Analysis of GPCR Activity on the EIDAQ™ 100 High Throughput Microscopy System
This application note describes the successful deployment of the NORAK Transfluor™ assay for GPCR
activity on the EIDAQ™ 100 High Throughput Microscopy system from Q3DM. The Norak Transfluor™
assay measures GPCR activation by the formation of GFP labeled pits, or vesicles, depending on the
GPCR that is being expressed by a cell. The EIDAQ™ 100 is a novel High Throughput Microscopy
(HTM) system that delivers accurate, quantitative, imaging, and analysis of cell populations, at high
speeds, directly from microtiter plates. For accurate reading/measurement, the EIDAQ™ 100 quantifies
distribution of fluorescence emanating from the GPCR associated arrestin-GFP fusion protein and in
sub-cellular compartments. Definitive measurements are quantified with a proprietary, multi-scale
vesicle definition algorithm developed at Q3DM.
Background and Significance
GPCRs mediate the activity of cell surface receptors and the transduction of a myriad of intra-cellular
responses. GPCRs have proven to be a highly amenable class of targets for successful therapeutic
intervention. In fact, of the approximately 500 drugs currently on the market today, more than 30% are
mediated through the activation of GPCRs. Because GPCRs are membrane-bound proteins, they have
been difficult to study in cell extracts, or to isolate and characterize. To directly ascertain GPCR activity,
intact cell-based assays are quickly becoming a method of choice in high-throughput screening.
GPCRs transduce extracellular signals through the formation of protein complexes that effect both
activation and subsequent desensitization of a cell surface receptor. Agonist binding to a receptor at the
cell surface initiates a conformational change in the intracellular domain of the receptor that results in the
phosphorylation of the receptor and subsequent binding of arrestin to the receptor. The arrestin-receptor
complex is then transported to clathrin-coated pits and internalized to clathrin-coated vesicles. Finally,
the entire complex is delivered to the endosomes. Some GPCRs dissociate from arrestin at or near the
plasma membrane, while others remain associated and traffic into endocytic vesicles.
Norak Transfluor™ GPCR Assay – Q3DM EIDAQ100™ HTM Analysis
This validation report describes the performance of the NORAK Transfluor™ assay for GPCR
activity on the EIDAQ™ 100 High Throughput Microscopy (HTM) system from Q3DM. The
EIDAQ™ 100 HTM system performs accurate reading & measurement of GPCR activity by
quantification of GPCR associated formation of GFP labeled pits or vesicles using a
proprietary, multi-scale vesicle definition algorithm. The performance of this algorithm in
combination with the EIDAQ™ 100 HTM system was evaluated by repeatably determining EC50
& IC50 values for agonist and antagonist well plates. The results showed very good repeatability
of the GPCR analysis using the EIDAQ™ 100 HTM system and CytoShop™ software suite with
the proprietary GPCR algorithm proving that the Q3DM system in combination with the Norak
Transfluor™ assay presents a powerful quantification tool for GPCR activity.
Background and Significance
GPCRs mediate the activity of cell surface receptors and the transduction of a myriad of intracellular responses. They have proven to be a highly amenable class of targets for successful
therapeutic intervention. In fact, of the approximately 500 drugs currently on the market today,
more than 30% are mediated through the activation of GPCRs. Because GPCRs are membranebound proteins, they have been difficult to study in cell extracts, or to isolate and characterize.
To directly ascertain GPCR activity, intact cell-based assays are quickly becoming a method of
choice in high-throughput screening.
GPCRs transduce extracellular signals through the formation of protein complexes that effect
both activation and subsequent desensitization of a cell surface receptor. Agonist binding to a
receptor at the cell surface initiates a conformational change in the intracellular domain of the
receptor that results in the phosphorylation of the receptor and subsequent binding of arrestin to
the receptor. The arrestin-receptor complex is then transported to clathrin-coated pits and
internalized to clathrin-coated vesicles. Finally, the entire complex is delivered to the
endosomes. Some GPCRs dissociate from arrestin at or near the plasma membrane, while others
remain associated and traffic into endocytic vesicles.
The combination of automated sub-micron imaging, proprietary image processing and the Norak
Transfluor™ assay enables accurate measurement of GPCR activity through vesicle response
and pit formation. Validated results of GPCR activity using the EIDAQTM 100 HTM system are
presented with fluorescent images and dose response curves.
Experimental Methods
Norak Transfluor™ Assay Plates
In the Norak Transfluor™ assay, a cell line is developed to monitor the interaction of a given
GPCR and a GFP fused to ß-arrestin. When each GPCR is activated, the ß-arrestin will bind to
the membrane associated GPCR. The activated ß-arrestin-GPCR complex then enters clathrincoated pits and migrates to intracellular vesicles via the endosomal pathway. Some GPCRs retain
the arrestin molecule throughout this process, so that vesicles will fluoresce with GFP. Other
GPCRs will dissociate from the arrestin such that the GFP remains with the pits, or is released to
the intracellular space, and the receptors recycle back to the cell surface and bind arrestin again.
VALIDATION REPORT
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Negative
Positive
Figure 1: Cell analysis for GPCR - example
images from a well plate scan; negative response
(top) compared to positive response (bottom);
pseudo-color overlay of nuclear dye Hoechst
(blue) and GFP ß-arrestin (green) shown in left
column, additionally masks for GPCR
measurement (red) shown in right column.
Two different plates were used, one containing a wild type cell line, the other containing an
enhanced cell line. The layout of the plates was identical and is shown in Table 1.