Gerard, Craig

Gerard, Norma P.

Frontiers in Immunology

Norma P. Gerard

Craig Gerard

Frontiers - Publisher Connector

OPINION
published: 01 September 2015
doi: 10.3389/fimmu.2015.00445
Edited by:
Bernhard Moser,
Cardiff University, UK
Reviewed by:
Israel Charo,
ChemoCentryx, Inc., USA
*Correspondence:
Craig Gerard
craig.gerard@childrens.harvard.edu
Specialty section:
This article was submitted to
Chemoattractants, a section of the
journal Frontiers in Immunology
Received: 29 June 2015
Accepted: 17 August 2015
Published: 01 September 2015
Citation:
Gerard NP and Gerard C (2015)
Cloning of the human C5a
anaphylatoxin receptor, and more.
Front. Immunol. 6:445.
doi: 10.3389/fimmu.2015.00445
Cloning of the human C5a
anaphylatoxin receptor, and more
Norma P. Gerard1,2 and Craig Gerard1*
1 Ina Sue Perlmutter Laboratory, Division of Respiratory Diseases, Department of Medicine, Children’s Hospital, Harvard Medical
School, Boston, MA, USA, 2 Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA
Keywords: C5aR/C5R1, 7-TMS receptors, complement anaphylatoxin, C5a, G protein coupled receptor
The initial observation in 1973 that the complement C5 activation product, C5a, has the ability
to stimulate human neutrophils led to the concept of a specific C5a receptor (1). In the mid-
1986, a groundbreaking paper published in Nature announced the cloning of the beta-adrenergic
receptor, and for the first time established seven transmembrane (7TM)G protein-coupled receptors
as members of the rhodopsin superfamily (2). Almost simultaneously, Feltner and colleagues
demonstrated that the FMLP, C5a, and LTB4 activities on rabbit neutrophils could be inhibited
by pertussis toxin, indicating coupling to GTP binding proteins (3). In 1987, Masu and colleagues
used an oocyte expression cloning system to isolate a cDNA encoding the neuropeptide substance
K receptor; and later, the same group identified the related substance P receptor (4, 5).
In 1989, we realized that the pertussis sensitivity of the fMLP, C5a, and LTB4 receptors suggested
that they would also be members of the rhodopsin superfamily. When we aligned the handful
of structures for the then known 7TM receptors (adrenergic, serotonin, dopamine, FSH/LH, and
substance P and K receptors), we recognized homologies in both the transmembrane segments
and intracellular loops, which presumably facilitated interactions with G proteins. This observation
actually presented us with an opportunity to attempt to clone receptors by homology to the
superfamily as “orphan receptors.”
We constructed an antisense oligonucleotide withminimal degeneracy that encompassed a highly
conserved NPXXY motif in the seventh transmembrane segment of the known rhodopsin family
members. In order to enrich in C5a receptors, we took advantage of the fact that the receptors
were induced by cyclic-AMP in U937 cells, and in retinoic acid differentiated human HL60 cells.
By summer of 1990, we had isolated ~20 cDNAs using this approach from the cAMP induced
U937 cell library. About half of these clones were an identical cDNA that we named NPIIY-18.
Using this as a probe, we demonstrated that NPIIY-18 recognized a ~2.2 kb mRNA only in cAMP
differentiated U937 cells. Northern blot analyses showed that NPIIY-18 was present only in cells
known to express the C5a receptor. As NPIIY-18 was not a full-length cDNA, we then probed the
retinoic acid differentiated HL60 cell library. We isolated a full-length DNA from this library that
encoded a 7TM receptor with 25% homology to the substance K receptor and 35% homology to
the human fMLP receptor (FPR1), which was cloned by Francois Boulay in May 1990 (6). When
expressed in COS cells, we showed that NPIIY-18 encoded a high-affinity receptor for human C5a
