Two serines in the distal C-terminus of the human ß₁-adrenoceptor determine ß-arrestin2 recruitment

<div><p>G protein-coupled receptors (GPCRs) undergo phosphorylation at several intracellular residues by G protein-coupled receptor kinases. The resulting phosphorylation pattern triggers arrestin recruitment and receptor desensitization. The exact sites of phosphorylation and their function remained largely unknown for the human β₁-adrenoceptor (ADRB1), a key GPCR in adrenergic signal transduction and the target of widely used drugs such as β-blockers. The present study aimed to identify the intracellular phosphorylation sites in the ADRB1 and to delineate their function. The human ADRB1 was expressed in HEK293 cells and its phosphorylation pattern was determined by mass spectrometric analysis before and after stimulation with a receptor agonist. We identified a total of eight phosphorylation sites in the receptor’s third intracellular loop and C-terminus. Analyzing the functional relevance of individual sites using phosphosite-deficient receptor mutants we found phosphorylation of the ADRB1 at Ser461/Ser462 in the distal part of the C-terminus to determine β-arrestin2 recruitment and receptor internalization. Our data reveal the phosphorylation pattern of the human ADRB1 and the site that mediates recruitment of β-arrestin2.</p></div

The β₁-adrenoceptor is phosphorylated at multiple sites in the third intracellular loop and at the C-terminus.

<p>(<b>A</b>) Phosphorylation of the ADRB1 with and without stimulation with NE (100 μM) for 5 min. Cells stably expressing the human ADRB1 were cultured in medium containing 1% FCS and ³²P. After cell lysis and IP of the ADRB1, the amount of phosphorylated ADRB1 (pADRB1) was quantified by phosphor imager analysis. Total ADRB1 was subsequently visualized by western blotting. (<b>B</b>) Quantification of ADRB1 phosphorylation from (A), n = 3. (<b>C</b>) Preparation of samples for mass spectrometry. (<b>D</b>) Western blot after crosslink IP of the ADRB1. HEK: untransfected HEK293 cells (negative control); ADRB1: HEK293 cells stably expressing the β₁-adrenoceptor. (<b>E</b>) Silver staining of SDS gel showing the ADRB1 after IP. (<b>F</b>) Phosphorylation pattern of the ADRB1. Amino acids marked in light grey and black indicate phosphorylation under basal conditions and after stimulation with 100 μM NE for 5 min, respectively. Pool of five experiments. Annotated spectra of the detected phosphosites are presented in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176450#pone.0176450.s001" target="_blank">S1 Fig</a>.</p

Phosphorylation at the distal C-terminus of the ADRB1 determines receptor internalization, but does not alter stimulation-dependent MAP kinase activation.

<p>(<b>A</b>) Representative Western blots using antibodies directed against activated MAPK1/3 (pMAPK1/3), MAPK1/3 and ADRB1. HSP90 was used as a loading control. Lysates were prepared from HEK293 cells stably expressing the indicated ADRB1 mutants and stimulated with NE (100 μM for 5 min) or control. (<b>B</b>) Quantification of MAPK1/3 activity from (A). Data are means+SEM, n = 3–9. (<b>C</b>) Internalization of ADRB1 mutants expressed in HEK293 cells (together with ARRB2) as determined by loss of cell surface receptor labeling with ³H-CGP-12177. Time points indicate duration of treatment with NE prior to labeling with ³H-CGP-12177. Data are means+SEM of n = 5 independent experiments. ** p ≤ 0.01, *** p ≤ 0.001 as determined by two-way ANOVA.</p

Phosphorylation at Ser461/Ser462 in the distal C-terminus determines arrestin binding.

