A Structural Homologue of the N-Formyl Peptide Receptor

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C5a, leukotriene B4, and histamine were purchased from Sigma. .... and then with a full-length cDNA encoding the FPRLl receptor (lane. 2). In each case the blot ...
Val. 267, No. 11, Issue of April 15, pp. 7637-7643, 1992 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY

A Structural Homologue of the N-Formyl Peptide Receptor CHARACTERIZATION AND CHROMOSOME MAPPING OF A PEPTIDE CHEMOATTRACTANT RECEPTOR FAMILY* (Received for publication, October 23,1991)

Philip M. Murphy$#,Tayfun Ozgelikn, Richard T. KenneyS,H. Lee Tiffany$, David McDermott8, and UtaFrancken I( From the $Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes ofHealth, Bethesda, Marylnnd 20892, the nHoward Hughes Medical Institute, and the IlDepartments of Genetics and Pediatrics, Stanford University Medical Center, Stanford, California 94305-5428

Phagocytic cells of many higher species express calcium mobilizing G protein-coupled receptors for bacterial N-formyl peptides which mediate chemotaxis, degranulation, and the respiratory burst. cDNA encoding an N-formyl peptide receptor (FPR) has been reported. We now report theisolation of a closely related cDNA, 2.6 kilobase pairs in length, which we have designated as the FPRLl receptor cDNA (FPRL1 = formyl peptide receptor like-1). FPR and the FPRLl receptor derive fromsmall, single-copy genes, both of which are located on human chromosome 19. The gene loci are designated FPRl and FPRLl, respectively. Both FPR and FPRLl cDNA cross-hybridize under high stringency conditions with a third gene, designated as FPRL2, which does notappear to be expressed in neutrophils. In contrast, transcripts for both the FPRLl receptor and FPR are detected only in differentiated myeloid cells; the distribution of N-formyl peptide binding sites is also restricted to mature myeloid cells. FPRLl cDNA encodes a 351-amino acid polypeptide whose sequence is 69%identical to thatof FPR. G protein-coupled receptors that exhibit this degree of structural similarity typically possess a conserved ligandspecificity. However, the FPRLlreceptor does not bind prototype N-formyl peptide ligands when expressed in heterologous cell types. These results suggest that FPRl may be the only gene that is expressed by neutrophils that encodes a receptor capable of binding prototype N-formyl peptides. Moreover, discovery of the FPRLl receptor indicates the existence of another as yet unidentified peptide that may recruit neutrophils to sitesof inflammation.

The N-formyl peptide receptor is a classic calcium mobilizing G protein-coupled receptor expressed by neutrophils and other phagocytic cells of the mammalian immune system (reviewed in Ref. 1).N-Formyl peptides of bacterial origin specifically

* This study was supported by National Institutes of Health Grant HG00298 (to U. F.) and by the Howard Hughes Medical Institute (of which T. 0.is an associate and U. F. is an investigator). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to theGenBankTM/EMBL Data Bankwith accession number(s) M84562. § T o whom correspondence should be addressed Bldg. 10, Rm. llN113, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-480-2037;Fax: 301-402-0789.

bind to thereceptor and engage a complex activation program that results in directed cell movement, release of inflammatory granule contents, and activation of a latent NADPH oxidase important for the production of metabolites of molecular oxygen. These and other cell functions play critical roles in protecting the host from pyogenic infections. Similar, perhaps identical, signal transduction occurs with host-derived inflammatory peptides such as C5a and IL-8l (2, 3). Analysis of phagocyte membrane preparations has revealed high and low affinity binding sites for N-formyl peptides that are interconvertible by guanine nucleotides, as well as two Nformyl peptide affinity-labeled spots as detected by two-dimensional SDS-polyacrylamide gel electrophoresis (4, 5 ) . At present, it is unclear whether to attribute these findings to the product of a single receptor gene or to the products of two or more structurally related genes. Cloning of human phagocyte cDNA encoding an N-formyl peptide receptor (FPR) that mobilizes calcium has been reported (6-8). COS cells transfected with this cDNA acquire N-formyl peptide binding characteristics similar to those reported on phagocytic cells. Scatchard transformation of the binding data identifies high and low affinity binding sites, suggesting a cooperative mechanism of affinity conversion of a single receptor polypeptide (6, 7). The present study supports the existence of only one N-formyl peptide receptor gene expressed in neutrophils, based on molecular cloning of a close structural homologue of FPR that hasdiverged functionally. EXPERIMENTALPROCEDURES

