Table 1.
Effect of Gαq Point Mutants on RH domain binding. The table summarizes the effects of the Gαq point mutants on binding to GRK2, RGS4 and RGS2. Pull-down assays were performed with GST-GRK2-(45–178), GST-RGS4 or GST-RGS2 on cell lysates that had been transfected with Gαq containing the different point mutations, as described in Experimental Procedures. There is no detectable binding of Gαq or Gαq mutants to GST alone. Binding to AlF4− activated forms of the mutants and to mutants in the Gαq-RC background was assessed for GRK2, RGS4 and RGS2; however, RGS4 does not bind to Gαq-QL so the effects of the point mutants in the QL form on binding could only be tested with GRK2 and RGS2. (+++) indicates that similar amounts (81–100% of control as described in Experimental Procedures) of the Gαq point mutant and wt Gαq bound to GRK2, RGS4 or RGS2, (++) indicates 51–80% of control, (+) indicates 21–50% of control and (−) indicates 0–20% of control. ND- not determined. Data are from 2 to 6 experiments.
| GST-GRK2-(45–178)
|
GST-RGS2
|
GST-RGS4
|
||||||
|---|---|---|---|---|---|---|---|---|
| Gαq construct1 | AlF4− | QL | RC | AlF4− | QL | RC | AlF4− | RC |
| wt-Gαq | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ |
| K77A2 | +++ | − | +++ | +++ | − | +++ | +++ | +++ |
| K77P | −3 | − | ND | ND | ND | ND | − | ND |
| L78D | ++4 | +++ | ND | ND | ND | ND | +++ | ND |
| Q81A2 | ++ | +++ | +++ | + | +++ | + | ++ | +4 |
| R92A2 | +++ | + | +++ | + | + | ++ | − | +++ |
| V118A | +++ | +++ | ND | ND | ND | ND | +++ | ND |
| Q152A2 | + | + | ++ | + | ++ | ++ | − | +++ |
| V184D | ++ | +++ | ND | ND | ND | ND | − | ND |
| P185K2 | − | − | − | +++ | − | − | +++ | ND |
| T187K2 | +++ | +++ | +++ | − | − | + | − | +4 |
| V240A | ++ | +++ | ND | ND | ND | ND | +++ | ND |
| D243A | − | +++ | ND | ND | ND | ND | ++ | ND |
Mutants are expressed at levels similar to Gαq-wt, Gαq-Q209L or Gαq- R183C, respectively, with the following exceptions: K77A-QL (39% of Gαq-QL), P185K (59% of Gαq), P185K-QL (72% of Gαq-QL), P185K-RC (30% of Gαq-RC) and Q152A-RC (75% of Gαq-RC).
The statistical significance of the difference between the indicated mutants and control is indicated in Figures 2 and 4 for GRK2 and RGS2, respectively.
For mutants indicated by a (−), statistical analysis could not be performed because there generally was no observable pull-down.
Binding of Gαq-L78D to GSTGRK2- RH is significantly different than binding of Gαq to GST-GRK2-RH (p < 0.001) and binding of Gαq-Q81A-RC and Gαq-T187K-RC to GST-RGS4 is statistically different then binding of Gαq-RC to GST-RGS4 (p < 0.001).