Gene. 118 (1992) 303-304 0 1992 Elsevier Science Publishers
GENE
B.V. All rights reserved.
303
0378-l 119/92/$05.00
06554
Cloning of a cDNA encoding receptor of human neutrophils* (Inflammation;
chemotaxis,
G-protein;
a receptor
” Department of Medicine.
Received
Biosciences.
University
of Cahfomia
South San Francisco.
by J.D. Ernst: 23 January
to the formyl
peptide
HL-60 cells; bone marrow)
H. Daniel Perez a, Richard Holmes a, Edward
ogy. Berlex
related
San Francisco,
CA 94080,
Kelly”, John McClaryb
San Francisco,
CA 94143.
USA.
and William H. Andrews b
Tel. (415)206-8189
and h Department
of MolecularBiol-
USA
1992; Revised/Accepted:
23 February/2
March
1992; Received
at publishers:
13 April 1992
SUMMARY
We cloned a cDNA (RFP) encoding a receptor (RFP) related (70% overall nucleotide homology) to the formyl peptide receptor of human neutrophils (hFPR). RFP is a seven-transmembrane-domain receptor and its distribution is limited to myeloid cells. Domain sequence comparison with hFPR reveals highly conserved regions and provides clues to putative domains involved in ligand binding and receptor desensitization.
Binding of formyl peptides to specific receptors present on human neutrophils stimulates these cells to migrate in a directed fashion, secrete a portion of their lysosomal contents and generate oxygen-derived free radicals (Perez et al. 1991). The FPR is a seven-transmembrane-domain, G-protein-linked receptor (Boulay et al., 1990). To determine if more than one hFPR exists (Perez et al., 1991), we screened a single donor human bone marrow cDNA library in AgtlO (4.5 x lo5 plaques). As a probe, we used an FPR ORF and hybridization was performed under lowstringency conditions (Perez et al., 1992); 22 clones were isolated. The size of one clone (RFP) cDNA was 1.8 kb
Correspondence
to: Dr. H. Daniel
nia, San Francisco.
Perez, Box 0868, University CA 94143, USA. Tel. (415)206-8189;
of Califor-
Fax (415)648-8425. * On request,
the authors
the conclusions
reached
Abbreviations: receptor;
will supply detailed
experimental
aa, amino acid(s); bp, base pair(s);
FPR, cDNA
evidence
for
in this Brief Note.
encoding
or 1000 bp; nt, nucleotide(s); related to FPR; RFP, cDNA mains.
FPR; formyl peptide
FPR, hFPR, human FPR; kb, kilobase ORF, open reading frame; RFP, receptor coding for RFP; TM, transmembrane do-
(FPR = 1.45 kb). Northern-blot analysis using mRNA derived from differentiated HL-60 cells (Perez et al., 1992) demonstrated that RFP hybridized strongly to a 1.8-kb band indicating that the cDNA was full length. Expression of RFP mRNA must be restricted to myeloid cells, since no message could be detected using mRNA from either human lymphocytes, lung, brain, heart, pancreas, or testes. COS7 cells transfected with RFP (using the expression vector pSG5) failed to bind formyl peptide, human C5a, recombinant interleukin 8 or leukotriene B,. Computer analysis of RFP cDNA sequence revealed 70% nt homology overall with FPR within the ORF. RFP coded for a 351-aa, seventransmembrane-domain receptor (RFP; Kyte and Doolittle, 1982; Fig. 1). Homology comparison of each particular domain (Table I) revealed that, within the extracellular domains, domain II had 90% homology and a Cyscontaining region in domain III (aa 175-180) was completely conserved. Computer modeling suggests that a disulfide bridge between Cys9’ (extracellular domain 2) and CYS”~ (extracellular domain 3) may contribute to form a ligand-binding pocket in FPR. These observations suggest that the ligand for RFP is a small peptide also. The cytoplasmic domains were highly conserved, indicating that they use similar (if not identical) signal transduction ma-