Materials-fMLP, f-nLLFnLYK, ATP,UTP, recombinant human C5a, leukotriene B4, and histamine were purchased from Sigma. Platelet activating factor (C18) wasfrom Bachem Bioscience (Philadelphia, PA). Interleukin 8 was from Genzyme (Cambridge, MA). cDNA Library Screening“ cDNA library was constructed in the unidirectional transcription competent vector UniZAP (Stratagene, La Jolla, CA) from a sucrose gradient fraction of HL60 neutrophil poly(A)+ RNA that possessed an average chain length of 2 kb. This fraction was chosen because of its ability to confer fMLP responsiveness to microinjected oocytes from Xenopus laeuis (8,9). Thecloning of cDNA encoding FPR has been previously described (6-8). Seventy thousand plaque lifts of the library were prepared by standard methods (10). Plaques were hybridized with FPR cDNA labeled with [a3ZP]dCTP(Amersham Corp.) by the random primer method (11)to The abbreviations used are: IL, interleukin; bp, base pair(s); cRNA, RNA synthesized in vitro from cDNA; fMLP, N-formylmethionyl-leucyl-phenylalanine;f-nLLFnLYK, N-formyl-norleucylleucyl-phenylalanyl-norleucyl-tyrosyl-lysine; G protein, guanine nucleotide-binding regulatory protein; kb, kilobase(s) or kilobase pair(s); SDS, sodium dodecyl sulfate; UTR, untranslated region; ORF, open reading frame; TMS, transmembrane segment(s).

7637

7638

Chemoattractant Family Peptide Gene Receptor -713 -653 -593 -533 -473 -413 -353 -293 -233 -173 -113 -53

0

28s

7 67

0

s1 s1

18s

-

127 187 247

- .

307 367 427 487 547 607 667 727 787 847

B

A

FIG. 2. Expression of FPR and FPRLl transcripts in myeloid cells. Each lane contains 10 pgof poly(A)+ RNA from the indicated source. Pan& A and B derive from the identical blot hybridized to FPR cDNA ( A ) , stripped and then rehybridized to FPRLl cDNA ( B ) .The positions of endogenous 18 and 28 S ribosomal RNA are indicated. Identical hybridization and washing conditions were used for both probes (final wash at 68 "C in0.1 X SSPE for 1 h). The blot was exposed to XAR-2 film with an intensifying screen at -80 "C for 2 ( A ) and 6 (€3) days. As a positive control, the blot was hybridized with PM1 cDNA (46) which recognizes a 1.2-kb transcript in all lanes (not shown).

907 967 1027

500Q

1087

0

n47 1207 1267 l327 x387 1441 1507 1567 1627 1687 1747 1807 1867

3

400-

300-

:a

200-

c.

2

1000

CF:

cRNA Injected: none

+ .

2 FPRLl

FPR

FIG. 3. Functional expression analysis of FPR and FPRLl cRNA in Xenopus oocytes. Oocytes were injected with the following amounts of R N A 10 ng of FPR cRNA, 25 ng of CF, 25 ng of FPRLl cRNA. CF designates a sourceof FPR complementary factor the A in the first codon which is defined as position 0. The box activity, undifferentiated HL60cell poly(A)+RNA in this experiment. encloses the predicted ATG initiation sitefor protein translation and It is important to note that FPR and CF are by themselves devoid of flanking sequences that conform to theconsensus sequencefor trans- calcium mobilizing formyl peptide receptor activity (8). Two days lationinitiation sites (20). A polyadenylationsignalsequenceis after injection, oocytes were stimulated with a mixture of fMLP and underlined. f-nLLFnLYK, 1 p~ each). At least five oocytes were tested per condition. The data are representative of five independent experia specific activity of 5 X loRcpm/pg DNA. Prehybridization condi- ments.Baseline calcium efflux and calcium loading were similar tions were as follows: buffer N (12), 37 "C for 60 min. Hybridization among all experimental conditions. conditions were as follows: fresh buffer N containing 10" cpm/ml probe, 42 "C, 12 h. Plaques which corresponded to duplicate hybridi- as a single appropriately sized band by denaturing agarose gel eleczation signals from nitrocellulose replicas washed a t 44 "C in 5 X trophoresis prior to microinjection. In addition, all cRNA were tested SSPE for 1 h (low stringency) and which then lost greater than 80% for the ability to direct the synthesis of a polypeptide chain in uitro of their signal intensity when rewashed a t 68 "C in 0.1 X SSPE for 1 using rabbit reticulocyte lysate (Promega, Madison,WI). The method h (high stringency)were identified as potential structural homologues of microinjection and thematerials and methods used for the calcium of FPR.Candidate cDNAs were then subcloned into Bluescript efflux assay were exactly as described previously in detail (14). RNA Suruey-Total cellular poly(A)' RNA was prepared as preplasmids as described (8)and sequenced with 17-mer oligonucleotides using a T7 DNA polymerase-based sequencing kit (Pharmacia LKB viously described (12, 14) from: 1) human peripheral blood T cells and tonsillarB cells; and 2) the following transformed cell lines Biotechnology Inc.) by the dideoxynucleotide chaintermination derived from human blood U937 promonocyte, THP-1 promonocyte, method (13). Receptor Reconstitution in the Xenopus Oocyte-Capped sense HL60 promyelocytes, Jurkat T lymphocytes and HL60 neutrophils RNA was synthesized by first linearizing plasmid constructs with (HL60 cells cultured for 48 h in 750 p~ dibutyryl cyclic adenosine XhoI followed by in uitro transcription with T 3 RNA polymerase as monophosphate). RNA blot hybridization was performed as previpreviously described (8).All cRNA transcripts were shown to migrate ously described (8).

