Molecular aspects of the tachykinin receptors

Gerard, Norma P.; Liu, Bao; He, Xiaolong; Gérard, Craig

doi:10.1016/0167-0115(93)90404-v

Cited by 107 publications

(58 citation statements)

References 55 publications

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“…The folded C5a structure is similar to that of C3a as determined previously by X-ray analysis (Huber et al, 1980). Three intrachain disulfide bonds contribute stability to C5a tertiary structure, which is of critical importance for optimal receptor binding (Gerard et al, 1979).…”

supporting

confidence: 48%

Site‐specific mutations in the N‐terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor

Carney

Hugli

1993

Protein Science

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C5a is an inflammatory mediator that evokes a variety of immune effector functions including chemotaxis, cell activation, spasmogenesis, and immune modulation. It is well established that the effector site in C5a is located in the C‐terminal region, although other regions in C5a also contribute to receptor interaction. We have examined the N‐terminal region (NTR) of human C5a by replacing selected residues in the NTR with glycine via site‐directed mutagenesis. Mutants of rC5a were expressed as fusion proteins, and rC5a was isolated after factor Xa cleavage. The potency of the mutants was evaluated by measuring both neutrophil chemotaxis and degranulation (β‐glucuronidase release). Mutants that contained the single residue substitutions Ile‐6 → Gly or Tyr‐13 → Gly were reduced in potency to 4–30% compared with wild‐type rC5a. Other single‐site glycine substitutions at positions Leu‐2, Ala‐10, Lys‐4, Lys‐5, Glu‐7, Glu‐8, and Lys‐14 showed little effect on C5a potency. The double mutant, Ile‐6 → Gly/Tyr‐13 → Gly, was reduced in potency to <0.2%, which correlated with a correspondingly low binding affinity for neutrophil C5a receptors. Circular dichroism studies revealed a 40% reduction in α‐helical content for the double mutant, suggesting that the NTR contributes stabilizing interactions that maintain local secondary or tertiary structure of C5a important for receptor interaction. We conclude that the N‐terminal region in C5a is involved in receptor binding either through direct interaction with the receptor or by stabilizing a binding site elsewhere in the intact C5a molecule.

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supporting

confidence: 48%

Site‐specific mutations in the N‐terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor

Carney

Hugli

1993

Protein Science

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“…P (415) 3085 C_ COO CCT CrA CGA OCT CCC CCC AGG OCT CTT CCC GAT GAO GAC CCT CCC ACT CCC TCC ATT GCT TOO CTO TCCr AC, 435 The interrupted open reading frame of the gene exhibited receptor genes display a relatively high homology to that of 88% and 100% nucleotide identity with canine gastrin recepthe gastrin/CCKB receptor and also contain four introns (19). tor and human CCKB receptor cDNA sequences, respecAlternative Splicing of Exon 4.…”

Section: Resultsmentioning

confidence: 99%

The human gastrin/cholecystokinin type B receptor gene: alternative splice donor site in exon 4 generates two variant mRNAs.

Song

Brown

Wiltshire

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

103

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Gastrin and its carboxyl-terminal homolog cholecystokinin (CCK) exert a variety of biological actions in the brain and gastrointestinal tract that are mediated in part through one or more G protein-coupled receptors which exhibit similar affinity for both peptides. Genomic clones encoding a human gastrin/CCKB receptor were isolated by screening a human EMBL phage library with a partial-length DNA fragment which was based on the nucleotide sequence of the canine gastrin receptor. The gene contained a 1356-bp open reading frame consisting of five exons interrupted by 4 introns and was assigned to human chromosome llpl5.4. A region of exon 4, which encodes a portion of the putative third intracellular loop, appears to be alternatively spliced to yield two different mRNAs, one containing (452 amino acids; long isoform) and the other lacking (447 amino acids; short isoform) the pentapeptide sequence Gly-Gly-Ala-Gly-Pro. The two receptor isoforms may contribute to functional differences in gastrin-and CCK-mediated signal transduction. (12,13). They encode homologous proteins of similar length (453 and 450 aa, respectively) with seven deduced transmembrane regions, comparable to other G protein-coupled receptors. The recombinant receptors, when transfected into COS-7 cells, manifest high (nanomolar) affinities for both gastrin and CCK and are coupled to an increase in intracellular calcium (12,13). Structurally, the canine gastrin receptor displays nearly 90% amino acid identity with the putative CCKB receptors from human and rat (9-11).The sequence and organization of the full-length genes encoding members of the gastrin/CCK receptor family have not been identified. Such information is essential for analysis of their transcriptional regulation and structural relationships to other receptor genes. We have cloned and sequenced a gastrin/CCKB receptor gene from a human genomic library**. The gene appears to be alternatively spliced to yield two different receptor isoforms which differ by 5 aa in the putative third intracellular loop. The gastrin/CCKB receptor gene was assigned to human chromosome 11p15.4 by fluorescence in situ hybridization. METHODSGenomic Library Screening and DNA Sequencing. A human genomic library in the bacteriophage AEMBL-3 (Clontech) was screened with a 32P-labeled cDNA fragment generated by the polymerase chain reaction (PCR) with primers based on the cDNA sequence of the canine gastrin receptor (see below). Two positive clones were digested with Sst I and the resulting restriction fragments of length 0.7-3.2 kb (Fig. 1) were subcloned into phage M13mpl8 or -mpl9 and sequenced in both directions by the dideoxy method (14). In Abbreviation: CCK, cholecystokinin. tPresent address:

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“…Although only one gene encoding the NK1 receptor has been isolated thus far (Gerard et al, 1993), the possibility exists that different forms of the receptor protein are generated from the same gene (Fong et al, 1992b;Kage et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

Comparison of tachykinin NK₁ and NK₂ receptors in the circular muscle of the guinea‐pig ileum and proximal colon

Maggi

Patacchini

Meini

et al. 1994

British J Pharmacology

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1 The aim of this study was the pharmacological characterization of tachykinin NKI and NK2 receptors mediating contraction in the circular muscle of the guinea-pig ileum and proximal colon. The action of substance P (SP), neurokinin A (NKA) and of the synthetic agonists [ Furthermore, an intraspecies heterogeneity of the NK2 receptor in the circular muscle of the guinea-pig ileum and colon is suggested.

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Molecular aspects of the tachykinin receptors

Cited by 107 publications

References 55 publications

Site‐specific mutations in the N‐terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor

Site‐specific mutations in the N‐terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor

The human gastrin/cholecystokinin type B receptor gene: alternative splice donor site in exon 4 generates two variant mRNAs.

Comparison of tachykinin NK₁ and NK₂ receptors in the circular muscle of the guinea‐pig ileum and proximal colon

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Molecular aspects of the tachykinin receptors

Cited by 107 publications

References 55 publications

Site‐specific mutations in the N‐terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor

Site‐specific mutations in the N‐terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor

The human gastrin/cholecystokinin type B receptor gene: alternative splice donor site in exon 4 generates two variant mRNAs.

Comparison of tachykinin NK1 and NK2 receptors in the circular muscle of the guinea‐pig ileum and proximal colon

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Comparison of tachykinin NK₁ and NK₂ receptors in the circular muscle of the guinea‐pig ileum and proximal colon