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PathHunter® GPCR Explorer
The PathHunter® GPCR Explorer Kit is a simple and easy-to-use method for building on-demand functional GPCR assays in just 3 simple steps – Clone. Transfect. Read. Whether you have a panel of mutant GPCR cDNAs or a favorite GPCR heterodimer you’d like to functionally test, the simple, optimized transfection protocol combined with PathHunter® EFC-based chemiluminescent detection makes it the ideal platform for exploring novel GPCR receptor biology.
Product Features
• Based on the G-protein independent β-Arrestin read-out
• Contains highly transfectable, ready-to-assay CHO-K1 cells
• Fast and simple protocol
• Chemiluminescent detection used with any standard luminometer
• Choice of vectors optimized for maximal expression in mammalian cells
Clone It. Transfect It. Read It.
Like the PathHunter® Arrestin GPCR assays, PathHunter® GPCR Explorer kits monitor GPCR activity by detecting the interaction of β-Arrestin with activated GPCRs using β-galactosidase (β-gal) enzyme fragment complementation. Because Arrestin recruitment occurs independent of G-protein coupling status, these assays provide a direct, universal platform for measuring receptor activation.
Express Any GPCR of Interest
First clone your GPCR of interest into one of our four ProLink Cloning Vectors. All ProLink vectors are designed such that the GPCR of interest is cloned in frame with the weakly complementing fragment of β-gal, termed ProLink (PK). All cloning vectors utilize a minimal CMV promoter for effective expression in mammalian cells.
*G-protein receptor kinases (GRKs) phosphorylate GPCRs, facilitating the recruitment of Arrestin. The Arrestin Recruitment Modifying Sequence (ARMS) is a GRK consensus phosphylation site deduced from analysis of GPCR which generate high S:B in the PathHunter assay.
Expedite Your Research
When you are ready to assay, plate your PathHunter® GPCR Explorer CHO-K1 cells
in the provided 96-well plates and transfect your ProLink-tagged vector 24 hours later.
The convenient, ready-to-assay format combined with a simple, optimized transient
transfection protocol and chemiluminescent detection lets you quickly and easily
generate high quality, proof-of-concept data to support your publication and grant
deadlines or hit important drug discovery milestones.
Click on figure to enlargeFigure 1. Build your own GPCR assays using the fast and simple PathHunter® GPCR Explorer protocol.
Explore Novel Receptor Biology
PathHunter® GPCR Explorer cells can be readily transfected with a wide variety of nucleic acids – mutant cDNAs, short interfering RNA (siRNA), DNA encoding GPCR accessory proteins (RAMPs or GRKs) or even a second GPCR for heterodimerization studies. By eliminating lengthy and time consuming cell culture, GPCR Explorer kits enable interrogation of multiple GPCR targets in only a few hours.
Click on figure to enlargeFigure 2. Creation of GPCR Biosensor Cell Lines
(A) CHO-K1 GPCR Explorer cells were plated at 10,000 cells per well and incubated overnight
at 37°C. The following day, cells were transfected with 2 µg of pEGFP vector and 48 hr
post-transfection, greater than 60% of the GPCR Explorer cells were transfected. (B) PathHunter
GPCR Explorer CHO-K1 cells were transiently transfected with 2 µg of the pCMV-SSTR2-PK vector
(+ SSTR2 DNA) or an empty vector (- SSTR2 DNA). Twenty-four hours later, cells were treated with
increasing concentrations of Somatostatin 28, a control agonist. Activation of the SSTR2 receptor
stimulates binding of Arrestin resulting in an increase in β-gal enzyme activity that can be
measured using chemiluminescent PathHunter Detection Reagents.
Figure 3. Study novel receptor biology
Several Orphan GPCRs (oGPCRs) are suspected to modulate the function of other GPCRs
co-expressed in the cell. In this experiment, an untagged oGPCR, CCBP2, was co-expressed
with Chemokine Receptor 2, CCR2. Co-expression of CCBP2 resulted in a decrease in the
responsiveness of the cell line to the CCR2 ligand indicating that CCBP2 negatively regulates
CCR2 signaling.
Applications
• GPCR Heterodimerization / Co-receptor expression studies
• GPCR profiling (mutants, orthologs)
• siRNA
• Novel receptor biology
Kit Contents
• PathHunter GPCR Explorer CHO-K1 cells
• PathHunter Detection Reagents
• Cell Plating Reagent
• Control pCMV-PK2 vector & ligand
• 96-well plate (only for 100 dp kit)
Download the user manual 