FIG. 1. Nucleotide and deduced amino acid sequence of FPRLl cDNA. The nucleotide sequence is enumerated relative to

Peptide Chemoattractant Receptor 1 Origin

kb 23 9.4 6.6

-

2

aa

-

4.4

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2.3 2.0

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7639

in 1 X SSC, 1% SDS at 55-65 "C for 10-15 min. Autoradiography was carried out for 16-48 h at -70 "C with Kodak X-Omat AR film and two intensifying screens. Sequence Analysis-DNA and protein sequences were compiled and analyzedusing the softwarepackagefrom the University of Wisconsin Genetics Computer Group (17) on a Cray supercomputer maintained by the National Cancer Institute Advanced Scientific Computing Laboratory, Frederick Cancer Research Facility, Frederick, MD. The evolutionary dendrogram was constructed by the Parsimony After Progressive Alignment(PAPA) methodof Doolittle and Feng (18, 19). Protein sequences were obtained from the GenBank and Protein IdentificationResource data bases. The group of sequences were progressively aligned by the distance matrix method which maximizes the similaritiesbetween multiple sequences. Aligned sequences were then examined for evidence of common ancestry by a column-by-column character analysis, and thephylogeny constructed on the basis of nearest neighbors. Branch lengths were calculated from the resultsof the phylogeny comparison. Programs were run on a Convex C220 computer maintained by the Division of Computing Resources and Technology a t theNationalInstitutes of Health, Bethesda, MD.

I

. .

