We have provided the first evidence for specific heteromerization between the

We have provided the first evidence for specific heteromerization between the α1A-adrenoceptor (α1AAR) and CXC chemokine receptor 2 (CXCR2) in live cells. SB265610. Furthermore Labetalol which is marketed for hypertension as a nonselective β-adrenoceptor antagonist with α1AR antagonist properties was identified as a heteromer-specific-biased agonist exhibiting partial agonism for inositol phosphate production but essentially full agonism for β-arrestin recruitment at the α1AAR-CXCR2 heteromer. Finally bioluminescence resonance energy transfer studies with both receptors tagged suggest that α1AAR-CXCR2 heteromerization occurs constitutively and is not modulated by ligand. These findings support the concept of GPCR heteromer complexes exhibiting distinct pharmacology thereby providing additional mechanisms through which GPCRs can potentially achieve their diverse biological functions. This has important implications for the use and future development of pharmaceuticals targeting these receptors. luciferase (Rluc) also kindly provided by Aron Chakera. Similarly the α1AAR/Rluc8 and orexin receptor 1 (OxR1)/Rluc8 cDNA constructs were generated from α1AAR/Rluc and OxR1/Rluc respectively (formerly produced by CGK 733 PCR amplification of receptor cDNA to remove the stop codon and ligation into pcDNA3 containing Rluc). CGK 733 OxR1/Rluc was generated previously in our laboratory by Matthew Dalrymple from OxR1 cDNA kindly provided by Masashi Yanagisawa (Howard Hughes Medical Institute). With all of these constructs the Rluc coding region was replaced with Rluc8 cDNA from pcDNA3.1-Rluc8 kindly provided by Andreas Loening and Sanjiv Gambhir (Stanford CGK 733 University) (32) as described previously for other GPCR constructs (33). Vasopressin receptor 2 (V2R)/Rluc8 was generated as described previously from V2R cDNA kindly provided by Brian Feldman (Stanford University) (34). The β-arrestin2/Venus cDNA construct was prepared previously from pcC2-Venus kindly provided by Atsushi Miyawaki (RIKEN Brain Science Institute Wako-city Japan) (33). CXCR2/Venus and V2R/Venus were prepared by replacing Rluc8 cDNA with Venus cDNA in the CXCR2/Rluc8 and V2R/Rluc8 constructs respectively as described CGK 733 previously for V2R/Venus (34). Ligands used were NE CXCL8 (Interleukin-8) CCL2 (MCP-1) arginine vasopressin Terazosin and Labetalol (Sigma) as well as SB265610 (Tocris) and Orexin A (American Peptide Co.). Cell Culture and Transfection HEK293FT cells were maintained at 37 °C in 5% CO2 and complete media (Dulbecco’s modified Eagle’s medium (DMEM) containing 0.3 mg/ml glutamine 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen)) supplemented with 10% fetal calf serum (FCS) and 400 μg/ml Geneticin (Invitrogen). Transient transfections were carried out 24 h after seeding about 550 0 cells/well of a 6-well plate. Genejuice (Novagen) transfection reagent was used according to the manufacturer’s instructions. Cells were harvested with 0.05% trypsin-EDTA (Invitrogen). For testing the CXCR2 antibody specificity Chinese hamster ovary (CHO) cells were seeded at a density of 40 0 cells/well of a 12-well plate and transiently transfected using Lipofectamine (Invitrogen) as per the manufacturer’s instructions. Cells were maintained at 37 °C in 5% CO2 and high glucose (25 mm) DMEM (supplemented with 4 mm Glutamax (Invitrogen) 5 FCS 16 mm HEPES 100 IU/ml penicillin 100 μg/ml streptomycin 1 mg/ml hygromycin B and 1 mm sodium pyruvate). Assessment of CXCR2 Antibody Rabbit polyclonal to KCNC3. Specificity CHO cells were immunostained 36 h after transfecting with 1 μg of CXCR2 or CCR2 cDNA per well of a 12-well plate. Cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature (RT). After three 10-min washes in PBS the cells were blocked and permeabilized (30 min RT) with 5% donkey serum in PBS containing 0.1% Triton X-100 and incubated overnight at 4 °C with rabbit polyclonal anti-CXCR2 antibodies (1:200; Abcam ab14935) in PBS containing 5% donkey serum 0.1% sodium azide and 0.1% Triton X-100. Cells were then washed as above and incubated with Alexa Fluor 594 donkey anti-rabbit antibodies CGK 733 (1:200; Invitrogen; 1 h RT). Cells were washed again as above CGK 733 and the nucleus was stained with TO-PRO-3 Iodide (1:3000 in PBS Invitrogen). Images were acquired using a Nikon A1R confocal microscope with 561 and 640 lasers and 595/50 and 700/75 emission filters respectively as well as a Nikon S Plan Fluor.