Alanine scanning mutagenesis of CCR3 reveals that the three intracellular loops are essential for functional receptor expression

Auger, Graham A.; Pease, James E.; Shen, Xiaofeng; Xanthou, Georgina; Barker, Michael D.

doi:10.1002/1521-4141(200204)32:4<1052::aid-immu1052>3.0.co;2-l

Cited by 36 publications

(29 citation statements)

References 23 publications

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“…The tyrosine residue mutation often does not (Arora et al, 1997;Lu et al, 1997;Ohyama et al, 2002;Gaborik et al, 2003) or only marginally (Zhu et al, 1994;Rhee et al, 2000;Auger et al, 2002;Hawtin, 2005) affects receptor function.…”

Section: Other Considerationsmentioning

confidence: 99%

The Highly Conserved DRY Motif of Class A G Protein-Coupled Receptors: Beyond the Ground State

Rovati

Capra²,

Neubig³

2007

Molecular Pharmacology

332

309

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Despite extensive study of heptahelical G protein-coupled receptors (GPCRs), the precise mechanism of G protein activation is unknown. The role of one highly conserved stretch of residues, the amino acids glutamic acid/aspartic acid-argininetyrosine (i.e., the E/DRY motif), has received considerable attention with respect to regulating GPCR conformational states. In the consensus view, glutamic acid/aspartic acid maintains the receptor in its ground state, because mutations frequently induce constitutive activity (CA). This hypothesis has been confirmed by the rhodopsin ground-state crystal structure and by computational modeling approaches. However, some class A GPCRs are resistant to CA, suggesting alternative roles for the glutamic acid/aspartic acid residue and the E/DRY motif. Here, we propose two different subgroups of receptors within class A GPCRs that make different use of the E/DRY motif, independent of the G protein type (G s , G i , or G q ) to which the receptor couples. In phenotype 1 receptors, nonconservative mutations of the glutamic acid/aspartic acid-arginine residues, besides inducing CA, increase affinity for agonist binding, retain G protein coupling, and retain an agonist-induced response. In contrast, in second phenotype receptors, the E/DRY motif is more directly involved in governing receptor conformation and G protein coupling/recognition. Hence, mutations of the glutamic acid/aspartic acid residues do not induce CA. Conversely, nonconservative mutations of the arginine of the E/DRY motif always impair agonist-induced receptor responses and, generally, reduce agonist binding affinity. Thus, it is essential to look beyond the rhodopsin ground-state model of conformational activation to clarify the role of this highly conserved triplet in GPCR activation and function.

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Section: Other Considerationsmentioning

confidence: 99%

The Highly Conserved DRY Motif of Class A G Protein-Coupled Receptors: Beyond the Ground State

Rovati

Capra²,

Neubig³

2007

Molecular Pharmacology

332

309

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“…Staining was carried out as previously described [30] using either mouse anti-human CXCR3 mAb (3 ? g/ml) or mouse anti-human CXCR1 mAb (10 ?…”

Section: Flow Cytometric Analysis Of Chemokine Receptor Expressionmentioning

confidence: 99%

Molecular characterization of the chemokine receptor CXCR3: evidence for the involvement of distinct extracellular domains in a multi‐step model of ligand binding and receptor activation

2003

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CXCR3 is a chemokine receptor predominantly expressed on T lymphocytes, and binds the chemokines CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (I-TAC). Here, we have investigated the role of the extracellular domains of CXCR3 in ligand selectivity and receptor activation by assessing the ligand binding and chemotactic responses of chimeric CXCR3/CXCR1 constructs. Our data reveal that the second extracellular loop of CXCR3 is essential for receptor activation in response to all CXCR3 ligands. In contrast, the N terminus and first extracellular loop of CXCR3 play some role in CXCL10-and CXCL11-mediated activation but are dispensable for CXCL9-induced signaling. The third extracellular loop of CXCR3 is important only for CXCL9-and CXCL10-induced chemotaxis. Binding studies suggest that the CXCR3 ligands bind to distinct sites composed of multiple domains of CXCR3 and that high-affinity binding and receptor activation are disparate functions. Collectively, our data support a multi-site model for CXCR3 interactions with its agonists, in which several extracellular domains of CXCR3 contribute to ligand binding and the induction of receptor activation. The development of antagonists targeting the second extracellular loop of CXCR3 should impede receptor activation and aid the treatment of several human inflammatory disorders.

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“…Staining was carried out as previously described [44] using either rat anti-human-CCR3 mAb (10 ? g/ml) or mouse antihuman-CXCR3 mAb (3 ?…”

Section: Facs Analysis Of Chemokine Receptor Expressionmentioning

confidence: 99%

CCR3 functional responses are regulated by both CXCR3 and its ligands CXCL9, CXCL10 and CXCL11

et al. 2003

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The chemokine receptor CXCR3 is predominantly expressed on T lymphocytes, and its agonists CXCL9, CXCL10 and CXCL11 are IFN-+ -inducible chemokines that promote Th1 responses. In contrast, the CCR3 agonists CCL11, CCL24 and CCL26 are involved in the recruitment of cells such as eosinophils and basophils during Th2 responses. Here, we report that although CCL11, CCL24 and CCL26 are neither agonists nor antagonists of CXCR3, CCL11 binds with high affinity to CXCR3. This suggests that, in vivo, CXCR3 may act as a decoy receptor, sequestering locally produced CCL11. We also demonstrate that the CXCR3 ligands inhibit CCR3-mediated functional responses of both human eosinophils and CCR3 transfectants induced by all three eotaxins, with CXCL11 being the most efficacious antagonist. The examination of CCR3-CCR1 chimeric constructs revealed that CCL11 and CXCL11 share overlapping binding sites contained within the CCR3 extracellular loops, a region that was previously shown to be essential for effective receptor-activation. Hence, eosinophil responses mediated by chemokines acting at CCR3 may be regulated by two distinct mechanisms: the antagonistic effects of CXCR3 ligands and the sequestration of CCL11 by CXCR3-expressing cells. Such interplay may serve to finely tune inflammatory responses in vivo.

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Alanine scanning mutagenesis of CCR3 reveals that the three intracellular loops are essential for functional receptor expression

Cited by 36 publications

References 23 publications

The Highly Conserved DRY Motif of Class A G Protein-Coupled Receptors: Beyond the Ground State

The Highly Conserved DRY Motif of Class A G Protein-Coupled Receptors: Beyond the Ground State

Molecular characterization of the chemokine receptor CXCR3: evidence for the involvement of distinct extracellular domains in a multi‐step model of ligand binding and receptor activation

CCR3 functional responses are regulated by both CXCR3 and its ligands CXCL9, CXCL10 and CXCL11

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