RESULTS AND DISCUSSION

Cloning of FPRLI cDNA-After combined high and low stringency screening of the HL60 neutrophil cDNA library with an FPR cDNA probe, 26 independent clones were isolated.Eleven were identical to FPR based on a common restriction endonuclease digestion pattern. cDNA from the FIG. 4. Members of the FPR family: genomic analysis. The remaining 15clones all corresponded to the same gene product same Nytran blotof an EcoRI digest of human genomic DNA (3 pg) as determined by the DNA sequence of the 3"untranslated was hybridized first with a full-length cDNA encoding FPR (lane 1 ) and thenwith a full-length cDNA encodingthe FPRLlreceptor (lane region (UTR). The longest of these, designated the FPRLl 2). In each case the blot was washed at 68 "C in 0.1 X SSPE for 1 h. receptor cDNA, was sequenced completely on both strands, It was then exposed to Kodak XAR-2 film in a Quanta I11 cassette a t and confirmatory sequence was obtained from an additional -80 "C for 4 (lane 1) and 6 (lane 2 ) days. The FPRprobe wasstripped six independent recombinants. Clones corresponding to this from the blot by washing in 50% formamide, 6 X SSPE at80 "C for structural homologue of FPR were as abundant as FPR clones 45 min priorto hybridization with the FPRLlprobe. The position of inthe library, i.e. approximately 1 in 5000 plaques were chain length standards is indicated in kilobases at the left. positive for each. FPRLl cDNA is 2657 bp in length (Fig. 1). The open reading frame (ORF) begins with the putative translation initiation sequence A A G m G . This conforms favorably to the consensus sequence found for translation initiation sites (20). The ORF is 1053 nucleotides in length and encodes a - 2.8 predicted protein of 351 amino acids. It ispreceded by a 772bp 5'-UTR andis followed by a 832-bp 3'-UTR that contains the polyadenylationsignal ATTAAA beginning 20 bp upFIG.5. Chromosomal localization. Chromosomal mapping of stream from the 26-bp poly(A) tail. Although the nucleotide the human FPRZ ( A ) and FPRLI ( B ) genes. '*P-Labeled full-length sequence of the ORFs of FPR and the FPRLl receptor are FPR and FPRLlcDNA probes were hybridized to Southern blotsof highly related, there is no detectable sequence similarity in EcoRI-digestedDNAfrom human X rodent hybrid cell lines and controls. A: lane 1, human lymphoblastoid cell line; lane 2, Chinese the flankingregions, as would be expected forthe products of hamster cell line V79/380-6; lanes 3 and 5, hybrid cells positive for distinct genes. Moreover, the lengths of the flanking regions human fragment; and lane 4, negative for the human fragment. B: are very different: 93 uerszu 772 bp in the 5'-UTR and 189 lane I , human lymphoblastoid cell line; lane 2, Chinese hamster cell uersus 832 bp in the3'-UTR. line V79/380-6; lanes 3-5, human X Chinese hamster cell hybrids of FPRLl Expression Analysis-Two size classes of FPRL1which lanes 4 and 5 are positive and lane 3 is negative for the human specific transcripts, 2.6 and 3.5 kb in length,were detected in fragment. HL60 neutrophil RNA by blot hybridization analysis. TranGenomic DNA Analysis-High molecular weight human genomic scripts were not detectable in poly(A)+ RNA from undifferDNAwas prepared from an adult Caucasian female by standard entiated HL60cells (Fig. 2); from two other myeloid precursor cell lines, U937 and THP-1; from human B and T lymphomethods (10). DNA (3 pgllane) was digested to completion with 6 units of EcoRI restriction endonuclease (Boehringer-Mannheim) and cytes; or from ten tissues of human cadaveric origin (not then was fractionated by electrophoresis on a 1% agarosegel. The gel shown). This restricted pattern of expression of FPRLl tranwas then denatured, and DNA was transferred to a Nytran filter by scripts isidentical to thatobserved for FPR andis consistent capillarity.Hybridization conditions were as describedabove for with theknown distribution of N-formyl peptide binding sites. cDNA library screening. RNA made in vitro (cRNA) from both FPR and FPRLl Chromosomal Localization-The chromosomal assignments were made by using panels of somatic cell hybrids that contain different cDNAdirected the synthesis of 32-kilodalton polypeptide subsets of human chromosomes from fusion series XII, XV, XVII, chains in vitro (not shown). This is similar to the size of the XVIII, XXI, and 31 (15). Ten pg of genomic DNA samples extracted affinity-labeled deglycosylated N-formylpeptidereceptor from parental control cells, and hybrid cell lines were digested with from neutrophilstreated with endoglycosidase F (5). The restriction enzymes, electrophoresed, transferred to Hybond nylon Xenopus oocyte was used in an attempt to identify the ligand membranes (Amersham Corp.), and hybridized to "P-labeled probes as described (16). Filters were rinsed twice in 2 X SSC and washed specificity of these receptor proteins. Functional expression '. I

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Chemoattractant Peptide

7640

Receptor Gene Family

TABLE I FPRl sequences and human chromosomes in rodentX human somatic cell hybrids Human FPRl sequencea~chromosome

1

2

3

4

5

6

7

8

9

10

Human chromosome 11 12 13 14 181517 16

1921 20

22

X

6 2

3 0

1 2 3 11

1 1 2 5

Concordant hybrids

+/+ 3 3 3 4 3 3 1 5 2 0 4 5 4 6 6 4 -/4 4 0 2 3 2 3 4 4 2 2 3 3 1 2 2 Discordant hybrids +/4 5 4 3 5 4 6 3 6 8 2 3 4 2 2 4 -/+ 0 0 4 0 1 2 1 0 0 2 2 1 1 2 2 1 Total discordant hybrids 4 5 8 3 6 6 7 3 6 1 0 4 4 5 4 4 5 Total informative hvbrids" 11 12 11 9 12 11 11 12 12 12 10 12 12 11 12 11 a Chromosomes with rearrangements or present at a frequency of 0.1 or less were excluded.

3 4

4 3

5 0

4 1

8 4

5 3

5 2

0 3 2 0 1 2 5 5 0 4 4 12 12 12 12 11

TABLE I1 FPRLl sequences and human chromosomes in rodentX human somatic cell hybrids Human FPRLl sequences/chromosome

Human chromosome 1

2

3

4

5

6

7

8 18 917 161015 14 1113 12

3 4

3 3

3 1

4 3

3 4

3 2

1 3

5 4

2 4

0 3

4 0 4 11

5 1 6 12

4 3 7 11

3 0 3 10

5 0 5 12

4 2 6 11

6 1 7 11

3 0 3 12

6 0 6 12

8 1 9 12

X

22 1921 20

Concordant hybrids

+/+ -/-

Discordant hybrids +/-

-/+

4 1

5 2

4 3

6 1

6 2

4 2

3 4

4 3

8 4

5 3

2 3 3 2 5 5 10 12

4 1 5 12

2 2 4 11

2 2 4 12

4 1 5 11

5 0 5 12

4 1 5 12

0 0 0 12

3 1 4 12

Total discordant hybrids Total informative hvbrids" Chromosomes with rearrangements or present at a frequency of 0.1 or less were excluded.