304 Hydrophilicity
Window
Size =
7
Scale
5.00 4.00 D .t: 3.00 .z 2.00 E 1.00 CL 0.00 e -1.00 0 -2.00 z -3.00 -4.00 -5.00 50
RFP
1
100
150
= Kyle-Doolittle
200
250
300
TM I TM II TM IIIMETNFSTPLNEYEEVSYESAG~VLRILPLWLGVTFVLGVLGNGLVIWVAGF~RTVTTICYLNLALADFSFTATLPFLIVSMAMGEKWPFGWFLCKLIHIWDINLFGSVFLIGFIA **** * * * **** c * * ********************** ***** **** *** ** t*** * ** ********* ************ ** l
hFPR
350
l
1 METNSSLPTNISGGTPAVSAGYLFLDIITYLVFAVTFVLGVLGNGLVIWVAGF~HTVTTISYLNLAVADFCFTSTLPFFMVRKAMGGHWPFGWFLCKFFTIVDINLFGSVFLIALIA TM IV TM V 121 LDRCICVLHPVWAQNHRTVSLAMKVIVGPWILALVLTLPVFLFLTTVTIPNGDTYCTFNFASWGGTPEERLKVAITMLTARGIIRFVIGFSLPMSIVAICYGLIAAKIHKKGMIKSSRPL **** ******* ********* ** *** ** ***** *** * ***** * ** ** *x* ****** **** ****** ***** **** * ******
RFP
l
hFPR
l
l
121 LDRCVCVLHPWJTQNHRTVSLAKKVIIGPWVMALLLTLPVIIRVTTVPGKTGTVACTFNFSPWTNDPKERINVAVAMLTVRGIIRFIIGFSAPMSIVAVSYGLIATKIHKQGLIKSSRPL _TM VI TM VII 241 RVLTAWASFFICWFPFQLVALLGTVWLKEMLFYGKYKIIDILVNPTSSLAFFNSCLNPMLYVFVGQDFRERLIHSLPTSLERALSEDSAPTN~AANSASPPAETELQAM *** * * ** * * *** ** * * ** * * t ** ************t** *******t** ** ***** *** * *** *f * ** **** 241 RVLSFVAAAFFLCWSPYQWALIATVRIRELLQ GMYKEIGIAVDVTSALAFFNSCLNPMLWFMGQDFRERLIHALPASLERALTEDSTQTS~ATNSTLPSAEVELQAK
RFP
l
hFPR
Fig. 1. Hydrophilicity plasmic)
domains
l
and aa sequence
and negative
of RFP and FPR. Asterisks
TABLE
l
encoded
values represent
represent
l
by RFP. (Top) Hydrophilicity
plot of RFP.
hydrophobic
domains.
aa identities.
TM I-VII
(transmembrane) are indicated.
Positive values represent
(Bottom)
GenBankiEMBL
Alignment
accession
hydrophilic
of the deduced
(extracellular
aa sequences
or cyto-
of the ORFs
No.: X63819.
I
Homology
comparison
Domains”
between
ORFs
by RFP and FPR Transmembrane
Cytoplasmic
Extracellular Homology
encoded
(O,,)h
I
47
80
65
II
90
83
69
III
50
83
70
IV V
41
74’ -
60 72
VI
-
-
60
VII
-
-
16
’ Domains membrane
refer to their topographical (Kyte and Doolittle,
h aa identities. ’ Conserved Ser/Thr
residues
RFP
EDSAPTNDTAANSA
hFPR
ED;TQ:SD;ATN;T
location
with respect
REFERENCES to the cell
1982). (asterisks)
in aa
in receptor desensitization via serine/threonine kinases. Thus, RFP codes for a receptor for an, as yet, unidentified ligand. This ligand may play an important role in the mediation of the acute inflammatory response. Current efforts are directed at identifying the ligand for RFP. Supported by National Institutes of Health grants AR28566 and AI-28290 to H.D.P.
328-341:
Boulay, F., Tardiff, M., Brouchon, L. and Vignais, P.: The human n-formyl peptide receptor. Characterization of two cDNA isolates and evidence for a new subfamily of G-protein istry 29 (1990) 11123-11133. Kyte, J. and Doolittle, pathic character
R.F.: A simple method
of a protein.
Perez, H.D.. Kelly, E., Elfman,
chinery. Ofinterest was the homology observed in the fourth cytoplasmic domain (aa 328-341). A conserved stretch of Ser and Thr residues were present, each separated by 2-3 aa (Table I). Its location suggests that they play a role
coupled
tive polymorphonuclear
receptors.
for displaying
Biochemthe hydro-
J. Mol. Biol. 157 (1982) 105-132. F., Armitage,
leukocyte
G. and Winkler. J.: Defec-
formyl peptidc
receptor(s)
nile periodontitis. J. Clin. Invest. 87 (1991) 971-976. Perez, H.D., Holmes, R. and Kelly, E.: Regulation of formyl receptor expression and its mRNA levels during HL-60 cells. J. Biol. Chem. 267 (1992) 358-363.
in juvcpeptidc
differentiation
of
GENE
B.V. All rights reserved.
