G Protein Activation by the Leukotriene B4 Receptor Dimer

Abstract

There is compelling evidence that G protein-coupled receptors exist as homo-and heterodimers, but the way these assemblies function at the molecular level remains unclear. We used here the purified leukotriene B 4 receptor BLT1 stabilized in its dimeric state to analyze how a receptor dimer activates G proteins. For this, we produced heterodimers between the wild-type BLT1 and a BLT1/ALXR chimera. The latter is no longer activated by leukotriene B 4 but is still activated by ALXR agonists. In this heterodimer, agonist binding to either one of the two protomers induced asymmetric conformational changes within the receptor dimer. Of importance, no G protein activation was observed when using a dimer where the ligand-loaded protomer was not able to trigger GDP/GTP exchange due to specific mutations in its third intracellular loop, establishing that the conformation of the agonist-free protomer is not competent for G protein activation. Taken together, these data indicate that although ligand binding to one protomer in the heterodimer is associated with cross-conformational changes, a trans-activation mechanism where the ligand-free subunit would trigger GDP/GTP exchange cannot be considered in this case for G protein activation. This observation sheds light into the way GPCR dimers, in particular heterodimers, could activate their cognate G proteins. GPCRs2 are versatile biological sensors that are responsible for the majority of cellular responses to hormones and neurotransmitters as well as for the senses of sight, smell, and taste (1, 2). Signal transduction is associated with a set of changes in the tertiary structure of the receptor that are recognized by the associated intracellular partners, in particular the G proteins (3).It has been recently shown that receptor monomers can efficiently activate their G protein partners (4 -7). However, both homo-and heterodimers have been described for many GPCRs in transfected cells, although more work is needed to extend these observations to native tissues (8). The exact molecular mechanisms governing the functioning of GPCR dimers/oligomers are not clear so far. There is increasing evidence that the two protomers in a dimer are not totally equivalent. In the case of the LTB 4 receptor BLT1 homodimer, we have evidence that only one of the protomers is activated at one time (9). In the same way, only about half of the rhodopsin present in lipid nanodiscs containing two rhodopsins is available to interact with transducin, suggesting an asymmetric functioning in this case also (10). A similar explanation has been provided for the differences in G protein activation between the neurotensin receptor monomer and dimer (7). Finally, for class C receptors, several data also support a model where the receptor dimer functions in an asymmetrical way, a single heptahelical domain being activated at a time (11, 12). In the same way, only one subunit (GB2) is able to activate G proteins in the heterodimeric GABA(B) receptor (13,14).The existence of GPCR dimers has led to the concept...

Allostery at G Protein-Coupled Receptor Homo- and Heteromers: Uncharted Pharmacological Landscapes

“…Furthermore, the D 2 -D 2 homomer findings support observations from various receptor dimers, including the glutamate Hlavackova et al, 2005) and LTB 4 BLT 1 receptors (Damian et al, 2006(Damian et al, , 2008, where maximal receptor signal is achieved only when one protomer is occupied by agonist or, in the case of CXCR2-␦, functionally reconstituted heteromers, where antagonism of CXCR2 by SB225002 enhances the action of ␦ opioid agonists (Parenty et al, 2008). However, such studies indicating asymmetry or single occupancy of homoand heteromers are in contrast to several other reports in which co-occupancy is required.…”

“…The technologies described above are based upon the transmission of allosteric signals from a receptor to effector in trans; however, there is still much debate over the mode of signal propagation at homomers and heteromers (Carrillo et al, 2003;Damian et al, 2008) or, indeed, the minimal signaling unit of a dimer (Lambert, 2010). For example, it is unclear whether occupancy of both elements of a homomer or heteromer results in maximal efficacy or instead is less energetically favorable.…”

“…Although the advent of heteromerspecific screening strategies (discussed in section V) should make identification of heteromer-selective ligands more practical, a number of measures are already available to researchers for defining or identifying allosteric interaction at dimers. For example, measures of conformational changes in protomer A in response to ligands acting at protomer B are indicative of allosterism across a dimer-such changes can be monitored as alterations in radioligand binding kinetics (El-Asmar et al, 2005;Urizar et al, 2005;Springael et al, 2006;Sohy et al, 2007) or in fluorescence (Damian et al, 2006(Damian et al, , 2008. Allosterism can also be manifest as changes in signaling properties, whether as subtle as differences in efficacy (Ciruela et al, 2001;Parenty et al, 2008) or as FIG.…”

Asymmetric perturbations of signalling oligomers

“…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).…”

“…ii) Several reports have concluded that ligand binding can elicit not only cis but also trans activation via the TM helices. However, fluorescence labelling and radioligand binding have revealed only cis activation for chimeric heterodimers of rhodopsinlike leukotriene B 4 receptors (Damian et al, 2008). Asymmetric activation was reported for G protein-fused dopamine D 2 receptor homodimers via functional complementation experiments (Han et al, 2009).…”