Soluble Mimics of the Cytoplasmic Face of the Human V1-Vascular Vasopressin Receptor Bind Arrestin2 and Calmodulin

Wu, Nan; Macion-Dazard, Rosemarie; Nithianantham, Stanley; Xu, Zhen; Hanson, Susan M.; Vishnivetskiy, Sergey A.; Gurevich, Vsevolod V.; Thibonnier, Marc; Shoham, Menachem

doi:10.1124/mol.105.018804

Cited by 5 publications

(1 citation statement)

References 41 publications

(31 reference statements)

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“…When the key phosphorylation sites of CXCR2 that are involved with its desensitization were mutated to alanine residues (4A mutant), the receptor mutant bound weakly to β‐arrestin1 (41% reduction), but exhibited normal binding to AP2 in comparison with WT‐CXCR2 (Figure B). The CXCR2 receptor with both IL/AA (mutant deficient in AP2 binding) and 4A (mutant with reduced binding to β‐arrestin) mutations, designated ‘4A/IL’ mutant, displayed reduced binding to AP2 (64% reduction) as expected and even further reduced binding of β‐arrestin1 (90% reduction), in comparison to the 4A mutant (Figure B) even though β‐arrestin is known to bind with low affinity to GPCRs through the intracellular loops of the receptor . The deficiency of binding of the AP‐2 complex to IL/AA‐CXCR2 mutant has been shown previously .…”

Section: Resultsmentioning

confidence: 81%

Adaptor Protein2 (AP2) Orchestrates CXCR2‐Mediated Cell Migration

Raman

Sai

Hawkins

et al. 2014

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The chemokine receptor CXCR2 is vital for inflammation, wound healing, angiogenesis, cancer progression, and metastasis. Adaptor protein 2 (AP2), a clathrin binding heterotetrameric protein comprised of α, β2, μ2, and σ2 subunits, facilitates clathrin-mediated endocytosis. Mutation of the LLKIL motif in the CXCR2 carboxyl-terminal domain (CTD) results in loss of AP2 binding to the receptor and loss of ligand mediated receptor internalization and chemotaxis. AP2 knockdown also results in diminished ligand-mediated CXCR2 internalization, polarization and chemotaxis. Using knockdown/rescue approaches with AP2-μ2 mutants, the binding domains were characterized in reference to CXCR2 internalization and chemotaxis. When in an open conformation, μ2 Patch 1 and Patch 2 domains bind tightly to membrane PIP2 phospholipids. When AP2-μ2 is replaced with μ2 mutated in Patch 1 and/or Patch 2 domains, ligand-mediated receptor binding and internalization are not lost. However chemotaxis requires AP2-μ2 Patch 1, but not Patch 2. AP2-σ2 has been demonstrated to bind dileucine motifs to facilitate internalization. Expression of AP2-σ2 V88D and V98S dominant negative mutants resulted in loss of CXCR2 mediated chemotaxis. Thus, AP2 binding to both membrane phosphatidylinositol phospholipids and dileucine motifs is crucial for directional migration or chemotaxis. Moreover, AP2-mediated receptor internalization can be dissociated from AP2-mediated chemotaxis.

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Section: Resultsmentioning

confidence: 81%

Adaptor Protein2 (AP2) Orchestrates CXCR2‐Mediated Cell Migration

Raman

Sai

Hawkins

et al. 2014

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Arrestin Binding to Calmodulin: A Direct Interaction Between Two Ubiquitous Signaling Proteins

Hanson

Francis

et al. 2006

Journal of Molecular Biology

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103

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SummaryArrestins serve as multi-functional regulators of G-protein coupled receptors, interacting with hundreds of different receptor subtypes and a variety of other signaling proteins. Here we identify calmodulin as a novel arrestin interaction partner using three independent methods in vitro and in cells. Arrestin preferentially binds calcium-loaded calmodulin with a K d of ~7μM, which is within range of endogenous calmodulin concentrations. The calmodulin binding site is localized on the concave side of the C-domain and a loop in the center of the arrestin molecule, significantly overlapping with receptor-and microtubule-binding sites. Using purified proteins, we found that arrestins sequester calmodulin, preventing its binding to microtubules. Nanomolar affinity of arrestins for their cognate receptors makes calmodulin an ineffective competitor for arrestin binding at relatively high receptor concentrations. The arrestin-calmodulin interaction likely regulates the localization of both proteins and their availability for other interaction partners.