(Figure 1) (7). This work was accepted for publication in Nature in December 1990. In the summary
paragraph of this manuscript, we pointed out that our approach should be helpful to clone the
receptors for the leukotrienes, platelet activating factor, interleukin-8, and adenosine receptors as
these are all present on cAMP differentiated U937 cells. Almost simultaneously, Francois Boulay
confirmed our identification of the human C5aR, which his group accomplished by expression
cloning of differentiated HL-60 cells (8). His work was published in March 1991, some 4months
after ours.
In November 1990, within months of the identification of the human FPR1, Thomas et al.
reported the cloning of the rabbit receptor for fMLP, F3R (9), which had almost no significant
homology to the human receptor. This was troubling, because we knew that when the ligand
Frontiers in Immunology | www.frontiersin.org September 2015 | Volume 6 | Article 4451
Gerard and Gerard Cloning the C5a anaphylatoxin receptor
FIGURE 1 | Final steps in cloning and identifying the human C5a receptor. (A) Tertiary screen of clone NPIIY-18 using 32P-labeled oligonucleotide probe.
(B) NPIIY-18 was transfected into COS7 cells and tested for binding of 125 I-C5a under competitive binding conditions. (C) The resulting binding isotherm and
Scatchard analysis proving the clone’s identity.
was identical from species to species, the receptors were generally
highly conserved. Thus, the adrenergic, dopamine, serotonin, and
histamine receptors are >90% identical across species. Curiously,
the cDNA was reported to bind the FPR1 radioligand and trans-
duce calcium transients. Because the expression of the claimed
rabbit F3R formyl peptide receptor was restricted to neutrophils,
we wondered if, in fact, the Navarro lab had misidentified an
interleukin-8 receptor. One of us (Craig Gerard) actually traveled
to the Navarro lab to obtain the F3R cDNA to establish a collabo-
ration and test its identity as a receptor for IL-8. At that time, there
was no radioligand IL-8 commercially available. Henry Showell, of
Pfizer Central Research, was able to provide us with a custom iod-
inated IL-8, which we demonstrated to bind F3R. Unfortunately,
we did not have sufficient quantities of the reagent to perform
comprehensive studies to publish our findings. We disclosed our
result to Javier Navarro, but were left in silence. Unbeknownst to
us, DanWitt, at Repligen, had reportedly approached the Navarro
lab with a similar idea. Thomas et al. went on to publish F3R
as an IL8 receptor, without retracting the previous paper (10).
During this time, Tom Schall and I met at a FASEB meeting with
Phil Murphy, and suggested to him that he use F3R to clone a
human homolog from HL60 cells and test it against IL8. The
landmark Murphy and Tiffany paper resulted (11). Phil offered
one of us (Craig Gerard) coauthorship for the helpful suggestion
but because of intellectual property concerns at our institution, we
requested an acknowledgment instead.
Over the next decade, the orphan receptor approach led to
the identification of a wide variety of chemoattractant recep-
tors, including most of the chemokine receptor system. The
most notable events in the area of chemokines occurred when
CXCR4 and CCR5 were identified as HIV coreceptors. It was
known from the work of Ed Berger that CXCR4 was the obli-
gate coreceptor with CD4 for laboratory-adapted strains of HIV
(12). However, the wild type, the so-called macrophage tropic
strain used an unknown coreceptor. In December 1995, it was
reported in the New York Times that the Gallo laboratory had
identified Mip1α, Mip1β, and RANTES as substances that inhib-
ited HIV infections (13). Coincidentally, at the Fourth Inter-
national Chemokine Symposium, held June 27–30, 1995, Izzy
Charo described an orphan receptor identified as CCR5, linked
