GoKawiilCell culture, antibodies, and reagents
HEK-293 cells (ATCC), including transient and stable lines, as well as CRISPR–Cas9 βARR1/2-knockout and parental lines61, were maintained in Eagle’s Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C and 5% CO 2 . U2OS cells (DiscoveRx PathHunter) were cultured in MEM containing 2 mM l-glutamine, 10% FBS, and 1% penicillin-streptomycin under similar conditions. U2OS-based β-arrestin recruitment and internalization assays were performed per the manufacturer’s protocol (DiscoveRx). For chemokine-induced migration assays, leukocytes were isolated from wild-type mice as described previously18,37,61. Escherichia coli strains DH5α and BL21 (DE3) (New England Biolabs) were cultured in LB or Terrific Broth (Fisher Scientific) at 37 °C. DH5α was used for plasmid amplification, and BL21 was used for recombinant protein expression. Sf9 insect cells (Spodoptera frugiperda, Expression Systems, 94-001F) were cultured in ESF 921 medium (Expression Systems) at 27 °C. Baculoviruses were generated and amplified according to the manufacturer’s instructions. For serum starvation experiments, cells were cultured in serum-free medium supplemented with 0.1% BSA, 10 mM HEPES, and 1% penicillin-streptomycin. Transfections were performed using FuGene 6 (Promega) or Lipofectamine 3000 (Invitrogen), according to manufacturers’ protocols. All cell lines were confirmed mycoplasma-free by routine testing through the Duke University cell culture facility. Monoclonal anti-Flag M2–horseradish peroxidase (HRP) (A8592) and anti-ERK1/2 (ABS44) antibodies were obtained from Sigma-EMD Millipore. HRP-conjugated secondary antibodies (NA9340-1ML and NA9310-1ML) were purchased from Cytiva. Protease and phosphatase inhibitor tablets (cOmplete, PhosSTOP) were obtained from Roche. Anti–phospho-p44/42 MAPK (Thr202/Tyr204) antibody (9101L) was sourced from Cell Signaling Technology.
Recombinant protein expression and purification
The expression and purification of β-arrestins have been described previously44,62,63,64. In brief, E. coli BL21 (DE3) pLysS cells (New England Biolabs: C2527I) carrying pGEX4T1-Rattus norvegicus βARR1 or βARR2 construct, with their C terminus truncated (at amino acid 393 or 394, respectively), were cultured in Terrific Broth (Teknova) medium at 37 °C. After OD 600 reached 0.6–0.8, the cells were induced with 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 18 °C overnight. Bacteria were collected by centrifugation (4,000 rpm), and cell pellets were resuspended in lysis buffer (20 mM HEPES, pH 8, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 0.2 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine) at 4 °C for 1 h. Cell suspensions were sonicated, centrifuged (14,000 rpm, 30 min, 4 °C), and the supernatant was loaded onto pre-equilibrated glutathione–agarose resin (GoldBio). After 3 h of binding at 4 °C, the beads were washed, and thrombin was added for overnight cleavage. The proteins were further purified by anion exchange chromatography followed by size-exclusion chromatography using a Superdex 200 Increase 10/300 GL column on an ÄKTA FPLC system (GE Healthcare). Eluted fractions were analysed by SDS–PAGE, pooled, concentrated using 30 kDa MWCO Amicon Ultra-15 Centrifugal Filter devices, flash-frozen in liquid nitrogen, and stored in aliquots at −80 °C. The same rat βARR1 constructs with mutations (L129A, L129S, L129G, P131A, P133A, E134A, D135A, K138A, C140A, Y249A, C251A, E283A, K284A and R285A) were generated and purified in a similar manner. Recombinant Gα subunits (Gα s , Gα q and Gα i ) were purified as GST fusion proteins using glutathione–agarose affinity chromatography as previously described65. The heterotrimeric G s protein complex, composed of Gα s , Gβ1 and Gγ2 subunits, was expressed in Sf9 cells using the baculovirus expression system and purified as previously described66. M 2 R expression, purification and reconstitution into high-density lipoprotein (HDL) particles were performed as previously described58. ERK2, SRC, p38α and JNK3 kinases were expressed and purified using previously established protocols6,16. Protein concentrations for each protein were determined by ultraviolet absorption at 280 nm and extinction coefficients estimated using the ExPASy ProtParam tool67.
Expression of β 2 AR constructs in Sf9 cells using the baculoviral system
The N-terminal Flag-tagged T4 lysozyme fusion-β 2 V 2 R construct (β 2 AR residues 1–341 fused to V 2 R residues 328–372), bearing a TEV cleavage site, was co-expressed with GRK2–CAAX (membrane-anchored GRK2) or wild-type β 2 AR in Sf9 insect cells using the Baculovirus Expression System, as described previously44,62,63,68,69. At 66 h after infection, cells expressing T4L–β 2 V 2 R were stimulated with 20 μM isoproterenol for 20 min at 37 °C to induce receptor phosphorylation (pβ 2 V 2 R), while β 2 AR-expressing cells remained untreated. All cells were washed extensively to remove residual agonist. For membrane preparation from these cells, briefly, cells were resuspended in cold homogenization buffer (75 mM Tris-HCl, pH 7.4, 2 mM EDTA, and cOmplete protease inhibitor) and collected by centrifugation at 500g for 5 min at 4 °C. After 2 additional rounds of centrifugation at 500g for 5 min at 4 °C, the supernatant was centrifuged at 21,000g for 30 min at 4 °C to collect crude membrane fractions. Pellets were then washed with resuspension buffer (75 mM Tris-HCl, pH 7.4, 2 mM EDTA, 12.5 mM MgCl 2 , cOmplete protease inhibitor, and PhosSTOP phosphatase inhibitor), passed through a 0.4 mm gauge needle 30 times using a syringe on ice, aliquoted, flash-frozen in liquid nitrogen, and stored at −80 °C.
Small-molecule library and reagents
A collection of structurally diverse, drug-like small-molecule libraries used in this work was obtained from the NCI/DTP Open Chemical Repository. The compound library comprised approximately 3,500 compounds, representing the structural diversity of over 250,000 unique small molecules. This DTP library included the NCI Diversity Set, natural products, and FDA-approved oncogenic drugs. Most compounds were certified as >95% pure by the supplier (NCI DTP Discovery Services). Powdered compounds were dissolved in 100% DMSO and stored at −20 °C. Isoproterenol (Sigma, I2760-1G), ICI-118,551, carvedilol, angiotensin II (ANGII; Sigma, A9525), and human epidermal growth factor (EGF) were purchased from Sigma-Aldrich, and AVP was obtained from GenScript. All small-molecule compounds, including BI-167107 (ref. 70), were dissolved in DMSO and stored at −20 °C as 100 mM stock solutions. The C-terminal peptide of the GPCR vasopressin-2 receptor (V 2 R), known as V 2 Rpp, was synthesized by the Tufts University Analytical Core Facility. Cellular agonist stimulations were performed at 37 °C, as described in the figure legends.
Differential scanning fluorimetry
DSF assay was performed to identify β-arrestin-binding small molecules from the drug-like compound library (DDLC) described above. The screen was conducted using the StepOnePlus Real-Time PCR System (Applied Biosystems) with the fluorescent reporter probe SYPRO Orange (Thermo Fisher Scientific) in a 96-well format. Proteins were buffered in 20 mM HEPES (pH 7.5) with 100 mM NaCl. Small molecules (100 μM) were screened with either βARR1 or βARR2 (5 μM). DMSO was used as a vehicle control, and V 2 Rpp (50 μM) served as a positive control.
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