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Dual Role of the β2-Adrenergic Receptor C Terminus for the Binding of β-Arrestin and Receptor Internalization

Krasel

Zabel²,

Lorenz³

et al. 2008

Journal of Biological Chemistry

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Homologous desensitization of ␤ 2 -adrenergic and other G-protein-coupled receptors is a two-step process. After phosphorylation of agonist-occupied receptors by G-protein-coupled receptor kinases, they bind ␤-arrestins, which triggers desensitization and internalization of the receptors. Because it is not known which regions of the receptor are recognized by ␤-arrestins, we have investigated ␤-arrestin interaction and internalization of a set of mutants of the human ␤ 2 -adrenergic receptor. Mutation of the four serine/threonine residues between residues 355 and 364 led to the loss of agonist-induced receptor-␤-arrestin2 interaction as revealed by fluorescence resonance energy transfer (FRET), translocation of ␤-arrestin2 to the plasma membrane, and receptor internalization. Mutation of all seven serine/threonine residues distal to residue 381 did not affect agonist-induced receptor internalization and ␤-arrestin2 translocation. A ␤ 2 -adrenergic receptor truncated distal to residue 381 interacted normally with ␤-arrestin2, whereas its ability to internalize in an agonist-dependent manner was compromised. A similar impairment of internalization was observed when only the last eight residues of the C terminus were deleted. Our experiments show that the C terminus distal to residue 381 does not affect the initial interaction between receptor and ␤-arrestin, but its last eight amino acids facilitate receptor internalization in concert with ␤-arrestin2.The interaction of ␤-arrestins with G-protein-coupled receptors is a prerequisite for at least three different processes: homologous desensitization (1), activation of tyrosine kinasemediated signaling pathways (2), and receptor internalization (3). This interaction requires the phosphorylation of the receptor by G-protein-coupled receptor kinases (GRKs) 3 and (at least for some receptors) the continuous presence of agonist (4). GRKs are unique among serine/threonine kinases in that they do not recognize a well-defined consensus sequence but instead show high specificity for agonist-activated receptors. This lack of a consensus sequence has made mapping of phosphorylated residues in G-protein-coupled receptors difficult. For example, the residues phosphorylated by GRK2 in the ␤ 2 -adrenergic receptor have been mapped to the C terminus, either between amino acids 384 and 411 (5) or between amino acids 355 and 364 (6 -10). It is now clear from a variety of studies (see Ref.11 for a review) that ␤-arrestins do not simply act as "phosphoreceptor-specific antibodies." Rather, the interaction of ␤-arrestins with GRK-phosphorylated receptors is believed to lead to a conformational change in the ␤-arrestin molecule, which enables it to bind to other parts of the receptor with higher affinity. Furthermore, it has also been demonstrated that ␤-arrestins sense the activated conformation of the receptor (4, 12). For example, ␤-arrestin mutants have been described that do not require GRK-mediated phosphorylation to bind to a receptor but still only interact with a receptor when...

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Soluble Mimics of the Cytoplasmic Face of the Human V1-Vascular Vasopressin Receptor Bind Arrestin2 and Calmodulin

Cited by 5 publications

References 41 publications

Adaptor Protein2 (AP2) Orchestrates CXCR2‐Mediated Cell Migration

Adaptor Protein2 (AP2) Orchestrates CXCR2‐Mediated Cell Migration

Arrestin Binding to Calmodulin: A Direct Interaction Between Two Ubiquitous Signaling Proteins

Dual Role of the β2-Adrenergic Receptor C Terminus for the Binding of β-Arrestin and Receptor Internalization

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