR1: P55919 thebasicligand,IL-8,alaninesubstitutionsateightof
Chimpanzee CXCR1: P55920 the nine charged positions of the CXCR1 N-terminal
Human CXCR2: P25025 domainhadnoeffectonbinding.Ligandbindingwas
Mouse CXCR2: P35343 only affected when one residue, Asp11, was mutated
Rabbit CXCR2: P35344 to Ala. As predicted, IL-8 binding was retained when
Rat CXCR2: P35407 Asp11 was replaced with another acidic residue, such
Gorilla CXCR2: Q28422 as Glu, or with Lys, found at position 11 in CXCR2
Rhesus CXCR2: Q28519 (Hebert et al., 1993). These data suggest that the
Chimpanzee CXCR2: Q28807 acidic nature of the N-terminal segment of the IL-8
Bovine CXCR2: Q28003 receptors is not functionally important.
Additional residues in extracellular domains of
CXCR1 were also shown to be important for IL-8
Sequence
binding and subsequent signaling (Figure 4). Cells
transformed with constructs containing alanine sub-
See Figure 3. stitutionsatresiduesArg199andArg203,inthesecond
extracellular loop, as well as Asp265, in the third
extracellular loop, exhibited markedly diminished or
Description of protein
absentIL-8bindingcomparedwithwild-typeCXCR1
(Leong et al., 1994). These positions are also con-
The human CXCR1 and CXCR2 proteins are highly served in the sequence of CXCR2 and are thus
homologousattheaminoacidlevel(76%)(Figure3). unlikely to play a role in receptor subtype specificity.
Both proteins also have a significant degree of Ala substitution of each of another two residues,
homology to other chemokine receptors (25–30%). Glu275 and Arg280, both in the third extracellular
Residues important for ligand binding and receptor loop of CXCR1, also resulted in a dramatic loss of
signalinghavebeenelucidatedusingalaninescanning IL-8bindingandsignaling(Hebertetal.,1993).These
mutagenesis as well as the construction of receptor two residues are conserved in CXCR1 and CXCR2,
chimeras. Sequence differences between CXCR1 and as well as rabbit and mouse homologs of CXCR1.
CXCR2 appear to be clustered at the N-terminus, as Glu275 and Arg280 are thought to interact with the
wellasinthesecondextracellularloop(Murphy,1997). positively charged Arg6 and Glu4 residues of IL-8,
These regions may thus be important in conferring respectively. The Arg6 residue of IL-8 is itself critical
differential ligand specificity to each receptor. for receptor binding: Ala or Lys substitution of this
The key residues of CXCR1 important for ligand single residue causes a 1000-fold decrease in IL-8
binding were defined by extensive site-directed muta- binding affinity (Hebert et al., 1991).
genesis, in which extracellular acidic residues were Site-directed mutagenesis studies demonstrated
replaced with alanine (Figure 4) (Hebert et al., 1993; that binding occurs via determinants on multiple
Leong et al., 1994). While it was originally thought extracellular domains of CXCR1, in addition to the
that acidic residues at the N-terminus interact with N-terminus. However, others have shown that an
Figure 3 Alignment of the sequences of human CXCR1 and CXCR2. Identical
residuesaremarkedbyanasterisk.Conservativesubstitutionsaremarkedbyadot.
