Table 1

Kinetic data of ligand-regulated GTPγS binding of β2AR-G fusion proteins: comparison with nonfused β2AR, β2AR coexpressed with Giα2, FPR-Giα2, and FPR coexpressed with Giα2

ConstructApparent Kd of Agonist-Stimulated GTPγS BindingApparent Kd of Inverse Agonist-Inhibited GTPγS BindingCoupling Factor
nM
β2AR2.4  ± 2.31-a 0.01  ± 0.01
β2AR-GsαL 0.4  ± 0.14.2  ± 0.51.12  ± 0.10
β2AR-GsαS 0.7  ± 0.20.95  ± 0.08
β2AR-Giα2 63.9  ± 5.71.05  ± 0.09
β2AR + Giα2 18.9  ± 5.80.91  ± 0.35
β2AR-Giα3 79.8  ± 9.00.95  ± 0.10
β2AR-G16α 4.4  ± 2.10.53  ± 0.02
β2AR-G 19.1  ± 1.40.08  ± 0.03
FPR-Giα2 0.9  ± 0.61.2  ± 0.3∼11-b
FPR + Giα2 0.8  ± 0.21.8  ± 0.7∼1.0–1.51-b

GTPγS binding was determined as described under Experimental Procedures. Sf9 membranes (10 μg protein/tube) expressing β2AR-G (3.7–8.6 pmol/mg) were incubated for 45 min in the presence of 0.1 to 1 nM [35S]GTPγS plus unlabeled GTPγS at different concentrations to give the final GTPγS concentrations of 0.1 to 10 nM. Experiments with membranes expressing nonfused β2AR (7.6–12.2 pmol/mg; 30 μg protein/tube) were performed under the same conditions as experiments with β2AR-G fusion proteins. GTPγS binding was stimulated with ISO (10 μM) or inhibited by ICI (1 μM). GTPγS saturation binding of β2AR-G was performed in membranes (42–71 μg protein/tube) expressing fusion proteins at 4.0 to 5.4 pmol/mg with GTPγS at 1 to 300 nM (1 nM [35S]GTPγS plus various concentrations of unlabeled GTPγS). The incubation time was 60 min. GTPγS saturation with membranes (40 μg protein/tube) expressing β2AR (2.3 pmol/mg) plus Giα2 (∼300 pmol/mg) was performed as for membranes expressing β2AR-Giα2. GTPγS saturation binding of β2AR-G16/qα was performed in membranes (41–81 μg/tube) expressing fusion proteins at 3.7–13.0 pmol/mg with GTPγS at 1–300 nM (1 nM [35S]GTPγS plus various concentrations of unlabeled GTPγS). The incubation time was 3 h. β2AR ligand-regulated GTPγS-binding saturation curves were analyzed by nonlinear regression and fit best to a one-site-binding hyperbola. The coupling factor is defined as the ratio of the B maxof ligand-regulated GTPγS binding and the B max of receptor antagonist binding of a given membrane. Data shown are the means ± SD of two to three experiments performed in triplicates. The data for FPR-Giα2 and FPR + Giα2 were taken from Wenzel-Seifert et al. (1999). In case of the FPR, cyclosporin H was used as inverse agonist.

    • 1-a  —, ICI-inhibited GTPγS binding was minimal. Therefore, apparent K d values could not be calculated.

    • 1-b  Coupling factors for FPR-Giα2 interaction can only be given approximately because FPR expression level cannot be precisely determined by receptor antagonist binding (Wenzel-Seifert et al., 1999).