Mutations inducing divergent shifts of constitutive activity reveal different modes of binding among catecholamine analogues to the β₂‐adrenergic receptor

Abstract

1 We compared the changes in binding energy generated by two mutations that shift in divergent directions the constitutive activity of the human b 2 adrenergic receptor (b 2 AR). 2 A constitutively activating mutant (CAM) and the double alanine replacement (AA mutant) of catechol-binding serines (S204A, S207A) in helix 5 were stably expressed in CHO cell lines, and used to measure the binding a nities of more than 40 adrenergic ligands. Moreover, the e cacy of the same group of compounds was determined as intrinsic activity for maximal adenylyl cyclase stimulation in wild-type b 2 AR. 3 Although the two mutations had opposite e ects on ligand a nity, the extents of change were in both cases largely correlated with the degree of ligand e cacy. This was particularly evident if the extra loss of binding energy due to hydrogen bond deletion in the AA mutant was taken into account. Thus the data demonstrate that there is an overall linkage between the con®guration of the binding pocket and the intrinsic equilibrium between active and inactive receptor forms. 4 We also found that AA mutation-induced a nity changes for catecholamine congeners gradually lacking ethanolamine substituents were linearly correlated to the loss of a nity that such modi®cations of the ligand cause for wild-type receptor. This indicates that the strength of bonds between catechol ring and helix 5 is critically dependent on the rest of interactions of the bethanolamine tail with other residues of the b 2 -AR binding pocket.

G Protein–Coupled Receptor Endocytosis Confers Uniformity in Responses to Chemically Distinct Ligands

“…Norepi for G protein signaling ( Fig. 2A), which is consistent with previous studies for these compounds (January et al, 1998;Del Carmine et al, 2002;Baker, 2005;Moore et al, 2007). Next, we measured b2-AR internalization as the loss of cell surface receptors after agonist exposure using flow cytometry in cell expressing a flag-tagged receptor.…”

Constitutive Activation of CCR5 and CCR2 Induced by Conformational Changes in the Conserved TXP Motif in Transmembrane Helix 2

“…As reported for CXCR4, substitution of critical residues with different amino acids was associated with different levels of G protein coupling in mammalian cells (28). Moreover, some CAMs had ligand binding similar to the wild-type receptor, whereas others had decreased ligand affinities, indicative of shifts in the conformation of extracellular domains involved in binding sites (47). The CXCR4-CAMs were found to be phosphorylated and chronically desensitized while AT 1A -CAM had a basal level of phosphorylation similar to WT receptor even upon ligand stimulation (28,48).…”

Ligand Function at Constitutively Active Receptor Mutants Is Affected by Two Distinct Yet Interacting Mechanisms

“…In the work by Del Carmine et al (2002), it was found that mutations of residues within the putative binding pocket of the ␤2-adrenoceptor (S204,207A, predicted to project adjacent to our S219H mutation in the CCK-2R) decreased ligand affinities by shifting the receptor toward a less active conformation. For a subset of compounds that was investigated, the latter effect (i.e., loss of affinity secondary to a shift in receptor conformation) seemed to be superimposed with a mutation-induced disruption of direct receptor-ligand interactions.…”