5 1

6 3

3 0

2 2 3 1 5 3 11 12

1 1 2 5

of FPR inXenopus oocytesrequired coinjection of FPR cRNA addition, the 5.7-kb band that hybridized weakly to both with RNA encoding a complementary human factor that is probes likelyrepresents a third gene that hasrecently diverged from FPRl and FPRLl. We have designated this gene FPRL2. expressed in both differentiated and undifferentiated HL60 cells (8).When oocytes were injected with FPRLl cRNA with The absence of a distinct weakly hybridizing band on HL60 or without a source of FPR complementary factor RNA and neutrophil RNA blots that is recognized by both FPR and were then screened with two different prototype N-formyl FPRLl cDNA probes suggests that FPRL2 may not be expeptides, fMLP and f-nLLFnLYK, acquired ligand-dependpressed in this cell. type (Fig. 2). Although it is tempting to ent calcium mobilizing activity was not detected (Fig. 3). As conclude based on our analysis of the FPRLl receptor that previously reported (8), oocytesinjected with FPR cRNA FPR is the only neutrophil receptorfor fMLPandfresponded to either prototype N-formyl peptide only when nLLFnLYK encoded within the human genome, the struccomplementary factor RNAwas coinjected. Under conditions tural relationship of the adrenergic receptors with the seroin which we were able to detect specific binding of T - f - tonin receptors indicates that this may not be the case. If 3T3 cells additional neutrophil fMLP receptor isotypes do exist, hownLLFnLYKtohumanneutrophilsandtoNIH transfected withFPR cDNA, we were unable to detect bindingever, they will be structurally more divergent from FPR than is the FPRLl receptor. to heterologous cell typesexpressing FPRLl RNA(not Chromosomal Localization of FPRl andFPRL1-In EcoRIshown). When oocytes injected with FPRLl cRNA, with or without a source of complementary factor RNA, were stimu- digested human DNA, theFPR cDNA probe hybridized lated with eight other ligands that mobilize calcium in phag- strongly with a 2.8-kb and weakly with a 3.8-kb fragment; it ocyticcells (histamine, C5a, interleukin 8, leukotriene B4, did not cross-hybridize with Chinese hamster DNA. The 2.8was present inhuman:rodent somatic cell ATP, UTP, prostaglandin E2, and platelet activating factor) kb human fragment no response was detected. As expected,oocytes expressing hybrids that contain human chromosome 19 (Fig. 5, lunes 3 and 5)and absent inhybrids that lack this chromosome (lune FPR were also unresponsive to these eightligands. 4 ) . The human FPRLl cDNA probe also hybridized only with Genomic Analysis of FPR and FPRLl-High stringency blot hybridization analysis of human genomic DNA digested human DNA and revealed a 3.8-kb and a very weak 2.8-kb EcoRI fragment. The 3.8-kb fragment was concordant with with EcoRI is shown in Fig. 4. When either an FPR or an human chromosome 19 in all the hybrids tested. All other FPRLl cDNAprobe was hybridized to the identical blot, three bands of identical size were revealed 1) a 2.8-kb band human chromosomes were excluded by a t least two discordant that hybridized strongly to FPR but weakly to FPRLlcDNA; hybrids for both probes (Tables I and 11). Therefore we map both the FPRl and the FPRLl genes to human chromosome 2) a 3.8-kb bandthat hybridized stronglytoFPRLlbut weakly to FPR cDNA; and 3) a 5.7-kb band that hybridized 19. The 5.7-kb fragment which was detected by cDNA probes weakly to bothprobes. This isa complementary cross-hybrid- corresponding to both of these genes in human DNA could izing banding pattern consistent with small, single-copy genes not be assigned to a chromosome since the signal was too that cross-hybridize to one another under high stringency weak to score in thehybrids. The colocalization of FPRl and conditions. This result is consistent with the cDNA cloning FPRLl genes to chromosome 19 suggestthat theirdifferences have arisen by divergent evolution after a relatively recent results described above. small size of the FPRl and The locus of the gene encoding FPR is designated by the gene duplicationevent.The intron content, which is a gene symbol FPRl. Thelocus of the gene encoding the human FPRLl genesreflectsalimited FPRLl receptor is designated by the gene symbol FPRLl. In common feature of G protein-coupled receptor genes (21-23).