<p>(<b>A</b>) Schematic of the human ADRB1 highlighting the phosphosites mutated in the distal C-terminus: ADRB1(Ala459/461/462) (light blue), ADRB1(Ala461/462) (orange) and ADRB1(Ala473/475) (dark grey). (<b>B</b>) Averaged FRET tracings comparing ADRB1(Ala459/461/462), ADRB1(Ala461/462) and ADRB1 (Ala473/475). Data are means±SEM of n ≥ 6 representative tracings. (<b>C</b>) Quantification of NE-induced changes of receptor-arrestin FRET obtained for the ADRB1 variants depicted in (B). Data are means+SEM of n ≥ 9 cells. *** p ≤ 0.001 vs. wild-type and n.s. = not significant. (<b>D</b>) Conservation of serine 459 (light blue), serine 461 and serine 462 (orange) among twelve species.</p

Phosphorylation sites in the C-terminus are crucial for β-arrestin2 recruitment.

<p>(<b>A</b>) Schematic of the FRET assay for β-arrestin2 recruitment (left) and confocal microscopy of representative cells expressing Cerulean-tagged ADRB1 and YFP-tagged β-arrestin2 (right, scale = 10 μm). (<b>B</b>) Representative tracings (left) and their ratiometric representation (right) of simultaneous recording of emissions from YFP (535 nm) and Cerulean (480 nm) upon stimulation of cells with norepinephrine (NE, 10 μM). (<b>C, D</b>) Schematics of the human ADRB1 highlighting the phosphosites mutated in the third intracellular loop and the C-terminus. (<b>E</b>) Averaged FRET tracings comparing wild-type ADRB1, ADRB1Δ3rdloop^a (= ADRB1(Ala260), light green) and a mutant devoid of all phosphosites in the third intracellular loop and C-terminus (ADRB1Δphos). (<b>F</b>) Quantification of NE-induced changes of receptor-arrestin FRET obtained for the ADRB1 variants depicted in (C). (<b>G, H</b>) Analogous experiments as in (E, F) for C-terminal mutants. Data are means±SEM from ≥10 tracings (E, G) and ≥ 30 cells (F, H). Kruskal-Wallis-Test with Dunn‘s post test. *** p ≤ 0.001 vs. wild-type and n.s. = not significant.</p

Composition of CXCL10-mucin-GPI as an example for a novel class of GPI-anchored chemokine fusion proteins.

<p>The mucin domain of CX3CL1 was combined with a GPI anchor and a CXCL10 chemokine head to generate a flexible tool for the modification of tumor micromilieus capable of selectively stimulating the recruitment of CXCR3⁺ leukocytes. The chemokine head directs the specificity towards CXCR3⁺ leukocytes, while the mucin domain assists in the recruitment process and lowers the requirement for other adhesion molecules. Inclusion of a GPI anchor allows the protein to integrate into the cell membranes of tumor, stromal and endothelial cells when applied exogenously, thus superseding the transfer of genetic material into the tumor.</p

CXCL10-mucin-GPI induces rolling and tight adhesion of CXCR3⁺ NK cells under conditions of physiologic flow.

<p>Laminar flow assays were performed to test if resting primary human microvascular endothelial cells treated with the GPI-anchored CXCL10 fusion proteins could recruit freely flowing CXCR3⁺ NK cells (YT) under conditions of physiologic flow. <b>A</b>: Resting primary human endothelial cells from fetal foreskin, selected for blood vessel endothelial cells, were treated with 0.34 nM of GPI-anchored CXCL10 fusion proteins or with identically diluted sEGFP-GPI protein for 1 h. Other slides were treated with commercially available CXCL10 at 1000-fold higher concentration as additional control (rhCXCL10). All samples contained the same percentage of chromatography buffer and detergent. Subsequently, YT cells were perfused over the endothelial cells with 1 dyn/cm² and the number of cells accumulating on the endothelial cells was counted. Data shown here are derived from six independent experiments, each performed using independent protein preparations and separate batches of cells. Bars represent the average numbers of cells adhering to the endothelium, +/− SEM. Statistical significance was calculated using the Kruskal-Wallis test (P = 0.0022) followed by Dunńs post test; ** = P<0.01. <b>B</b>: Experiments were performed as detailed in A. Tight adhesion was defined as an event in which a particular NK cell adhered to the endothelium and did not move further than one cell diameter within 30 sec. Rolling adhesion was defined as an event in which the NK cell adhered to the endothelium, but was dragged along the endothelium by the shear forces exerted by the buffer at a higher speed than one cell diameter per 30 sec or detached again. Cells displaying both rolling and tight adhesion were counted only as tightly adherent. Bars represent averaged values derived from four independent experiments +/− SEM. Statistical significance was calculated using the Kruskal-Wallis test (P = 0.0038 for rolling adhesion and 0.0021 for tight adhesion) followed by Dunńs post test; * = P<0.05, ** = P<0.01.</p