to CCR2, which bound Mip1α, Mip1β, and RANTES (14). Thus,
an international race began as five chemokine labs partnered
with HIV labs to prove the hypothesis that CCR5 was the HIV
coreceptor.
References
1. Goldstein I, Hoffstein S, Gallin J, Weissmann G. Mechanisms of lysosomal
enzyme release from human leukocytes: microtubule assembly and membrane
fusion induced by a component of complement. Proc Natl Acad Sci U S A (1973)
70:2916–20. doi:10.1073/pnas.70.10.2916
2. Dixon RA, Kobilka BK, Strader DJ, Benovic JL, Dohlman HG, Frielle
T, et al. Cloning of the gene and cDNA for mammalian beta-adrenergic
Frontiers in Immunology | www.frontiersin.org September 2015 | Volume 6 | Article 4452
Gerard and Gerard Cloning the C5a anaphylatoxin receptor
receptor and homology with rhodopsin. Nature (1986) 321:75–9. doi:10.1038/
321075a0
3. Feltner DE, Smith RH,MarascoWA. Characterization of the plasmamembrane
bound GTPase from rabbit neutrophils. I. Evidence for an Ni-like protein
coupled to the formyl peptide, C5a, and leukotriene B4 chemotaxis receptors.
J Immunol (1986) 137:1961–70.
4. Masu Y, Nakayama K, Tamaki H, Harada Y, Kuno M, Nakanishi S. cDNA
cloning of bovine substance-K receptor through oocyte expression system.
Nature (1987) 329:836–8. doi:10.1038/329836a0
5. Yokota Y, Sasai Y, Tanaka K, Fujiwara T, Tsuchida K, Shigemoto R, et al.
Molecular characterization of a functional cDNA for rat substance P receptor.
J Biol Chem (1989) 264:17649–52.
6. Boulay F, Tardif M, Brouchon L, Vignais P. The human N-formylpeptide recep-
tor. Characterization of two cDNA isolates and evidence for a new subfamily
of G-protein-coupled receptors. Biochemistry (1990) 29:11123–33. doi:10.1021/
bi00502a016
7. Gerard NP, Gerard C. The chemotactic receptor for human C5a anaphylatoxin.
Nature (1991) 349:614–7. doi:10.1038/349614a0
8. Boulay F, Mery L, Tardif M, Brouchon L, Vignais P. Expression cloning of
a receptor for C5a anaphylatoxin on differentiated HL-60 cells. Biochemistry
(1991) 30:2993–9. doi:10.1021/bi00226a002
9. Thomas KM, Pyun HY, Navarro J. Molecular cloning of the fMet-Leu-Phe
receptor from neutrophils. J Biol Chem (1990) 265:20061–4.
10. Thomas KM, Taylor L, Navarro J. The interleukin-8 receptor is encoded by a
neutrophil-specific cDNA clone, F3R. J Biol Chem (1991) 266:14839–41.
11. Murphy PM, Tiffany HL. Cloning of complementary DNA encoding a func-
tional human interleukin-8 receptor. Science (1991) 253:1280–3. doi:10.1126/
science.1891716
12. Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: functional
cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science
(1996) 272:872–7. doi:10.1126/science.272.5263.872
13. Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P. Identifi-
cation of RANTES, MIP-1 alpha, andMIP-1 beta as the major HIV-suppressive
factors produced by CD8+ T cells. Science (1995) 270:1811–5. doi:10.1126/
science.270.5243.1811
14. Raport CJ, Gosling J, Schweickart VL, Gray PW, Charo IF. Molecular cloning
and functional characterization of a novel human CC chemokine recep-
tor (CCR5) for RANTES, MIP-1beta, and MIP-1alpha. J Biol Chem (1996)
271:17161–6. doi:10.1074/jbc.271.29.17161
Conflict of Interest Statement: The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Copyright © 2015 Gerard and Gerard. This is an open-access article distributed under
the terms of the Creative Commons Attribution License (CC BY). The use, distribution
or reproduction in other forums is permitted, provided the original author(s) or
licensor are credited and that the original publication in this journal is cited, in
accordance with accepted academic practice. No use, distribution or reproduction is
permitted which does not comply with these terms.
Frontiers in Immunology | www.frontiersin.org September 2015 | Volume 6 | Article 4453


