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Cloning of the Human C5a Anaphylatoxin Receptor, and More.

Gerard NP, Gerard C - Front Immunol (2015)

View Article: PubMed Central - PubMed

Affiliation: Ina Sue Perlmutter Laboratory, Division of Respiratory Diseases, Department of Medicine, Children's Hospital, Harvard Medical School , Boston, MA , USA ; Department of Medicine, Beth Israel Deaconess Medical Center , Boston, MA , USA.

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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... As NPIIY-18 was not a full-length cDNA, we then probed the retinoic acid differentiated HL60 cell library... His work was published in March 1991, some 4 months 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, which had almost no significant homology to the human receptor... 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 collaboration and test its identity as a receptor for IL-8... Henry Showell, of Pfizer Central Research, was able to provide us with a custom iodinated 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... 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... Thus, an international race began as five chemokine labs partnered with HIV labs to prove the hypothesis that CCR5 was the HIV coreceptor... The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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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 125I-C5a under competitive binding conditions. (C) The resulting binding isotherm and Scatchard analysis proving the clone’s identity.
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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 125I-C5a under competitive binding conditions. (C) The resulting binding isotherm and Scatchard analysis proving the clone’s identity.

Mentions: We constructed an antisense oligonucleotide with minimal 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 4 months after ours.


Cloning of the Human C5a Anaphylatoxin Receptor, and More.

Gerard NP, Gerard C - Front Immunol (2015)

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 125I-C5a under competitive binding conditions. (C) The resulting binding isotherm and Scatchard analysis proving the clone’s identity.
© Copyright Policy
Related In: Results  -  Collection

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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 125I-C5a under competitive binding conditions. (C) The resulting binding isotherm and Scatchard analysis proving the clone’s identity.
Mentions: We constructed an antisense oligonucleotide with minimal 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 4 months after ours.

View Article: PubMed Central - PubMed

Affiliation: Ina Sue Perlmutter Laboratory, Division of Respiratory Diseases, Department of Medicine, Children's Hospital, Harvard Medical School , Boston, MA , USA ; Department of Medicine, Beth Israel Deaconess Medical Center , Boston, MA , USA.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

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... As NPIIY-18 was not a full-length cDNA, we then probed the retinoic acid differentiated HL60 cell library... His work was published in March 1991, some 4 months 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, which had almost no significant homology to the human receptor... 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 collaboration and test its identity as a receptor for IL-8... Henry Showell, of Pfizer Central Research, was able to provide us with a custom iodinated 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... 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... Thus, an international race began as five chemokine labs partnered with HIV labs to prove the hypothesis that CCR5 was the HIV coreceptor... The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

No MeSH data available.