Chemokine receptors CXCR4 and CCR5: Cell surface expression, signaling and modulation by β-arrestin 2

G protein-coupled receptors which mediate a large variety of different cellular effects have always been a field of significant interest for pharmaceutical research. Since it was shown that the chemokine receptors CXCR4 and CCR5 act as essential co-receptors for HIV-1 entry the interest in understanding the regulation of cell surface expression of these specific GPCR increased even more. The receptor expression at the cell surface is regulated by different mechanisms such as agonist induced receptor endocytosis and subsequent receptor recycling, whereas boths effects are more or less distinct for different receptors. During recent years it was also shown that constitutive receptor endocytosis plays a prominent role in the circulation of receptors. Previous methods for the analysis of receptor cycling which used receptor-specific antibodies were not sensitive enough and lacked the potential to monitor constitutive receptor endocytosis in quantitative terms. In this work an antibody-independent method based on specific biotinylation of an acceptor peptide (AP) by the enzyme biotin ligase A was established. Exemplified by the quantification of the ligand induced internalization and recycling of CXCR4 and CCR5 the robustness and high sensivity of the assay was demonstrated. Furthermore, the assay was not influenced by receptors which were freshly translocated to the cell surface without prior ligand binding. As an additional feature the assay provided the possibility for a detailed quantification of constitutive receptor endocytosis. In order to detect all receptors within a cell regardless of whether they had previously been expressed at the cell surface AP-specific monoclonal antibodies were generated which can be used for double immunofluorescence microscopy. These antibodies allow discriminations of biotinylated and non biotinylated receptors or detection of transmembrane proteins lacking high specific antibodies. β-Arrestin 2 is a multivalent adaptor protein involved in receptor signaling as well as endocytosis which binds to various intracellular proteins. Recent reports challenged the classical concept of GPCR signaling via heterotrimeric G proteins and postulated a higher relevance of receptor homodimerization or binding of β-arrestins to the receptor. To circumvent G protein activation after ligand binding a chemical-induced dimerization system consisting of three components was used. Either a FKBP12 (DmrA) or FRB (DmrC) domain was fused to the C-terminus of CXCR4/CCR5 and β arrestin 2. Treatment of Rec DmrA/βArr DmrC cell lines with AP21967 led to dose- and time dependent recruitment of β arrestin 2 to the receptor in the absence of ligand stimulation. AP21967-induced translocation of β arrestin 2 to the receptor significantly decreased ligand-induced G protein mediated calcium release. In cell lines without βArr-DmrC expression no alterations were obtained. AP21967-binding also provoked a ligand-independent internalization of CXCR4/CCR5 which was on a comparable level as ligand-induced internalization. Interestingly, the AP21967 induced recruitment of β-arrestin 2 to the receptor was sufficient to mimic the specific, ligand-induced intracellular receptor distribution of either CXCR4 or CCR5. Whereas AP21967 treatment led to a β arrestin 2 receptor desensitization and internalization it was not sufficient to mediate receptor signaling via the MAP kinases ERK 1/2. AP20187-induced receptor homodimerization had no detectable effect on either receptor desensitization or the phsophorylation level of ERK 1/2. However AP20187 pretreament led to an enhanced ligand-induced internalization in Rec-DmrA cell lines. In summary, the results obtained within this work contribute to a more detailed understanding of β arrestin-mediated functions during chemokine receptor trafficking and demonstrated the applicability of a highly sensitive, biotin-based detection system for the analysis of trafficking of transmembrane proteins

Analysis of Chemokine Receptor Trafficking by Site-Specific Biotinylation.

Chemokine receptors undergo internalization and desensitization in response to ligand activation. Internalized receptors are either preferentially directed towards recycling pathways (e.g. CCR5) or sorted for proteasomal degradation (e.g. CXCR4). Here we describe a method for the analysis of receptor internalization and recycling based on specific Bir A-mediated biotinylation of an acceptor peptide coupled to the receptor, which allows a more detailed analysis of receptor trafficking compared to classical antibody-based detection methods. Studies on constitutive internalization of the chemokine receptors CXCR4 (12.1% ± 0.99% receptor internalization/h) and CCR5 (13.7% ± 0.68%/h) reveals modulation of these processes by inverse (TAK779; 10.9% ± 0.95%/h) or partial agonists (Met-CCL5; 15.6% ± 0.5%/h). These results suggest an actively driven internalization process. We also demonstrate the advantages of specific biotinylation compared to classical antibody detection during agonist-induced receptor internalization, which may be used for immunofluorescence analysis as well. Site-specific biotinylation may be applicable to studies on trafficking of transmembrane proteins, in general

Constitutive receptor internalization and its modulation by receptor ant-/agonists.