Human CXCR1:
1 MSNITDPQMW DFDDLNFTGM PPADEDYSPC MLETETLNKY VVIIAYALVF LLSLLGNSLV
61 MLVILYSRVG RSVTDVYLLN LALADLLFAL TLPIWAASKV NGWIFGTFLC KVVSLLKEVN
121 FYSGILLLAC ISVDRYLAIV HATRTLTQKR HLVKFVCLGC WGLSMNLSLP FFLFRQAYHP
181 NNSSPVCYEV LGNDTAKWRM VLRILPHTFG FIVPLFVMLF CYGFTLRTLF KAHMGQKHRA
241 MRVIFAVVLI FLLCWLPYNL VLLADTLMRT QVIQETCERR NNIGRALDAT EILGFLHSCL
301 NPIIYAFIGQ NFRHGFLKIL AMHGLVSKEF LARHRVTSYT SSSVNVSSNL
Human CXCR2:
1 MEDFNMESDS FEDFWKGEDL SNYSYSSTLP PFLLDAAPCE PESLEINKYF VVIIYALVFL
61 LSLLGNSLVM LVILYSRVGR SVTDVYLLNL ALADLLFALT LPIWAASKVN GWIFGTFLCK
121 VVSLLKEVNF YSGILLLACI SVDRYLAIVH ATRTLTQKRY LVKFICLSIW GLSLLLALPV
181 LLFRRTVYSS NVSPACYEDM GNNTANWRML LRILPQSFGF IVPLLIMLFC YGFTLRTLFK
241 AHMGQKHRAM RVIFAVVLIF LLCWLPYNLV LLADTLMRTQ VIQETCERRN HIDRALDATE
301 ILGILHSCLN PLIYAFIGQK FRHGLLKILA IHGLISKDSL PKDSRPSFVG SSSGHTSTTL
1986 Iris Roth and Caroline Hebert
Figure 3 (Continued)
antibody that maps to the N-terminal region of MGSA/GRO(cid:11) (Hoch et al., 1996; Norgauer et al.,
CXCR1 blocks IL-8 binding (Chuntharapai et al., 1996). While antibodies to the N-terminus inhibit
1994a). Similarly, an antibody that maps to the N- binding, peptides corresponding to the N-termini of
terminus of CXCR2 blocks binding of IL-8 and each receptor only weakly inhibit IL-8 binding to
CXCR1 and CXCR2 1987
Figure 4 Model of the secondary structures of human CXCR1 and CXCR2. As deduced by alanine scanning
mutagenesis, residues in CXCR1 that are important for receptor expression or for ligand binding are indicated in green
(Hebertetal.1993;Leongetal.1994).ResiduesinvolvedinGproteinsignalingareshowninblue(Damajetal., 1996b;
Xie et al., 1997). Important structural determinants conserved in both CXCR1 and CXCR2 are indicated in red. (Full
colour figure can be viewed online.)
CXCR1 and CXCR2 (K=17–2.2mM, and K (cid:136) suggest that, in contrast to previous reports, the N-
i i
150mM, respectively; Gayle et al., 1993). In addition, terminal segment of CXCR1 is not a dominant
a soluble peptide of the first 37 amino acids of determinantofreceptorsubtypespecificity.Inanother
CXCR1 is only capable of binding IL-8 with low report, the same chimeric receptor had a CXCR1
affinity (Gayle et al., 1993). Another peptide corres- phenotype: MGSA/GRO(cid:11) did not bind transfected
ponding to residues 1–40 of CXCR1 was also shown cells, nor did it induce chemotaxis (Wu et al., 1996).
to bind IL-8 with a dissociation constant of 170(cid:6) These observations reported by Wu et al. are more
50mM (Clubb et al., 1994). These results are in consistent with those previously reported by groups
agreementwithdatafromalanine-scanningmutagen- using the rabbit CXCR1 N-terminus in the chimera
esis of CXCR1, demonstrating that multiple extra- rather than the human CXCR1 (LaRosa et al., 1992;
cellular domains of the receptor are involved in Gayle et al., 1993; Wu et al., 1996).
high-affinity ligand interactions. Morerecently,Katanciketal.mappedthedomains
Using receptor chimeras, others have studied the involved in binding of the ligands IL-8, NAP-2, and
selectivity determinants in CXCR1 and CXCR2 MGSA/GRO(cid:11) to CXCR2 using peptides corres-
(LaRosa et al., 1992; Gayle et al., 1993; Ahuja et al., pondingtodistinctregionsofthisreceptor(Katancik
1996; Wu et al., 1996). Experiments with human/ et al., 1997). Peptides representing the N-terminus
rabbit chimeric receptors first implicated the N- and first extracellular loop of CXCR2 both inhibited
terminal domains in determining ligand specificities IL-8 binding to the receptor. However, NAP-2
(LaRosa et al., 1992; Gayle et al., 1993). Chimeras bindingwasinhibitedonlybythepeptidecorrespond-
containing the CXCR1 N-terminus were selective for ing to the first extracellular loop, and MGSA/GRO(cid:11)
IL-8, whereas those carrying the N-terminal domain binding was inhibited by portions of the N-terminus.