7641

Peptide Chemoattractant Receptor Gene Family

6

FIG. 6. Structural evolution of the G protein-coupled receptor superfamily. An evolutionary dendrogram was generated by the method of parsimony after progressive alignment (18, 19) for the indicated protein sequences (CAMPRI, cyclic AMP receptor 1; TSH R, thyrotropin receptor;LHCG R, luteinizing hormone-chorionic gonadotropin receptor;GRI, gastric peptide receptor 1; AR, adrenergicreceptor; HT,serotonin receptor; PI, adenosine receptor; M, muscarinic acetylcholine receptor; SKR, substance K receptor; SPR, substance P receptor; I U R A , IL-8 receptor A; I U R B , IL-8 receptor B; PAF R, platelet activating factor receptor; mas, mas oncogene. The species source for each sequence is designated as Dd,Dictyostelium; d, dog; h, human; r, rat; ham, hamster; b, bovine; rub, rabbit; and CMV, cytomegalovirus. The relative evolutionary distance between any two sequences is depicted as the linear distance to the common branch point.

hemoattractant

Juvenile periodontitis is an autosomal dominant disorder characterized by gingival infections that lead to tooth loss. The gene for this disorder has been localized to chromosome 4 and linked to dentinogenesis imperfecta (24). In one patient with juvenile periodontitis, formyl peptide receptor proteins have been found detective (25). The assignment of FPRl and FPRLl to chromosome 19 excludes involvement of these genes in the form of the disorder linked to markers on chromosome 4. However, there could be another form of juvenile periodontitis due to mutations in FPRl or FPRLl. Structural Analysis of the FPRLl Receptor-Neutrophil cDNAs have been isolated that encode the receptor for the peptide chemoattractant C5a and two distinct receptor subtypes for the peptide chemoattractant IL-8 (26-28). Fig. 6 highlights the unusual propertiesof the FPRLlreceptor when analyzed in the context of these and otherG protein-coupled receptors. The evolutionary distance between any two members of the G protein-coupled receptor superfamily was determined from the primary amino acid sequences by the algorithm of Feng and Doolittle (18, 19) and is depicted as the linear distance to the common branch point. It is apparent that clusters of structurally conserved receptors have evolved and that the dominant featurethat links the receptors ineach cluster is a similar ligand specificity. A sufficiently large membership has been identified for several of these families

to show that a substantial divergence of primary structure due to differences in species, effector system coupling, and pharmacology can be tolerated without altering the basic ligand preference of individual members of the family. M1 and M2 muscarinic receptor subtypes, for example, couple to distinct G proteins and are only 43% identical, yet are both able to bind acetylcholine analogues (21). 01 and p2 adrenergic receptor subtypes are 54% identical, yet binding of isoproterenol and related catecholamines is conserved (22, 29). The substance K and substance P receptors are 48% identical, yet canbe cross-activated, albeit at reduced potency, by the otherneuropeptide (30,31).Within thepeptide chemoattractant receptor family, human and rabbit receptors have evolved that bind IL-8 but that are only 69% identical in primary structure (28, 32). The FPRLl receptor possesses 69% overall amino acid identity to FPR after the imposition of a single one amino acid gap. In this context the inability of the FPRLl receptor to interact with two structurally heterogeneous N-formyl peptides, both of which activate FPR, is highly anomalous. The predicted primary structures of the FPRLl receptor, FPR and both human IL-8 receptor subtypes are aligned in Fig. 7. As with all other G protein-coupled receptors, these sequences possess seven discrete hydrophobic domains that are predicted to reside within the plasma membrane, thereby

Chemoattractant Peptide

7642

Receptor Gene Family

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R T ~ H R A M R V I F A ~ I F L ~ L P ~ ~ T ~ - R T ~ I ~ ~ ~ I D ~ D A ~ I L G I L E S C ~ L I Y ~ I ~ G L ~ I ~ I - - - E G355 L I S ~ S L P

