Emma L. Wise, PhD, Kandace T. Bonner, BSc, Timothy J

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Presentation transcript:

A single nucleotide polymorphism in the CCR3 gene ablates receptor export to the plasma membrane Emma L. Wise, PhD, Kandace T. Bonner, BSc, Timothy J. Williams, PhD, James E. Pease, PhD Journal of Allergy and Clinical Immunology Volume 126, Issue 1, Pages 150-157.e2 (July 2010) DOI: 10.1016/j.jaci.2010.04.015 Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 The L324P-CCR3 and C218S mutants exhibit reduced trafficking to the cell membrane but are detected intracellularly. A and B, Relative cell surface expression of CCR3 variants (n = 3) with representative examples of WT-CCR3, C218S-CCR3, and L324P-CCR3 staining. FL1-H, height of fluorescent intensity. C, Intracellular expression of L324P-CCR3 and C218S-CCR3 (n = 3). D, Western blot of WT-CCR3, L324P-CCR3, or C218S-CCR3 constructs with controls. Relative ODs are indicated. An α-tubulin loading control is indicated. Journal of Allergy and Clinical Immunology 2010 126, 150-157.e2DOI: (10.1016/j.jaci.2010.04.015) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Migratory reponses of L1.2 cells transiently transfected with either WT or CCR3 variant proteins as indicated in the panels A-G. Data are represented as means ± SEMs of 3 separate experiments. M, mol/L. Journal of Allergy and Clinical Immunology 2010 126, 150-157.e2DOI: (10.1016/j.jaci.2010.04.015) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 L324 perse is critical for CCR3 trafficking and function. A, Extracellular expression of L324A-CCR3 expressed as a percentage of WT-CCR3 staining (n = 3). B, Intracellular expression of L324A-CCR3 expressed as a percentage of WT staining (n = 3). C, Chemotaxis of WT- CCR3 and L324A-CCR3 transfectants in response to increasing doses of CCL11 (n = 3). Journal of Allergy and Clinical Immunology 2010 126, 150-157.e2DOI: (10.1016/j.jaci.2010.04.015) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 CCR3 is associated with the cell membrane fraction and undergoes constitutive degradation. A, Western analysis of CCR3 constructs after subcellular fractionation. Probing was with anti-HA (CCR3), anti-GAPDH (cytosol), anti-calnexin (ER), anti-GM130 (Golgi), and anti-lamin B (nuclear envelope). B, Western analysis of CCR3 constructs after cycloheximide treatment. Probing was with anti-HA or anti-tubulin-α antibodies. Relative ODs of CCR3 are shown. Journal of Allergy and Clinical Immunology 2010 126, 150-157.e2DOI: (10.1016/j.jaci.2010.04.015) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 CCR3 is associated with the ER, Golgi, and lysosomes. L1.2 WT or L324P-CCR3 transfectants were analyzed by using confocal microscopy after staining with the anti-HA antibody and the ER marker calnexin (A), the Golgi marker GM130 (B), or the lysosome marker LAMP-1 (C). Data shown are representative of several independent experiments. Scale bars denote 10 μm. Journal of Allergy and Clinical Immunology 2010 126, 150-157.e2DOI: (10.1016/j.jaci.2010.04.015) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

SNPs within the CCR3 gene and their corresponding protein product SNPs within the CCR3 gene and their corresponding protein product. A, Table showing non-synonymous SNPs of CCR3. Asth, asthmatics; Jap, Japanese; Brit, British. B, The CCR3 primary sequence with sites of point mutation shown in filled circles and annotated. Journal of Allergy and Clinical Immunology 2010 126, 150-157.e2DOI: (10.1016/j.jaci.2010.04.015) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Mutation of L324 in CCR1 is without effect Mutation of L324 in CCR1 is without effect. A, Alignment of the C-termini of human (h) CCR1 and CCR3. The conserved L324 residue is boxed. B, Relative cell surface expression of the L324P-CCR1 construct (n = 3). C, Migration of L1.2 transfectants expressing either WT-CCR1 or L324P-CCR1 in response to increasing amounts of CCL3 (n = 3). M, mol/L. Journal of Allergy and Clinical Immunology 2010 126, 150-157.e2DOI: (10.1016/j.jaci.2010.04.015) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions