Identification of a Motif in the Carboxyl Terminus of CXCR2 That Is Involved in Adaptin 2 Binding and Receptor Internalization

Fan, Guo-Huang; Yang, Wei; Wang, Xuejie; Qian, Qinghua; Richmond, Ann

doi:10.1021/bi001661b

Cited by 98 publications

(149 citation statements)

References 42 publications

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“…This is important because various examples demonstrate that β-arrestin2 recruitment and receptor internalization can be independent processes, as it is the case e.g. with the PAR1 thrombin receptor (Paing et al, 2002), the CXCR2 chemokine receptor (Fan et al, 2001;Zhao et al, 2004), the N-formyl peptide receptor (FPR) (Vines et al, 2003), or at high agonist concentration the AT 1 angiotensin receptor (Gaborik et al, 2001;Hunyady and Catt, 2006;Zhang et al, 1996). Therefore, it is important to study directly the β-arrestin dependence of CB 1 R internalization.…”

Section: Discussionmentioning

confidence: 99%

Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB1 cannabinoid receptor

Gyombolai

Boros

Hunyady

et al. 2013

Molecular and Cellular Endocrinology

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CB 1 cannabinoid receptor (CB 1 R) undergoes both constitutive and agonist-induced internalization, but the underlying mechanisms of these processes and the role of β-arrestins in the regulation of CB 1 R function are not completely understood. In this study, we followed CB 1 R internalization using confocal microscopy and bioluminescence resonance energy transfer measurements in HeLa and Neuro-2a cells. We found that upon activation CB 1 R binds β-arrestin2 (β-arr2), but not β-arrestin1. Furthermore, both the expression of dominant-negative β-arr2 (β-arr2-V54D) and siRNA-mediated knock-down of β-arr2 impaired the agonist-induced internalization of CB 1 R. In contrast, neither β-arr2-V54D nor β-arr2-specific siRNA had a significant effect on the constitutive internalization of CB 1 R. However, both constitutive and agonist-induced internalization of CB 1 R were impaired by siRNA-mediated depletion of clathrin heavy chain. We conclude that although clathrin is required for both constitutive and agonist-stimulated internalization of CB 1 R, β-arr2 binding is only required for agonist-induced internalization of the receptor suggesting that the molecular mechanisms underlying constitutive and agonist-induced internalization of CB 1 R are different.

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Section: Discussionmentioning

confidence: 99%

Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB1 cannabinoid receptor

Gyombolai

Boros

Hunyady

et al. 2013

Molecular and Cellular Endocrinology

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“…The plasmids of CXCR2, 331T, IL323,324/AA, enhanced green fluorescent protein (EGFP)-Rab11a, full-length EGFP-myosin Vb, EGFP-myosin Vb tail, full-length EGFP-Rab11-FIP2, EGFP-Rab11-FIP2 (129 -512), and DsRed2-myosin Vb tail were constructed as described previously (Mueller et al, 1995;Fan et al, 2001b;Lapierre et al, 2001;Hales et al, 2002).…”

Section: Plasmidsmentioning

confidence: 99%

“…We have demonstrated previously that a C-terminal truncated mutant of CXCR2-331T, which eliminates all of the phosphorylation sites, exhibited normal internalization in HEK293 cells in the absence of ligand-dependent phosphorylation of the receptor (Fan et al, 2001b). To determine whether CXCR2 phosphorylation is required for the receptor association with Rab11-FIP2 and myosin Vb, HEK293 cells stably expressing CXCR2 or CXCR2-331T were transfected with plasmids for EGFPmyosin Vb or EGFP-Rab11-FIP2.…”

Section: Association Of Cxcr2 With Myosin Vb and Rab11-fip2mentioning

confidence: 99%

“…For the receptor resensitization assay, cells were incubated with CXCL1 (200 ng/ml) for 30 min, followed by repeated wash with ligand-free incubation buffer, and the recovery of the cells was allowed in the reaction buffer for 60 min, before being loaded with Fluo-3. CXCL1-induced Ca 2ϩ mobilization was measured as described previously (Fan et al, 2001b). To compare the receptor resensitization between the control cells, cells expressing full-length myosin Vb, and cells expressing myosin Vb tail, the time taken to recover 80% of the mobilized Ca 2ϩ (t 0.80 ) (Fan et al, 2001b) and the potency of the Ca 2ϩ mobilization (peak Ca 2ϩ concentration Ϫ basal Ca 2ϩ concentration) were calculated.…”

mentioning

confidence: 99%

“…CXCL1-induced Ca 2ϩ mobilization was measured as described previously (Fan et al, 2001b). To compare the receptor resensitization between the control cells, cells expressing full-length myosin Vb, and cells expressing myosin Vb tail, the time taken to recover 80% of the mobilized Ca 2ϩ (t 0.80 ) (Fan et al, 2001b) and the potency of the Ca 2ϩ mobilization (peak Ca 2ϩ concentration Ϫ basal Ca 2ϩ concentration) were calculated. …”

mentioning

confidence: 99%

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Rab11-Family Interacting Protein 2 and Myosin Vb Are Required for CXCR2 Recycling and Receptor-mediated Chemotaxis

Fan

Lapierre

Goldenring

et al. 2004

MBoC

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127

130

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Agonist-stimulated internalization followed by recycling to the cell membrane play an important role in fine-tuning the activity of chemokine receptors. Because the recycling of chemokine receptors is critical for the reestablishment of the cellular responsiveness to ligand, it is crucial to understand the mechanisms underlying the receptor recycling and resensitization. In the present study, we have demonstrated that the chemokine receptor CXCR2 associated with myosin Vb and Rab11-family interacting protein 2 (FIP2) in a ligand-dependent manner. Truncation of the C-terminal domain of the receptor did not affect the association, suggesting that the interactions occur upstream of the C terminus of CXCR2. After ligand stimulation, the internalized CXCR2 colocalized with myosin Vb and Rab11-FIP2 in Rab11a-positive vesicles. The colocalization lasted for ϳ2 h, and little colocalization was observed after 4 h of ligand stimulation. CXCR2 also colocalized with myosin Vb tail or Rab11-FIP2 (129 -512), the N-terminal-truncated mutants of myosin Vb and Rab11-FIP2, respectively, but in a highly condensed manner. Expression of the enhanced green fluorescent protein-tagged myosin Vb tail significantly retarded the recycling and resensitization of CXCR2. CXCR2 recycling was also reduced by the expression Rab11-FIP2 (129 -512). Moreover, expression of the myosin Vb tail reduced CXCR2-and CXCR4-mediated chemotaxis. These data indicate that Rab11-FIP2 and myosin Vb regulate CXCR2 recycling and receptor-mediated chemotaxis and that passage of internalized CXCR2 through Rab11a-positive recycling system is critical for physiological response to a chemokine. INTRODUCTIONChemokine receptors belong to the large family of seventransmembrane G protein-coupled receptors (GPCRs) that function in immune and inflammatory response by regulating the activation and migration of leukocytes, immune cell development, and angiogenesis (Nagasawa et al., 1996;Luster 1998;Belperio et al., 2000;Murphy et al., 2000;Zlotnik and Yoshie, 2000). Some of them (e.g., CCR5 and CXCR4) participate in HIV infection of CD4 ϩ T lymphocytes as coreceptors (Berger et al., 1999). Ligand binding to the chemokine receptors triggers various signaling cascades, including activation of G proteins, phosphotidylinositol 3-kinase, Janus kinase/signal transducers and activators of transcription proteins, the Rho-p160 ROCK axis, and the MAPK pathway (Wu et al., 1993;Ganju et al., 1998;Mellado et al., 1998;Vicente-Manzanares et al., 1999;Vicente-Manzanares et al., 2002). Chemokine activation of these intracellular signals is often accompanied by chemokine receptor internalization and trafficking back to the cell membrane. The intracellular trafficking of chemokine receptors controls their activities, and the balance between the chemokine receptor recycling and degradation dictates the leukocyte responsiveness to chemokines (Sabroe et al., 1997;Asagoe et al., 1998;Khandaker et al., 1998;Mack et al., 1998).Ligand stimulated chemokine receptor internalization can be accomplished ...

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Intracellular trafficking of human CXCR1 and CXCR2: regulation by receptor domains and actin-related kinases

et al. 2002

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In this study we investigated the regulation of CXCR1 and CXCR2 intracellular trafficking. First, we produced a chimeric CXCR2 receptor that contained the internalization motifs of both CXCR2 and CXCR1 (CXCR2: LLKIL sequence; CXCR1: C‐terminal phosphorylation sites). Elevated levels of internalization were induced by different ELR‐expressing CXC chemokines on the chimeric receptor, as compared to wild‐type CXCR2. Analysis of inter‐relationships between CXCR1 and CXCR2 during internalization indicated that the exposure of cells that expressed both CXCR1 and CXCR2 to CXCL8 or CXCL6 resulted in decreased levels of CXCR1 internalization as compared to those in cells that expressed only CXCR1. To characterize the role of actin‐related components in CXCR1 and CXCR2 trafficking, wortmannin, a potent inhibitor of phosphatidylinositol kinases, was used. The presence of wortmannin during receptor recycling inhibited CXCR1 and CXCR2 re‐expression following CXCL8‐induced internalization, and resulted in a marked disruption of the proper organization of actin filaments. The kinase‐dependent recycling process required CXCR2 C‐terminal phosphorylation sites. Our results suggest that actin‐related kinases are required for the proper functionality of actin filaments, which are the instrumental factors needed for receptor recycling. In all, CXCR1 and CXCR2 internalization and recycling are tightly regulated by receptor domains and by actin‐related kinases.

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Identification of a Motif in the Carboxyl Terminus of CXCR2 That Is Involved in Adaptin 2 Binding and Receptor Internalization

Cited by 98 publications

References 42 publications

Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB1 cannabinoid receptor

Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB1 cannabinoid receptor

Rab11-Family Interacting Protein 2 and Myosin Vb Are Required for CXCR2 Recycling and Receptor-mediated Chemotaxis

Intracellular trafficking of human CXCR1 and CXCR2: regulation by receptor domains and actin-related kinases

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