Cloning of the Human C5a Anaphylatoxin Receptor, and More

Harvard University - DASH 

Cloning of the Human C5aAnaphylatoxin Receptor, and MoreThe Harvard community has made thisarticle openly available.  Please share  howthis access benefits you. Your story mattersCitation Gerard, Norma P., and Craig Gerard. 2015. “Cloning of the HumanC5a Anaphylatoxin Receptor, and More.” Frontiers in Immunology 6(1): 445. doi:10.3389/fimmu.2015.00445. http://dx.doi.org/10.3389/fimmu.2015.00445.Published Version doi:10.3389/fimmu.2015.00445Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:22857042Terms of Use This article was downloaded from Harvard University’s DASHrepository, and is made available under the terms and conditionsapplicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAAOPINIONpublished: 01 September 2015doi: 10.3389/fimmu.2015.00445Edited by:Bernhard Moser,Cardiff University, UKReviewed by:Israel Charo,ChemoCentryx, Inc., USA*Correspondence:Craig Gerardcraig.gerard@childrens.harvard.eduSpecialty section:This article was submitted toChemoattractants, a section of thejournal Frontiers in ImmunologyReceived: 29 June 2015Accepted: 17 August 2015Published: 01 September 2015Citation:Gerard NP and Gerard C (2015)Cloning of the human C5aanaphylatoxin receptor, and more.Front. Immunol. 6:445.doi: 10.3389/fimmu.2015.00445Cloning of the human C5aanaphylatoxin receptor, and moreNorma P. Gerard1,2 and Craig Gerard1*1 Ina Sue Perlmutter Laboratory, Division of Respiratory Diseases, Department of Medicine, Children’s Hospital, Harvard MedicalSchool, Boston, MA, USA, 2 Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USAKeywords: C5aR/C5R1, 7-TMS receptors, complement anaphylatoxin, C5a, G protein coupled receptorThe initial observation in 1973 that the complement C5 activation product, C5a, has the abilityto stimulate human neutrophils led to the concept of a specific C5a receptor (1). In the mid-1986, a groundbreaking paper published in Nature announced the cloning of the beta-adrenergicreceptor, and for the first time established seven transmembrane (7TM)G protein-coupled receptorsas members of the rhodopsin superfamily (2). Almost simultaneously, Feltner and colleaguesdemonstrated that the FMLP, C5a, and LTB4 activities on rabbit neutrophils could be inhibitedby pertussis toxin, indicating coupling to GTP binding proteins (3). In 1987, Masu and colleaguesused an oocyte expression cloning system to isolate a cDNA encoding the neuropeptide substanceK receptor; and later, the same group identified the related substance P receptor (4, 5).In 1989, we realized that the pertussis sensitivity of the fMLP, C5a, and LTB4 receptors suggestedthat they would also be members of the rhodopsin superfamily. When we aligned the handfulof structures for the then known 7TM receptors (adrenergic, serotonin, dopamine, FSH/LH, andsubstance P and K receptors), we recognized homologies in both the transmembrane segmentsand intracellular loops, which presumably facilitated interactions with G proteins. This observationactually presented us with an opportunity to attempt to clone receptors by homology to thesuperfamily as “orphan receptors.”We constructed an antisense oligonucleotide withminimal degeneracy that encompassed a highlyconserved NPXXY motif in the seventh transmembrane segment of the known rhodopsin familymembers. In order to enrich in C5a receptors, we took advantage of the fact that the receptorswere induced by cyclic-AMP in U937 cells, and in retinoic acid differentiated human HL60 cells.By summer of 1990, we had isolated ~20 cDNAs using this approach from the cAMP inducedU937 cell library. About half of these clones were an identical cDNA that we named NPIIY-18.Using this as a probe, we demonstrated that NPIIY-18 recognized a ~2.2 kb mRNA only in cAMPdifferentiated U937 cells. Northern blot analyses showed that NPIIY-18 was present only in cellsknown to express the C5a receptor. As NPIIY-18 was not a full-length cDNA, we then probed theretinoic acid differentiated HL60 cell library. We isolated a