Asymmetric conformational changes in a GPCR dimer controlled by G-proteins

Abstract

G-protein-coupled receptors (GPCRs) are key players in cell communication. Although long considered as monomeric, it now appears that these heptahelical proteins can form homo-or heterodimers. Here, we analyzed the conformational changes in each subunit of a receptor dimer resulting from agonist binding to either one or both subunits by measuring the fluorescent properties of a leukotriene B 4 receptor dimer with a single 5-hydroxytryptophan-labeled protomer. We show that a receptor dimer with only a single agonist-occupied subunit can trigger G-protein activation. We also show that the two subunits of the receptor dimer in the G-protein-coupled state differ in their conformation, even when both are liganded by the agonist. No such asymmetric conformational changes are observed in the absence of G-protein, indicating that the interaction of the G-protein with the receptor dimer brings specific constraints that prevent a symmetric functioning of this dimer. These data open new options for the differential signaling properties of GPCR dimers.

Structural basis for nucleotide exchange on Gα i subunits and receptor coupling specificity

“…1A Inset). Notably, combining KB-752 and D2N resulted in a rate of GTP␥S binding approaching that of a full-length receptor acting on the G␣␤␥ heterotrimer in reconstituted systems and cell membrane preparations (13). Peptide effects were predominantly seen in the initial reaction rate; modest effects on the overall magnitude of GTP␥S binding to G␣ i1 likely reflect the ability of these peptides to reduce the requirement on magnesium for GTP␥S binding similar to that displayed by activated receptors (9,14).…”

Mutually opposite signal modulation by hypothalamic heterodimerization of ghrelin- and melanocortin-3 receptors

“…(iii) Conformational changes between ␣ 2A -adrenergic and -opioid receptor heterodimers have been reported whereby the propagated conformational change from one receptor caused inactivation of the second receptor (45). (iv) Studies on homodimeric GPCR have shown that after agonist-mediated activation in the presence of G-proteins, only G-protein coupling at one protomer might occur, which leads to asymmetric conformations of the receptors (46), indicating that G-protein coupling prevents symmetric functioning of the dimer. Considering points i-iv for the MC3R/GHSR dimer with the mutual and opposite signaling effects on each other reported here, a structural interplay is to be assumed, where the receptor interfaces on one hand mediate constraints on GHSR and on the other hand improve signaling at MC3R.…”

Asymmetric perturbations of signalling oligomers

“…Fluorescent markers can be introduced into ligands and proteins by covalent modifications of specific functional groups (thiols, amines) engineered by site-directed mutagenesis allowing selective, single-site labelling. Recombinant protein expression techniques using auxotroph E. coli (Ross et al, 1992) and suppressor tRNA technology can be used for site-specific introduction of unnatural fluorescent amino acids (Mendel et al, 1995) allowing selective monitoring of conformational changes in homodimers (Damian et al, 2006). Semisynthetic techniques allowing the engineering of proteins with chemically defined site-specific modification have also been increasingly used (Focke and Valiyaveetil, 2010).…”

“…By monitoring the fluorescence of the ligand or that of the target protein, fluorescence spectroscopy is well suited to reveal phenomena such as binding cooperativity (Alvarado, 2010) and by the application of special labelling techniques, distinguish between cis and trans activation in GPCR dimers (Damian et al, 2006(Damian et al, , 2008. Site-directed fluorescence spectroscopy can also be used to identify different structural domains associated with asymmetric ligand binding (Sommer et al, 2012).…”

“…An inverse agonist enhanced signalling, whereas another agonist inhibited it together with symmetry restoration. iii) G protein binding to the IC side of only one subunit of GPCRs also results in asymmetric functioning as detected by fluorescence for a leukotriene B 4 receptor dimer with a single tryptophan-labelled protomer (Damian et al, 2006). The other subunit can bind other GPCR-interacting proteins, arrestins or regulators leading to further differences in down-stream signalling (Rovira et al, 2009).…”