<p>A: RBL cells stably expressing CXCR4-AP or CCR5-AP were enzymatically biotinylated and incubated in BM medium for up to four hours (37°C). Cells were stained with anti-AP (dashed lines) or streptavidin (straight lines) and analyzed by flow cytometry. B: CCR5-AP cells were either treated with (filled symbols) or without (open symbols) ant-/agonists (CCL5 0.05 μM; Met-CCL5 0.15 μM; TAK779 3 μM) and stained in accordance to A. All results represent the mean value +/- s.d. of at least three independent experiments.</p

Double immunoflourescence of CXCR4-/CCR5-expressing cells during ligand-induced internalization (30’) and recycling (60’).

<p>Prior to stimulation and staining CXCR4-AP (bottom) and CCR5-AP (top) expressing RBL cells were seeded on glass cover slips. Cells were enzymatically biotinylated and stimulated with the corresponding ligand (125 nM CCL5, CXCL12; 30’/37°C). Recycling was induced by acid wash and transfer into ligand-free medium containing receptor antagonists (30 μM AMD 3100; 3 μM TAK779). Cells were fixed with 3% PFA (15’/37°C) and permeabilized with 0.1% saponin (15’/37°C). 10 μg/ml anti-AP antibody (anti AP; with or without preincubation with 2mg /ml of the AP-peptide) and 2 μg/ml streptavidin-Alexa647 (Biotin) was used for staining (60’/on ice/dark). Samples were fixed with mounting medium and analyzed by confocal laser scanning microscopy. Scale bar 10 μM.</p

Binding of anti-AP and anti-receptor antibodies to RBL-CXCR4/CCR5-cells.

<p>RBL-CXR4-AP (right) or RBL-CCR5-AP (left) cells were stained (60’/4°C) with 10 μg anti-AP (upper panels) or 1.5 μg PE-labeled anti-receptor antibody (anti CXCR4 12G5, anti-CCR5 T21/8) (lower panels) after preabsorption of antibodies with 15 μg AP-peptide (30 ‘/rt) or pretreatment of receptor-expressing cells with 50 nM ligand (CCL5/CXCL12) (10’/4°C). Receptor-bound antibodies were detected with FITC-labeled anti-mouse IgG antibodies. RBL-2H3 cells served as negative control. Representative diagrams show the mean channel of fluorescence (MCF) in correlation to detected events.</p

Ligand-induced internalization and recycling in presence or absence of receptor antagonists.

<p>A: RBL cells stably expressing CXCR4- (left) or CCR5-AP (right) were enzymatically biotinylated and incubated in BM medium containing 125 nM ligand (30’/37°C). Recycling was triggered by acid wash to remove the ligand and transfer into ligand-free medium (30’/37°C). Cells were stained with anti-AP (dashed line) or streptavidin (straight line) and analyzed by flow cytometry. Each curve shows the mean percentage (+/- s.d.) of receptors expressed on the cell surface normalized to the MCF value of untreated cells. B: Cells were treated and stained accordingly to panel A (grey bars anti-AP, black bars streptavidin). During the recycling phase the corresponding antagonist was added (TAK779 3 μM; AMD3100 30 μM). C: RBL cells expressing CCR5-AP S/A (filled triangle up) or CCR5-AP WT (open square) were enzymatically biotinylated. Internalization was triggered as described in panel A. Staining was done with streptavidin-PE. Receptor recycling was calculated as percentage of the difference between cell surface expression of the receptor at time points 0 and 30 minutes Results represent mean +/- s.d. of at least three independent experiments. n.s., not significant; **, p < 0.001).</p

BirA-mediated biotinylation of AP-tagged CCR5 on RBL cells.

<p>RBL cells (2H3) expressing CCR5, CCR5-AP or no receptor (2H3) were either biotinylated with BirA (BirA-Biotin) or biotin-X-NHS (NHS-Biotin; 15’/rt). Cells were lysed (10’) and immunoprecipitated via streptavidin agarose (90’/4°C). Samples were probed by immunoblotting with anti-CCR5 (T21/8; left) or streptavidin peroxidase conjugates (right).</p