Evidence for a single heptahelical domain being turned on upon activation of a dimeric GPCR

Abstract

G-protein-coupled receptors (GPCRs) have been shown to form dimers, but the relevance of this phenomenon in G-protein activation is not known. Among the large GPCR family, metabotropic glutamate (mGlu) receptors are constitutive dimers. Here we examined whether both heptahelical domains (HDs) are turned on upon full receptor activation. To that aim, we measured G-protein coupling efficacy of dimeric mGlu receptors in which one subunit bears specific mutations. We show that a mutation in the third intracellular loop (i3 loop) known to prevent Gprotein activation in a single subunit decreases coupling efficacy. However, when a single HD is blocked in its inactive state using an inverse agonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), no decrease in receptor activity is observed. Interestingly, in a receptor dimer in which the subunit that binds MPEP is mutated in its i3 loop, MPEP enhances agonist-induced activity, reflecting a 'better' activation of the adjacent HD. These data are consistent with a model in which a single HD is turned on upon activation of such homodimeric receptors and raise important issues in deciphering the functional role of GPCR dimer formation for G-protein activation.

Surface expression of metabotropic glutamate receptor variants mGluR1a and mGluR1b in transfected HEK293 cells

“…The responses would thus represent only about half of the theoretical maximum obtained if wild type mGluR1a would be employed. This conclusion we draw based on our previous observations using such mutants Havlickova et al, 2002;Hlavackova et al, 2005). We hypothesize that in the heterodimeric complex the presence of the mGluR1a C-tail is dominant for modulation of the shape of the response.…”

“…In such functional heterodimers the two splice variants are therefore present, as the activation of VFT of the mGluR1aF/P has to be transferred on the YADAmGluR1b heptahelical domain to activate Gproteins. It was shown previously, that such trans-activation occurs in mGlu receptors (Brock et al, 2007;Goudet et al, 2005;Hlavackova et al, 2005). …”

“…We previously reported that the insertion of these tags after the signal peptide, and before the Venus fly trap (VFT) ligand binding domain does not alter functional properties of class C GPCRs (both mGlu and GABAb receptors) (Ango et al, 2000;Galvez et al, 2001;Havlickova et al, 2002;Hlavackova et al, 2005;Pagano et al, 2001). We used wild type receptors where possible in order to verify results obtained with our N-terminally tagged proteins.…”

“…Constructs HA-mGluR1 and HA-GB1a and HA-GB2 were used and described in our previous studies (Ango et al, 2000;Galvez et al, 2001;Havlickova et al, 2003;Hlavackova et al, 2005;Pagano et al, 2001). MGluR1b variant was tagged using the same strategy.…”

Asymmetric perturbations of signalling oligomers

“…free vs. receptor-bound ligands). For instance, with labelled antibodies raised against each subunit Hlavackova et al, 2005) or with selective fluorescent ligands (Albizu et al, 2010), time-resolved FRET could be employed to demonstrate the asymmetric activation of various GPCR dimers at the surface of living cells and correlate local structural changes with functional states of ion channels (Kusch et al, 2012).…”