303
0378-l 119/92/$05.00
06554
Cloning of a cDNA encoding receptor of human neutrophils* (Inflammation;
chemotaxis,
G-protein;
a receptor
” Department of Medicine.
Received
Biosciences.
University
of Cahfomia
South San Francisco.
by J.D. Ernst: 23 January
to the formyl
peptide
HL-60 cells; bone marrow)
H. Daniel Perez a, Richard Holmes a, Edward
ogy. Berlex
related
San Francisco,
CA 94080,
Kelly”, John McClaryb
San Francisco,
CA 94143.
USA.
and William H. Andrews b
Tel. (415)206-8189
and h Department
of MolecularBiol-
USA
1992; Revised/Accepted:
23 February/2
March
1992; Received
at publishers:
13 April 1992
SUMMARY
We cloned a cDNA (RFP) encoding a receptor (RFP) related (70% overall nucleotide homology) to the formyl peptide receptor of human neutrophils (hFPR). RFP is a seven-transmembrane-domain receptor and its distribution is limited to myeloid cells. Domain sequence comparison with hFPR reveals highly conserved regions and provides clues to putative domains involved in ligand binding and receptor desensitization.
Binding of formyl peptides to specific receptors present on human neutrophils stimulates these cells to migrate in a directed fashion, secrete a portion of their lysosomal contents and generate oxygen-derived free radicals (Perez et al. 1991). The FPR is a seven-transmembrane-domain, G-protein-linked receptor (Boulay et al., 1990). To determine if more than one hFPR exists (Perez et al., 1991), we screened a single donor human bone marrow cDNA library in AgtlO (4.5 x lo5 plaques). As a probe, we used an FPR ORF and hybridization was performed under lowstringency conditions (Perez et al., 1992); 22 clones were isolated. The size of one clone (RFP) cDNA was 1.8 kb
Correspondence
to: Dr. H. Daniel
nia, San Francisco.
Perez, Box 0868, University CA 94143, USA. Tel. (415)206-8189;
of Califor-
Fax (415)648-8425. * On request,
the authors
the conclusions
reached
Abbreviations: receptor;
will supply detailed
experimental
aa, amino acid(s); bp, base pair(s);
FPR, cDNA
evidence
for
in this Brief Note.
encoding
or 1000 bp; nt, nucleotide(s); related to FPR; RFP, cDNA mains.
FPR; formyl peptide
FPR, hFPR, human FPR; kb, kilobase ORF, open reading frame; RFP, receptor coding for RFP; TM, transmembrane do-
(FPR = 1.45 kb). Northern-blot analysis using mRNA derived from differentiated HL-60 cells (Perez et al., 1992) demonstrated that RFP hybridized strongly to a 1.8-kb band indicating that the cDNA was full length. Expression of RFP mRNA must be restricted to myeloid cells, since no message could be detected using mRNA from either human lymphocytes, lung, brain, heart, pancreas, or testes. COS7 cells transfected with RFP (using the expression vector pSG5) failed to bind formyl peptide, human C5a, recombinant interleukin 8 or leukotriene B,. Computer analysis of RFP cDNA sequence revealed 70% nt homology overall with FPR within the ORF. RFP coded for a 351-aa, seventransmembrane-domain receptor (RFP; Kyte and Doolittle, 1982; Fig. 1). Homology comparison of each particular domain (Table I) revealed that, within the extracellular domains, domain II had 90% homology and a Cyscontaining region in domain III (aa 175-180) was completely conserved. Computer modeling suggests that a disulfide bridge between Cys9’ (extracellular domain 2) and CYS”~ (extracellular domain 3) may contribute to form a ligand-binding pocket in FPR. These observations suggest that the ligand for RFP is a small peptide also. The cytoplasmic domains were highly conserved, indicating that they use similar (if not identical) signal transduction ma-