FIG. 7. Primary structural analysis of members of the peptide chemoattractant receptor family. Sequence sources are asfollows: FPRLlR (the FPRLlreceptor; this paper); FPR (6);I U R A (interleukin 8 receptor A, Ref. 28);and ZURB (interleukin 8 receptor B, Ref. 27). Vertical bars indicate identical residues for each adjacent sequence position. Solid lines indicate the locs$ion of predicted membrane spanning segments I through VI1 as determined by the Kyte-Doolittle algorithm (47). Open boxes designate predicted sites for N-linked glycosylation. Arabic numbers aboue the sequence blocks enumerate the FPRLl receptor sequence and are left justified. Dashes indicate gaps that were inserted to optimize the alignment. A

similar except for the sequence from TMS VI to VII, determinants of the ligand binding specificity for FPR and the FPRLl receptor may reside in thisregion. FPR and the FPRLlreceptor are each approximately 34% identical in overall sequence to the C5a receptor and 28% [LIRA RTLFKAHMGQKHRAMR identical to both IL-8 receptor subtypes. The sequence relatIIIIIIIIIIIIIIII edness of FPR and the FPRLl receptor with G proteinIL8RB RTLFKAHMGQKHRAMR B coupled receptors for completely different classes of ligands such as neurotransmitters or peptide hormones is 23% or less (8) and can be estimated for any given receptor from the dendrogram in Fig.6. Thusthe peptide chemoattractant receptor sequences constitute an evolutionary family within the superfamily of G protein-coupled receptors. Additional members of this family include the “orphan”receptors desigFIG. 8. Structural analysis of the thirdcytoplasmic loops of the peptide chemoattractant receptors. A, primary structural nated as US28 (33) and RDCl (23). Both of these sequences divergence of the predicted third cytoplasmic loops. The sequence possess a similar length and 28-35% overall amino acid sesegments between the limits of transmembrane domains V and VI quence identity to othermembers of the peptide chemoattracfor each designated protein are aligned. Sequence sources are the tant receptor family (Fig. 6). same as in Fig. 7. Vertical bars indicate identity between each adjacent All five deduced sequences of the peptide chemoattractant residue. B, secondary structure conservation of the predicted third receptors have an unusually short predicted third cytoplasmic cytoplasmic loops. The sequences of FPR, C5aR and IL-8 receptor loop between TMS V and VI, only 16 amino acids in length. aligned in panel A are arranged on ahelical wheel whoselong axis is perpendicular to the plane of the page (37). Charged residues are For other members of the receptor superfamily this loop has indicated by a circk enclosing the appropriate charge sign. Histidines been shown to contain sequences that determine the specificare assumed to be uncharged a t physiological pH. The arrangement ity of G protein coupling (34-36). Although the formyl peptide for the FPRLl receptor is similar to that of FPR. For the five receptor, the C5a receptor and theIL-8 receptor all couple to sequences, the charges are all positive and segregate to one face of pertussis toxin-sensitive G proteins, the predicted third cythe helix. toplasmic loops of the deduced receptor sequences possess no delimiting four extracellular and four cytoplasmic domains. significant primary structural similarity (Fig. &I). NevertheThe predicted extracellular amino-terminalsegment, the sec- less, when the sequences are arranged on a Shiffer-Edmundond extracellular loop between transmembrane segments son helical wheel diagram (37), a strongly amphipathic cati(TMS) IV and V, and thecarboxyl-terminal segment are only onic character is observed in each case (Fig. 8B). The premoderately conserved for each pair of receptor sequences dicted third cytoplasmic loop of the FPRLl receptor is 81% (approximately 30-45%). The sequence from the sixth TMS identical to thatof FPR andalso forms a strongly amphipathic to the first 8 residues of the seventh TMS of FPR and the cationic structure when examined on a helical wheel. A clue FPRLl receptor are also only 45% identical, whereas the IL- to thefunctional significance of this observation comes from 8 receptors are 95% identical in this segment. Since both the the properties of mastoparan, a 14-amino acid peptide comrapidity and location of the evolutionary changes between the ponent of wasp venom.Mastoparan is able to directly activate IL-8 receptors and between FPR and the FPRLl receptor are pertussis toxin-sensitive G proteins independently of surface FPRLl AKIHKKGMIKSSRPLR IIII I IIIIIIII FPR TKIHKQGLIKSSWLR I I C5aR LRTWSRRATRSTKTLK