Purified GPI-anchored proteins incorporate into cell membranes.

<p><b>A</b>: In order to test the capacity of the purified recombinant fusion proteins to reincorporate into cell membranes, non-transfected CHO cells were incubated with the purified GPI-anchored proteins (0.9 nM) for 1 h at 37°C. As controls, two samples were treated either with identically diluted chromatography buffer (buffer) or MEM alpha medium (medium). The soluble CXCL10-mucin-Stop protein served as an additional control as it lacks a GPI anchor. All samples except the medium control contained the same percentage of chromatography buffer and detergent. Following incubation, the cells were washed and tested for the presence of the proteins on their surface by FACS staining. Dead cells were identified by 7-AAD staining and the histograms shown are gated on viable cells. The black lines indicate staining with isotype-matched control antibodies, blue lines staining with anti c-myc antibodies. Mean fluorescence intensities (MFIs) are given for each sample. This experiment was performed routinely to monitor protein quality after purification and the data shown here therefore stand representative for over 20 independent experiments. <b>B</b>: To verify the subcellular localization of the incorporated proteins, immunofluorescence microscopy was performed. Primary microvascular endothelial cells were treated with purified CXCL10-GPI, CXCL10-mucin-GPI or a buffer control diluted in culture medium, with all samples containing the same percentage of buffer to exclude artifacts. After treatment, the cells were washed, fixed with Paraformaldehyde and incorporated proteins were detected using anti c-myc primary and biotinylated secondary antibodies followed by RPE-labeled streptavidin. The figure shows fluorescence images and corresponding bright field images from a representative experiment, which was performed three times. All images within each row were acquired using the same exposure time. The black bars indicate 50 µm.</p

Subcutaneously implanted 291 tumors are well vascularized and show a pronounced infiltration with T cells.

<p>Tumor cells were injected subcutaneously into the flanks of C57BL/6 mice. Grown tumors were excised, fixed and analyzed by hematoxylin/eosin (H/E) staining or immunohistology using antibodies against CD3 or NKp46. <b>A–F</b>: The respective magnifications are indicated at the top of each image. Panels A and B show the presence of numerous blood vessels (arrows) throughout the tumors in H/E staining. Panels D, E and F represent serial sections of the same position within a tumor. Various blood vessels are visible in H/E staining and the CD3 staining shows infiltration of the tumor with CD3⁺ cells (dark grey/black staining). Less NKp46⁺ cells could be detected in the respective staining of the same position (dark grey/black staining; one cell is marked by an arrow). Panel C depicts a cluster of NKp46⁺ cells, which was sometimes found at the margins of tumors (margin: upper part of the picture). <b>G</b>: Injection of CXCL10-mucin-GPI tends to increase endogenous NK cell infiltration of subcutaneous tumors. Purified CXCL10-mucin-GPI was injected into established 291 tumors. As controls, either the same or a 500×higher molar quantity of commercially available human CXCL10 (rhCXCL10) were injected. As additional controls, the same volume of identically purified sEGFP-GPI was injected or the tumors were left completely untreated. The animals were sacrificed 4 h after injection and infiltration of the tumors was assessed by FACS analysis. The figure shows the percentage of CD3^- NK1.1⁺ cells among the total lymphocyte count with each symbol representing one individual tumor and horizontal bars the average values. Statistical significance was calculated using the Kruskal-Wallis-test (P = 0.19) followed by Dunńs post test (not significant, N.S.).</p