ofCXCR2boundbothIL-8andMGSA/GRO(cid:11)with Theseresultssuggestthatdistinctextracellulardomains
high affinity. Further studies utilized chimeric forms of CXCR2 mediate the binding of each ligand to this
of both human receptors, yet observations of indi- receptor.
vidualgroupsusingthesameconstructsdidnotagree. Additional residues in both CXCR1 and CXCR2
For example, a chimera composed of the CXCR1 N- are important for maintaining correct tertiary struc-
terminusdisplayedaCXCR2phenotypeinonestudy: ture of the receptors. Scanning mutagenesis demon-
MGSA/GRO(cid:11) and NAP-2 bound with high affinity strated that amino acids Cys30 (in the N-terminus),
and induced a strong calcium flux (Ahuja et al., Cys187(inthesecondextracellularloop),andCys277
1996). The results of these studies by Ahuja et al. (in the third extracellular loop) of CXCR1 are
1988 Iris Roth and Caroline Hebert
sensitive to mutation to alanine (Leong et al., 1994). receptor family are: TD(X)YLLNLA(X2)DLLF(X2)
Cys30 and Cys277 are conserved in human CXCR1 TLP(X)WinTM2,PLL(X)M(X2)CYinTM5,W(X)-
and CXCR2, as well as rabbit and mouse IL-8 PYN in TM6, and HCC(X)NP(X)IYAF(X) G(X2)
receptors,andmaythusbecriticalforcorrectprotein FR in TM7.
folding (Hoch et al., 1996). These two cysteine A serine/threonine-rich C-terminal region is also a
residues likely form a disulfide bridge, bringing common feature of GPCRs, including CXCR1 and
Asp11, Glu275, and Arg280 into close spatial CXCR2 (Probst et al., 1992). This region may be a
proximity to one another. These three charged targetforphosphorylationbyserinethreoninekinases.
residues, each critical for ligand binding, thus form Phosphorylation of these residues at the C-terminus
a binding domain in CXCR1 where they likely make of the (cid:12)-adrenergic and rhodopsin receptors, both
contact with oppositely charged residues in IL-8 GPCRfamilymembers,leadstoreceptor desensitiza-
(Hebert et al., 1993). An additional feature common tion (Lefkowitz et al., 1990; Palczewski and Benovic,
to nearly all GPCRs includes Asp85, located in the 1991).
second transmembrane domain of CXCR1. This A series of truncation mutants further defined
residueisconservedinmorethan90%ofmembersof key determinants important for receptor signaling.
the GPCR superfamily, and is also thought to Because expression of CXCR2 requires an intact N-
maintaincorrecttertiarystructureofthereceptor.An terminal tail, the role of this domain could not be
Asp residue in the second transmembrane domain is determined (Gayle et al., 1993; Ahuja et al., 1996).
also conserved in almost all GPCRs and replacement Analysis of truncated forms of CXCR2 suggests that
ofthisresidueleadstoalossofligandbinding(Probst signaling requires the C-terminal sequence between
etal.,1992).WhenAsp85ofCXCR1isreplacedwith amino acids 317 and 324 (Ben-Baruch et al., 1995a).
Ala, the receptor is not expressed on the cell surface, Cells transfected with truncated mutant receptors
thus abolishing binding of IL-8 (Hebert et al., 1993). bound IL-8 with the same high affinity as those
The domains in CXCR1 and CXCR2 involving transfected with wild-type receptors, but were unable
ligand binding may not be important for receptor to chemotax in response to IL-8. In addition, the C-
signaling. A detailed study of chimeric forms of both terminus-truncated mutant of CXCR2 is not phos-
humanCXCR1andCXCR2demonstratedthatIL-8, phorylated by high doses of IL-8, as is the wild-type
GRO(cid:11), and NAP-2 signal via interactions with receptor (Ben-Baruch et al. 1997). Phosphorylation
multiplereceptordomains(Ahujaetal.,1996).Inthis analysis of the wild-type receptor further demon-
study,theauthorsdemonstratedthatallthreeligands strates that IL-8 induced higher levels of phosphor-
could elicit calcium flux via a CXCR2 chimeric ylationthandidNAP-2.ThegreaterabilityofIL-8to
receptor containing the N-terminus of human CC induce receptor phosphorylation may thus also con-
chemokine receptor 1 (CCR1), even though no IL-8 tribute to its more potent stimulation of chemotaxis
binding was detected. The structural determinants compared with NAP-2.
