Extended Data Figure 5: Y308A mutation abolishes the rate-slowing effects of Nb80. | Nature

Extended Data Figure 5: Y308A mutation abolishes the rate-slowing effects of Nb80.

From: Allosteric coupling from G protein to the agonist-binding pocket in GPCRs

Extended Data Figure 5

a, b, Time course of [3H]DHAP binding to wild-type (WT) β2AR (a) or β2AR(Y308A) (b) after pre-incubation of receptor with Nb80. Nb80 significantly slowed [3H]DHAP association to wild-type β2AR (−Nb80 observed rate constant, kobs = 0.45 ± 0.05 min−1 or association half-time, t½ = 1.5 ± 0.2 min, +Nb80 kobs = 0.20 ± 0.03 min−1 or t½ = 3.5 ± 0.5 min; P = 0.011 by an unpaired two-tailed t-test), but less effectively slowed [3H]DHAP association to β2AR(Y308A) (−Nb80 kobs = 0.50 ± 0.06 min−1 or t½ = 1.4 ± 0.2 min; +Nb80 kobs = 0.32 ± 0.01 min−1 or t½ = 2.2 ± 0.1 min; P = 0.05 by an unpaired two-tailed t-test. All data are shown as mean ± s.e.m. from n = 4 (−Nb80) or n = 3 (+Nb80) independent experiments performed in duplicate. c, d, Time course of [3H]formoterol binding to wild-type β2AR (c) or β2AR(Y308A) (d) after pre-incubation of receptor with Nb80. Nb80 slowed [3H]formoterol association to wild-type β2AR (0.1 μM Nb80 kobs = 0.68 ± 0.13 min−1 or t½ = 1.0 ± 0.2 min, 10 μM Nb80 kobs = 0.27 ± 0.05 min−1 or t½ = 2.6 ± 0.5 min; P = 0.031 by an unpaired two-tailed t-test). However, with β2AR(Y308A), Nb80 had little effect on the observed association rate constant but enhanced the amount of [3H]formoterol bound (0.1 μM Nb80 kobs = 0.37 ± 0.11 min−1 or t½ = 1.9 ± 0.6 min with a plateau of 10.1 ± 0.8 fmol, 10 μM Nb80 kobs = 0.53 ± 0.13 min−1 or t½ = 1.3 ± 0.4 min with a plateau of 21.3 ± 1.2 fmol; unpaired two-tailed t-test of the kobs values showed P = 0.4). All data are shown as mean ± s.e.m. from n = 4 independent experiments performed in duplicate.

Back to article page