full-length DNA from this library thatencoded a 7TM receptor with 25% homology to the substance K receptor and 35% homology tothe human fMLP receptor (FPR1), which was cloned by Francois Boulay in May 1990 (6). Whenexpressed in COS cells, we showed that NPIIY-18 encoded a high-affinity receptor for human C5a(Figure 1) (7). This work was accepted for publication in Nature in December 1990. In the summaryparagraph of this manuscript, we pointed out that our approach should be helpful to clone thereceptors for the leukotrienes, platelet activating factor, interleukin-8, and adenosine receptors asthese are all present on cAMP differentiated U937 cells. Almost simultaneously, Francois Boulayconfirmed our identification of the human C5aR, which his group accomplished by expressioncloning of differentiated HL-60 cells (8). His work was published in March 1991, some 4monthsafter ours.In November 1990, within months of the identification of the human FPR1, Thomas et al.reported the cloning of the rabbit receptor for fMLP, F3R (9), which had almost no significanthomology to the human receptor. This was troubling, because we knew that when the ligandFrontiers in Immunology | www.frontiersin.org September 2015 | Volume 6 | Article 4451Gerard and Gerard Cloning the C5a anaphylatoxin receptorFIGURE 1 | Final steps in cloning and identifying the human C5a receptor. (A) Tertiary screen of clone NPIIY-18 using 32P-labeled oligonucleotide probe.(B) NPIIY-18 was transfected into COS7 cells and tested for binding of 125 I-C5a under competitive binding conditions. (C) The resulting binding isotherm andScatchard analysis proving the clone’s identity.was identical from species to species, the receptors were generallyhighly conserved. Thus, the adrenergic, dopamine, serotonin, andhistamine receptors are >90% identical across species. Curiously,the cDNA was reported to bind the FPR1 radioligand and trans-duce calcium transients. Because the expression of the claimedrabbit F3R formyl peptide receptor was restricted to neutrophils,we wondered if, in fact, the Navarro lab had misidentified aninterleukin-8 receptor. One of us (Craig Gerard) actually traveledto the Navarro lab to obtain the F3R cDNA to establish a collabo-ration and test its identity as a receptor for IL-8. At that time, therewas no radioligand IL-8 commercially available. Henry Showell, ofPfizer Central Research, was able to provide us with a custom iod-inated IL-8, which we demonstrated to bind F3R. Unfortunately,we did not have sufficient quantities of the reagent to performcomprehensive studies to publish our findings. We disclosed ourresult to Javier Navarro, but were left in silence. Unbeknownst tous, DanWitt, at Repligen, had reportedly approached the Navarrolab with a similar idea. Thomas et al. went on to publish F3Ras an IL8 receptor, without retracting the previous paper (10).During this time, Tom Schall and I met at a FASEB meeting withPhil Murphy, and suggested to him that he use F3R to clone ahuman homolog from HL60 cells and test it against IL8. Thelandmark Murphy and Tiffany paper resulted (11). Phil offeredone of us (Craig Gerard) coauthorship for the helpful suggestionbut because of intellectual property concerns at our institution, werequested an acknowledgment instead.Over the next decade, the orphan receptor approach led tothe identification of a wide variety of chemoattractant recep-tors, including most of the chemokine receptor system. Themost notable events in the area of chemokines occurred whenCXCR4 and CCR5 were identified as HIV coreceptors. It wasknown from the work of Ed Berger that CXCR4 was the obli-gate coreceptor with CD4 for laboratory-adapted strains of HIV(12). However, the wild type, the so-called macrophage tropicstrain used an unknown coreceptor. In December 1995, it wasreported in the New York Times that the Gallo laboratory hadidentified Mip1α, Mip1β, and RANTES as substances that inhib-ited HIV infections (13). Coincidentally, at the Fourth Inter-national Chemokine Symposium, held June 27–30, 1995, IzzyCharo described