important for ligand binding and signaling have been Chemokine receptor structure, including that of
further dissected using various blocking antibodies CXCR1andCXCR2,isunknown.However,amodel
against the IL-8 receptors (Wu et al., 1996). Studies can be constructed based on analogy with another
showed that an antibody against CXCR1 can block GPCR, bacteriorhodopsin, whose crystal structure
IL-8-mediated functional responses, but is ineffective hasbeensolved(Straderetal.,1994;Luoetal.,1997;
at blocking ligand binding. Together, these findings Unger et al., 1997; Lomize et al., 1999). The hydro-
suggest that the structural requirements for IL-8 phobic regions of the receptor traverse the plasma
binding and signaling are distinct. This is consistent membrane, separating extracellular and cytoplasmic
with the idea that conformational changes of the loops of the protein and thus giving it a serpentine
receptor secondary to ligand binding are required to appearance (Figure 1). CXCR1 and CXCR2 are
elicit biological responses (Gilman, 1987). probably in the cell membrane with the N-terminal
Additional structural determinants are also impor- tail extracellular, and the C-terminus in the cyto-
tant for receptor signaling (Figure 4). All chemokine plasm, with the seven hydrophobic domains
receptors, including CXCR1 and CXCR2, contain a embeddedinthemembraneas(cid:11)helices.Theresulting
highly conserved DRYLAIVHA motif at the end of structure comprises a total of three extracellular and
the third transmembrane domain (TM3) (Baggiolini three intracellular loops connecting each of the seven
et al., 1997). As the DRY motif is necessary for G transmembrane domains, and free N- and C-termini.
proteinactivationinotherreceptors,thisdomainmay CXCR1 has five potential N-linked glycosylation
be important for signaling and biological activity of sites (Strosberg, 1991). The molecular size predicted
CXCR1andCXCR2(O’Dowdetal.,1989).Additional from the amino acid sequence is approximately
motifs conserved among members of the chemokine 40kDa(Holmesetal.,1991).However,thereceptoris
CXCR1 and CXCR2 1989
glycosylated, resulting in an observed mass of 55– much like that of the human receptor: IL-8 (K (cid:24)
d
69kDa (Samanta et al., 1989). CXCR2 is also 4nM) (cid:29)> MGSA>NAP-2 (Prado et al., 1994). No
glycosylated at its one N-linked glycosylation site, binding was observed with radiolabeled GRO(cid:11) or
togiveafinalmassofapproximately60kDa(Horuk, PF4(Thomasetal.,1994).IL-8alsostimulatescalcium
1994). flux of transfected cells, while fMLP has no effect.
Rabbit CXCR2 has an 80% amino acid identity to
human CXCR2, 74% to rabbit CXCR1, and 73% to
Relevant homologies and species human CXCR1 (Prado et al., 1994). Rabbit CXCR2
binds IL-8, NAP-2, and MGSA with apparent K
differences i
values of 4, 120, and 320nM, respectively. IL-8
induced calcium flux and desensitization in mamma-
The human CXCR1 and CXCR2 proteins are highly lian cells transfected with rabbit CXCR2.
homologous at the amino acid level (77%) with A mouse IL-8 receptor homolog was cloned by
the highest homology over the membrane-spanning screening a cDNA library for sequences homologous
regions and significant divergence at both N- and to human and rabbit receptors (Bozic et al., 1994;
C-termini (Kelvin et al., 1993). Both proteins also Harada et al., 1994). The cDNA corresponding to
have a significant degree of homology to other human CXCR2 hybridizes strongly with two restric-
chemokine receptors: sequence alignment of the tion fragments in mouse genomic DNA, suggesting
human receptors for the three neutrophil chemo- that there are two candidate murine homologs of the
attractants IL-8, fMLP, and C5a shows a 29–34% human IL-8 receptors (Bozic et al., 1994). However,
amino acid identity between these receptors. only one murine IL-8 receptor-like protein has been
Genes encoding CXCR1 and CXCR2 homologs isolated to date, most closely resembling human
from four nonhuman primates have been cloned and CXCR2initsbindingcharacteristics(Leeetal.,1995).
