Spatial Intensity Distribution Analysis (SpIDA): A New Tool for Receptor Tyrosine Kinase Activation and Transactivation Quantification - Methods in Cell Biology - page 5

FIGURE 1.1
SpIDA allows for pharmacological characterization of EGFR transactivation by NK1 GPCR.
Dose-response curves of EGFR dimer density 1 min after substance P stimulation in CHO-k1
cells. Cells were transfected with either 1.5

m g of GPCR DNA (n ¼ 120 cells/point from six

individual experiments) or 6 60 cells/point from three individual experiments). Cells

were cotransfected with Rab5 S34N to prevent receptor internalization. Data are
means

m g(n

¼

SEM.

resulting from the stimulation of a cotransfected GPCR, specifically stimulation
of neurokinin 1 receptors (NK-1R) by substance P . CHO-EGFR-eGFP cells
( Brock, Hamelers, & Jovin, 1999 ) were transiently transfected with NK-1R DNA,
and EGFR-eGFP dimerization was measured by SpIDA following agonist-specific
stimulation by increasing doses of substance P . Dimer densities were plotted using
the Hill-Langmuir binding isotherm model, and SpIDA results allowed us to extract
key parameters including D 50 (ligand concentration required to obtain 50% maximum
dimer density signal) and D max (maximal level of receptor dimer density signal).
The fitted parameters obtained, D 50 and D max , can be compared to similar pharma-
codynamic parameters like Bret50 and Fret50 commonlymeasured inRET-based phar-
macological studies ( Ayoub et al., 2007; Masri et al., 2008; Salahpour &Masri, 2007 ).
Therefore, application of SpIDA to CLSM image analysis allows for the direct
comparison of transactivation efficiencies across different cell types and tissues. As
demonstrated in Fig. 1.1 , SpIDA is able to reveal that the abundance of the GPCR
may be a limiting factor inRTK transactivation. Indeed, increasing the amount ofGPCR
cotransfected intocells results inan increased D max , similar towhatwouldbeobserved in
thecaseofa B max in a binding experiment ( Kenakin, 2009; Swift et al., 2011 ).
The analysis further allows for quantification of receptor internalization, as being
evaluated by a reduction in surface receptor density. In the case of RTK transactiva-
tion by GPCRs, receptor internalization may be prevented by transfection of a rab5
mutant DNA construct, rab5 S34N ( Bucci et al., 1992; Li & Stahl, 1993 ). Using a