an orphan receptor identified as CCR5, linkedto CCR2, which bound Mip1α, Mip1β, and RANTES (14). Thus,an international race began as five chemokine labs partneredwith HIV labs to prove the hypothesis that CCR5 was the HIVcoreceptor.References1. Goldstein I, Hoffstein S, Gallin J, Weissmann G. Mechanisms of lysosomalenzyme release from human leukocytes: microtubule assembly and membranefusion induced by a component of complement. Proc Natl Acad Sci U S A (1973)70:2916–20. doi:10.1073/pnas.70.10.29162. Dixon RA, Kobilka BK, Strader DJ, Benovic JL, Dohlman HG, FrielleT, et al. Cloning of the gene and cDNA for mammalian beta-adrenergicFrontiers in Immunology | www.frontiersin.org September 2015 | Volume 6 | Article 4452Gerard and Gerard Cloning the C5a anaphylatoxin receptorreceptor and homology with rhodopsin. Nature (1986) 321:75–9. doi:10.1038/321075a03. Feltner DE, Smith RH,MarascoWA. Characterization of the plasmamembranebound GTPase from rabbit neutrophils. I. Evidence for an Ni-like proteincoupled to the formyl peptide, C5a, and leukotriene B4 chemotaxis receptors.J Immunol (1986) 137:1961–70.4. Masu Y, Nakayama K, Tamaki H, Harada Y, Kuno M, Nakanishi S. cDNAcloning of bovine substance-K receptor through oocyte expression system.Nature (1987) 329:836–8. doi:10.1038/329836a05. Yokota Y, Sasai Y, Tanaka K, Fujiwara T, Tsuchida K, Shigemoto R, et al.Molecular characterization of a functional cDNA for rat substance P receptor.J Biol Chem (1989) 264:17649–52.6. Boulay F, Tardif M, Brouchon L, Vignais P. The human N-formylpeptide recep-tor. Characterization of two cDNA isolates and evidence for a new subfamilyof G-protein-coupled receptors. Biochemistry (1990) 29:11123–33. doi:10.1021/bi00502a0167. Gerard NP, Gerard C. The chemotactic receptor for human C5a anaphylatoxin.Nature (1991) 349:614–7. doi:10.1038/349614a08. Boulay F, Mery L, Tardif M, Brouchon L, Vignais P. Expression cloning ofa receptor for C5a anaphylatoxin on differentiated HL-60 cells. Biochemistry(1991) 30:2993–9. doi:10.1021/bi00226a0029. Thomas KM, Pyun HY, Navarro J. Molecular cloning of the fMet-Leu-Phereceptor from neutrophils. J Biol Chem (1990) 265:20061–4.10. Thomas KM, Taylor L, Navarro J. The interleukin-8 receptor is encoded by aneutrophil-specific cDNA clone, F3R. J Biol Chem (1991) 266:14839–41.11. Murphy PM, Tiffany HL. Cloning of complementary DNA encoding a func-tional human interleukin-8 receptor. Science (1991) 253:1280–3. doi:10.1126/science.189171612. Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: functionalcDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science(1996) 272:872–7. doi:10.1126/science.272.5263.87213. Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P. Identifi-cation of RANTES, MIP-1 alpha, andMIP-1 beta as the major HIV-suppressivefactors produced by CD8+ T cells. Science (1995) 270:1811–5. doi:10.1126/science.270.5243.181114. Raport CJ, Gosling J, Schweickart VL, Gray PW, Charo IF. Molecular cloningand functional characterization of a novel human CC chemokine recep-tor (CCR5) for RANTES, MIP-1beta, and MIP-1alpha. J Biol Chem (1996)271:17161–6. doi:10.1074/jbc.271.29.17161Conflict of Interest Statement: The authors declare that the research was con-ducted in the absence of any commercial or financial relationships that could beconstrued as a potential conflict of interest.Copyright © 2015 Gerard and Gerard. This is an open-access article distributed underthe terms of the Creative Commons Attribution License (CC BY). The use, distributionor reproduction in other forums is permitted, provided the original author(s) orlicensor are credited and that the original publication in this journal is cited, inaccordance with accepted academic practice. No use, distribution or reproduction ispermitted which does not comply with these terms.Frontiers in Immunology | www.frontiersin.org September 2015 | Volume 6 | Article 4453

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Cloning of the Human C5a Anaphylatoxin Receptor, and More

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