sequenced (Alvarez et al., 1996). Both gorilla and Mouse CXCR2 binds the murine counterpart of
chimpanzee CXCR1 homologs show 98–99% simi- human GRO proteins, macrophage inflammatory
laritytohumanCXCR1(Figure5).Rhesusandorang protein 2 (MIP-2), with high affinity (K (cid:24)5nM),
d
utan CXCR1 homologs are pseudogenes, in which a activating both calcium flux and a chemotactic
2bp insertion has generated a sequence with several response in neutrophils (Bozic et al., 1994; Lee et al.,
stop codons. However, the CXCR2 genes from all 1995). Another murine GRO homolog, KC, is
four primate species are 95–99% identical to their approximately 10-fold less potent at triggering these
human homolog. responses. Mouse CXCR2 is 359 amino acids in
Human and rabbit CXCR1 proteins share a 84% length,andsharesa68and71%homologytohuman
aminoacididentity(Holmesetal.,1991).Theaffinity CXCR1 and CXCR2, respectively (Cerretti et al.,
profile and ligand specificity of rabbit CXCR1 is 1993b). The mRNA for this receptor is expressed in
peritonealneutrophils,andsouthernblottinganalysis
suggeststhatthisisasingle-copygene(Cerrettietal.,
Figure 5 Graphical representation of the sequence 1993b; Harada et al., 1994). A murine form of IL-8
homologyamongCXCR1andCXCR2proteinsfrom
has not been identified, but human IL-8 is an agonist
different animal species.
for mouse CXCR2 (K (cid:24)400nM) (Bozic et al., 1994;
d
Suzuki et al., 1994).
Tworatgenes,CXCR1-likeandCXCR2,havealso
been cloned (Dunstan et al., 1996). Both genes are
approximately 70% identical to their human homo-
logs at the amino acid level (Dunstan et al., 1996;
Gobl et al., 1997). Both rat genes also share a 65 and
86% identity, respectively, with murine CXCR2.
Southern blot analysis indicates that rat CXCR1-like
and CXCR2 are each single-copy genes. CXCR2
mRNA is detected in adult rat lung, spleen, and
neutrophils.CXCR1-likemRNAisexpressedinadult
rat lung and primary rat macrophages, but not in
neutrophils. Murine MIP-2 induces calcium flux in
mammalian cells expressing the rat CXCR2 receptor,
but not the CXCR1-like protein. Hybridization ana-
lysis of mouse genomic DNA suggests that the rat
1990 Iris Roth and Caroline Hebert
CXCR1-like gene is homologous to the second degranulation, but not PLD activation, in human
candidate murine IL-8 receptor homolog (Bozic neutrophils (L’Heureux et al., 1995). IL-8-induced
et al., 1994; Dunstan et al., 1996). Since the rat neutrophil chemotaxis and priming were predomi-
CXCR1-like protein is nearly equally homologous to nantly mediated by CXCR1, whereas priming by
both human CXCR1 and CXCR2 sequences, these GRO(cid:11)andENA-78ismediatedbyCXCR2(Hammond
unique rat and murine genes may represent a third et al., 1995; Green et al., 1996).
classofreceptors,distinctfromCXCR1andCXCR2. The biological significance of the differential
activity of CXCR1 and CXCR2 is unclear. For
ligands other than IL-8, the two receptors may be
involvedindifferentialspatialtransductionofchemo-
Affinity for ligand(s)
tactic signals: At low chemokine concentrations,
chemotaxismaybepreferentiallymediatedbyCXCR2.
