same high level of constitutive activity as that measured after heterologous expression in HEK cells
(Bazzacco et al. 2012). The GHS-R1a and melatonin MT1R receptors were extracted by SMA either
from asolectin vesicles, into which GHS-R1a had been integrated following expression in inclusion
bodies and in vitro folding in A8-35, or from native membranes from Pichia pastoris, into which
MT1R had been inserted in vivo. They were characterized by their ligand binding, conformational
transitions, G protein activation, and arrestin recruitment (Logez et al. 2016). The vasopressin V2R
receptor was expressed in insect cells under its native form, solubilized in a detergent mixture,
transferred to NAPols, and characterized by its ligand binding, conformational transitions, G protein
activation, and arrestin recruitment (Rahmeh et al. 2012). In short, it seems that GPCRs can be trapped
Fig. 5.25 Allosteric transitions of the nicotinic acetylcholine receptor in three different environments.
Kinetics of binding of a fluorescent ligand to nAChR in (A, B) native membrane fragments from Torpedo
marmorata electric organ; (C, D) after solubilization in detergent solution (CHAPS); (E, F) after addition
of A8-35 and dilution below the CMC of CHAPS. In its native membrane environment, the nAChR
pre-exists to the addition of ligands in an equilibrium between a low-affinity resting state and high-affinity,
inactive state(s), in a proportion of about 9:1. Upon addition of a low concentration of fluorescent agonist,
only the high-affinity state(s) binds the ligand (panel A), relaxation of the resting state conformation to
high-affinity ones occurring more slowly (panel B). After solubilization, the ratio between high- and
low-affinity states in the absence of ligand becomes about 1:1, explaining the higher level of fast binding
seen in Panel C. When most of CHAPS is replaced by A8-35 in the environment of the receptor, the
allosteric equilibrium comes back to a situation similar to that in the membrane (E, F) (From Martinez et al.
2002, # 2015 Federation of European Biochemical Societies).
288
5 Formation and Properties of Membrane Protein/Amphipol Complexes
(Bazzacco et al. 2012). The GHS-R1a and melatonin MT1R receptors were extracted by SMA either
from asolectin vesicles, into which GHS-R1a had been integrated following expression in inclusion
bodies and in vitro folding in A8-35, or from native membranes from Pichia pastoris, into which
MT1R had been inserted in vivo. They were characterized by their ligand binding, conformational
transitions, G protein activation, and arrestin recruitment (Logez et al. 2016). The vasopressin V2R
receptor was expressed in insect cells under its native form, solubilized in a detergent mixture,
transferred to NAPols, and characterized by its ligand binding, conformational transitions, G protein
activation, and arrestin recruitment (Rahmeh et al. 2012). In short, it seems that GPCRs can be trapped
Fig. 5.25 Allosteric transitions of the nicotinic acetylcholine receptor in three different environments.
Kinetics of binding of a fluorescent ligand to nAChR in (A, B) native membrane fragments from Torpedo
marmorata electric organ; (C, D) after solubilization in detergent solution (CHAPS); (E, F) after addition
of A8-35 and dilution below the CMC of CHAPS. In its native membrane environment, the nAChR
pre-exists to the addition of ligands in an equilibrium between a low-affinity resting state and high-affinity,
inactive state(s), in a proportion of about 9:1. Upon addition of a low concentration of fluorescent agonist,
only the high-affinity state(s) binds the ligand (panel A), relaxation of the resting state conformation to
high-affinity ones occurring more slowly (panel B). After solubilization, the ratio between high- and
low-affinity states in the absence of ligand becomes about 1:1, explaining the higher level of fast binding
seen in Panel C. When most of CHAPS is replaced by A8-35 in the environment of the receptor, the
allosteric equilibrium comes back to a situation similar to that in the membrane (E, F) (From Martinez et al.
2002, # 2015 Federation of European Biochemical Societies).
288
5 Formation and Properties of Membrane Protein/Amphipol Complexes