304 Hydrophilicity
Window
Size =
7
Scale
5.00 4.00 D .t: 3.00 .z 2.00 E 1.00 CL 0.00 e -1.00 0 -2.00 z -3.00 -4.00 -5.00 50
RFP
1
100
150
= Kyle-Doolittle
200
250
300
TM I TM II TM IIIMETNFSTPLNEYEEVSYESAG~VLRILPLWLGVTFVLGVLGNGLVIWVAGF~RTVTTICYLNLALADFSFTATLPFLIVSMAMGEKWPFGWFLCKLIHIWDINLFGSVFLIGFIA **** * * * **** c * * ********************** ***** **** *** ** t*** * ** ********* ************ ** l
hFPR
350
l
1 METNSSLPTNISGGTPAVSAGYLFLDIITYLVFAVTFVLGVLGNGLVIWVAGF~HTVTTISYLNLAVADFCFTSTLPFFMVRKAMGGHWPFGWFLCKFFTIVDINLFGSVFLIALIA TM IV TM V 121 LDRCICVLHPVWAQNHRTVSLAMKVIVGPWILALVLTLPVFLFLTTVTIPNGDTYCTFNFASWGGTPEERLKVAITMLTARGIIRFVIGFSLPMSIVAICYGLIAAKIHKKGMIKSSRPL **** ******* ********* ** *** ** ***** *** * ***** * ** ** *x* ****** **** ****** ***** **** * ******
RFP
l
hFPR
l
l
121 LDRCVCVLHPWJTQNHRTVSLAKKVIIGPWVMALLLTLPVIIRVTTVPGKTGTVACTFNFSPWTNDPKERINVAVAMLTVRGIIRFIIGFSAPMSIVAVSYGLIATKIHKQGLIKSSRPL _TM VI TM VII 241 RVLTAWASFFICWFPFQLVALLGTVWLKEMLFYGKYKIIDILVNPTSSLAFFNSCLNPMLYVFVGQDFRERLIHSLPTSLERALSEDSAPTN~AANSASPPAETELQAM *** * * ** * * *** ** * * ** * * t ** ************t** *******t** ** ***** *** * *** *f * ** **** 241 RVLSFVAAAFFLCWSPYQWALIATVRIRELLQ GMYKEIGIAVDVTSALAFFNSCLNPMLWFMGQDFRERLIHALPASLERALTEDSTQTS~ATNSTLPSAEVELQAK
RFP
l
hFPR
Fig. 1. Hydrophilicity plasmic)
domains
l
and aa sequence
and negative
of RFP and FPR. Asterisks
TABLE
l
encoded
values represent
represent
l
by RFP. (Top) Hydrophilicity
plot of RFP.
hydrophobic
domains.
aa identities.
TM I-VII
(transmembrane) are indicated.
Positive values represent
(Bottom)
GenBankiEMBL
Alignment
accession
hydrophilic
of the deduced
(extracellular
aa sequences
or cyto-
of the ORFs
No.: X63819.
I
Homology
comparison
Domains”
between
ORFs
by RFP and FPR Transmembrane
Cytoplasmic
Extracellular Homology
encoded
(O,,)h
I
47
80
65
II
90
83
69
III
50
83
70
IV V
41
74’ -
60 72
VI
-
-
60
VII
-
-
16
’ Domains membrane
refer to their topographical (Kyte and Doolittle,
h aa identities. ’ Conserved Ser/Thr
residues
RFP
EDSAPTNDTAANSA
hFPR
ED;TQ:SD;ATN;T
location
with respect
REFERENCES to the cell
1982). (asterisks)
in aa
in receptor desensitization via serine/threonine kinases. Thus, RFP codes for a receptor for an, as yet, unidentified ligand. This ligand may play an important role in the mediation of the acute inflammatory response. Current efforts are directed at identifying the ligand for RFP. Supported by National Institutes of Health grants AR28566 and AI-28290 to H.D.P.
328-341:
Boulay, F., Tardiff, M., Brouchon, L. and Vignais, P.: The human n-formyl peptide receptor. Characterization of two cDNA isolates and evidence for a new subfamily of G-protein istry 29 (1990) 11123-11133. Kyte, J. and Doolittle, pathic character
R.F.: A simple method
of a protein.
Perez, H.D.. Kelly, E., Elfman,
chinery. Ofinterest was the homology observed in the fourth cytoplasmic domain (aa 328-341). A conserved stretch of Ser and Thr residues were present, each separated by 2-3 aa (Table I). Its location suggests that they play a role
coupled
tive polymorphonuclear
receptors.
for displaying
Biochemthe hydro-
J. Mol. Biol. 157 (1982) 105-132. F., Armitage,
leukocyte
G. and Winkler. J.: Defec-
formyl peptidc
receptor(s)
nile periodontitis. J. Clin. Invest. 87 (1991) 971-976. Perez, H.D., Holmes, R. and Kelly, E.: Regulation of formyl receptor expression and its mRNA levels during HL-60 cells. J. Biol. Chem. 267 (1992) 358-363.
in juvcpeptidc
differentiation
of