Peptide Chemoattractant Receptor Gene Family

7643

(1990) 14. Mumhv. P. M.. Gallin, E. K., and Tiffany, - . H.L. . . J. Z m m k d 145,2227-2234 15. Francke. U.. Yane-Fene. T. L.. Brissenden. J. E.. and Ullrich. A. (1986)Coid SpGng furbor &mp. Quant.’Biol. 6 1,855-866 16. Hsieh, C.-L., Vogel, U. S., Dixon, R. A. F., and Francke, U.(1989) Somat. Cell Mol. Genet. 15,579-590 17. Devereux, J., Haeberli, P., and Smithies, 0.(1984)Nucleic Acids Res. 12,389-395 18. Feng, D.-F., and Doolittle, R. F. (1987)J. Mol. Euol. 25,351-360 19. Doolittle, R. F., and Feng, D.-F. (1990)Methods Enzymol. 183, 659-669 20. Kozak, M. (1984)Nucleic Acids Res. 12,857-872 21. Bonner, T.I., Young, A. C., Brann, M. R., and Buckley, N. J. (1988)Neuron 1,403-410 22. Kobilka, B. K., Dixon, R. A. F., Frielle, T., Dohlman, H. G., Bolanowski, M. A., Sigal, I. S., Yang-Feng, T. L., Francke, U., Caron, M. G., and Lefkowitz, R. J. (1987)Proc. Natl. Acad. Sci. U.S. A. 84,46-50 23. Libert, F., Parmentier, M., Lefort, A., Dinsart, C., Van Sande, J., Maenhaut, C., Simons, M.-J., Dumont, J. E., and Vassart, G. (1989)Science 244,569-572 24. Boughman, J. A., Halloran, S. L., Roulston, D., Schwartz, S., Suzuki, J. B., Weitkamp, L. R., Wenk, R. E., Wooten, R., and Cohen, M. M. (1986)J. Craniofacial Genet. Deu. Biol. 6, 341350 25. Perez, H. D., Kelly, E., Elfman, F., Armitage, G., and Winkler, J . (1991)J. Clin. Znuest. 87,971-976 26. Gerard, N. P., and Gerard, C. (1991)Nature 349,614-617 27. Holmes, W. E.,Lee, J., Kuang, W . - J . ,Rice, G. C., and Wood, W . I. (1991)Science 253,1278-1280 28. Murphy, P. M., and Tiffany, H. L. (1991)Science 253, 12801283 29. Frielle, T., Collins, S., Daniel, K. W., Caron, M. G., Lefkowitz, R. J., and Kobilka, B. K. (1987)Proc. Natl. Acad. Sci. U. S. A . Acknowledgments-We thank T. Let0 for the helical wheel dia84,7920-7924 grams, D.-F. Feng and R. F. Doolittle for the multisequence alignment 30. Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M., and Nakanishi, S . (1987)Nature 329,836-838 software, and H. L. Malech for critical reading of the manuscript. 31. Yokota, Y., Sasai, Y., Tanaka, K., Fujiwara, T., Tsuchida, K., REFERENCES Shigemoto, R., Kakizuka, A., Ohkubo, H., and Nakanishi, S. (1989)J. Biol. Chem. 264, 17649-17652 1. Snyderman, R., and Uhing, R. J. (1988)in Inflammation: Basic Principles and Clinical Correlates (Gallin, J. I., Goldstein, 1. M., 32. Thomas, K. M., Taylor, L., and Navarro, J . (1991)J. Biol. Chem. 266, 14839-14841 and Snyderman, R., eds) pp. 309-323,Raven Press, Ltd., New 33. Chee, M. S., Satchwell, S. C., Preddie, E., Weston, K. M., and York Barrell, B. G. (1990)Nature 344,774-777 2. Goldstein, I. M. (1988)in Inflammation: Basic Principles and 34. O’Dowd, B.F., Hnatowich, M., and Lefkowitz, R. J . (1991)Encycl. Clinical Correlates (Gallin, J . I., Goldstein, I. M., and SnyderHum. 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receptor activation (38, 39). Moreover, when mastoparan interacts with phospholipid membranes, it has been shown to form a cationic amphipathicCY helix (40). Taken together, this analysis strongly suggests that the FPRLl receptor is a G protein-coupled receptor that selectively links a distinct chemoattractantpeptide ligand to calcium mobilizing signal transduction pathways in the phagocyte. Host-derived N-formyl peptides have been identified in the mouse that bind tonon-classical major histocompatibility complex Class 1molecules and form the maternally transmitted antigen or Mta.Interestingly these N-formyl peptides are of mitochondrial origin, do not bind to neutrophils, and are not chemotactic for neutrophils (41, 42). Perhaps distinct host-derived or microbe-derived N-formyl peptides existthat bind specifically to the FPRLlreceptor but not to FPR or to Mta. To date, the specific structures of microbe-derived chemotactic N-formyl peptides have been defined for only three microorganisms (43-45). In conclusion, we have identified the FPRLl receptor, a functionally divergent structural homologue of FPR thatmay have arisen by a recent gene duplication on human chromosome 19. The immediate importance of this predicted protein is 8-fold. First, the sequence of the FPRLl receptor can be used to target the ligand binding site of FPR. Secondly, it indicates the existence of another asyet unidentified peptide ligand that may be capable of recruiting phagocytes to inflammatory foci.

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