IL-8 and GCP-2 are equipotent agonists at both Near the site of inflammation, where chemokine con-
CXCR1 and CXCR2, while the affinity for each centrations are high and CXCR2 is unable to signal,
receptor for other ligands is distinct. The rank order CXCR1 might be more important. However, both
ofpotencyofallagonistsofCXCR1andCXCR2has receptors bind IL-8 with similar affinity, thus this
been determined based on mean effective concentra- mechanismwouldnotbeusefulforIL-8gradients.In
tion values (EC ) in calcium flux assays (Ahuja and this case, CXCR2 may mediate chemotaxis at more
50
Murphy, 1996; Baggiolini et al., 1997; Van Damme distal points, while CXCR1 induces respiratory burst
et al., 1997; Wuyts et al., 1997, 1998; Wolf et al., at high chemokine concentrations in the inflamma-
1998). In the case of CXCR2, ligand potency is as tory focus. In fact, the two receptor subtypes are dif-
follows: GRO(cid:13) is the most potent (1nM)>IL-8 ferentially desensitized in human neutrophils. Higher
(4nM)(cid:24)GCP-2 (3nM)(cid:24)GRO(cid:11) (5nM)(cid:24)GRO(cid:12) concentrationsofIL-8arerequiredforinternalization
(4nM)(cid:24)NAP-2 (7nM)>ENA-78 (11nM). For of CXCR1 than CXCR2, thus downmodulating
CXCR1, potency was far more selective, with IL-8 receptor expressionon the cell surface (Chuntharapai
and GCP-2 being the most potent agonists (4 and and Kim, 1995; Sabroe et al., 1997).
3nM, respectively) (cid:29)> ENA-78 (40nM)(cid:24)NAP-2
(45nM) GRO(cid:11) (63nM)(cid:24)GRO(cid:13) (65nM) (cid:29) GRO(cid:12).
All six agonists for CXCR2 competed for high- Cell types and tissues expressing
affinity [125I]IL-8, [125I]GRO(cid:11), [125I]NAP-2, and
the receptor
[125I]ENA-78 binding to CXCR2. GRO(cid:11), GRO(cid:12),
GRO(cid:13), NAP-2, and ENA-78 each competed weakly
for high-affinity IL-8 binding to CXCR1. Although Using monoclonal antibodies specific for CXCR1
the affinity of CXCR1 for GRO(cid:11) and NAP-2 is and CXCR2, their expression pattern on various
almost100-foldlowerthanthatforIL-8,theseligands peripheral blood leukocytes have been studied by
can act as low-potency agonists in chemotaxis assays flowcytometry(Figure6)(Chuntharapaietal.,1994b;
(Loetscher et al., 1994; Ahuja and Murphy, 1996). Oin et al., 1996). A wide range of donor variation in
CXCR1 has also recently been shown selectively to expression levels was observed. All neutrophils, all
bind the N-terminal cytokine module of human monocytes, and 5–25% of CD8(cid:135) T cells and CD56(cid:135)
tyrosyl tRNA synthetase with an affinity similar to NK cells expressed both receptors (Chuntharapai
that of IL-8 (Wakasugi and Schimmel, 1999). This et al., 1994b). No CD20(cid:135) B cells or CD4(cid:135) T cells
ELR-containing enzyme is secreted under apoptotic expressed CXCR1 or CXCR2. Neutrophils expressed
conditions and may be involved in inflammatory the highest level of both CXCR1 and CXCR2 at an
signaling by apoptotic cells. approximately equal ratio, while CXCR2 expression
Selective blocking antibodies to CXCR1 and prevailedonotherleukocytesubsets,includingmono-
CXCR2 have been used to determine the role ofeach cytes and lymphocytes.
receptor in mediating specific neutrophil functions. Consistent with this report, Gerszten et al. recently
Responses such as chemotaxis, calcium flux, and the demonstrated the prevalence of CXCR2 expression
release of neutrophil granule enzymes appear to be onelutriatedmonocytes(Gersztenetal.,1999).Relative
independently mediated through both CXCR1 and prevalence varied among donors, with CXCR1
CXCR2, while the respiratory burst and PLD acti- expressed on 23–90% of monocytes, while 22–93%
vation depend exclusively on stimulation through of monocytes expressed CXCR2. These receptors on
CXCR1(Loetscheretal.,1994;Hammondetal.,1995; monocytes are functional, as antibodies to both
Jones et al., 1996). This is consistent with the ability receptors inhibited IL-8-induced monocyte chemo-
of GRO(cid:11) and NAP-2 to induce calcium flux and taxis as well as calcium flux.