Estimation of the receptor-state affinity constants of ligands in functional studies using wild type and constitutively active mutant receptors: Implications for estimation of agonist bias.

ABSTRACT: We describe a method for estimating the affinities of ligands for active and inactive states of a G protein-coupled receptor (GPCR). Our protocol involves measuring agonist-induced signaling responses of a wild type GPCR and a constitutively active mutant of it under control conditions and after partial receptor inactivation or reduced receptor expression. Our subsequent analysis is based on the assumption that the activating mutation increases receptor isomerization into the active state without affecting the affinities of ligands for receptor states. A means of confirming this assumption is provided. Global nonlinear regression analysis yields estimates of 1) the active (K_act) and inactive (K_inact) receptor-state affinity constants, 2) the isomerization constant of

SUBMITTER: Ehlert FJ 

PROVIDER: S-EPMC5769967 | biostudies-literature | 2017 Jan - Feb

REPOSITORIES: biostudies-literature