ReviewNovel pharmacological targets based on receptor heteromers
Introduction
An increasing number of G-protein-coupled heptaspanning-membrane receptors (GPCRs) are reported to be expressed on the plasma membrane as dimers (Carriba et al., 2007, Bouvier, 2001, Devi, 2001, Rios et al., 2001, Agnati et al., 2003, Franco, 2003, Terrillon and Bouvier, 2004, Bulenger et al., 2005, Prinster et al., 2005, Herrick-Davis et al., 2006, Milligan, 2006, Milligan, 2007, Milligan and Kostenis, 2006, Pin et al., 2007). Quite often dimers are constitutively expressed in a given cell and a given cell can express both homodimers and a variety of heterodimers. From our work and the work of other laboratories on homo and heteromerization of receptors present in striatal neurons it has been feasible to demonstrate some consequences of homo and heteromerization in terms of both, ligand pharmacology and receptor function.
Key roles for receptor homo and heterodimerization include effective quality control of protein folding prior to plasma membrane delivery and interactions with heterotrimeric G-proteins (Bulenger et al., 2005, Milligan, 2007). At the plasma membrane level, there are several examples showing that cells sense neurotransmitters (or hormones) in a different way when they express one or another set of receptors that assemble into heterodimers (see for instance Gines et al., 2000, Canals et al., 2003). One of such examples is given by the lack of desensitization of D1 receptors (D1R) interacting with adenosine A1 receptors (A1R) in striatal GABAergic neurons unless both receptors in the D1R/A1R receptor heteromer are activated simultaneously (Fig. 1, Gines et al., 2000). Another example is at the biochemical level. An antagonistic A2AR–D2R interaction have been discovered in which the stimulation of A2AR decreases the affinity of D2R for agonists in membrane preparations (Ferré et al., 1991). The intramembrane A2AR–D2R interaction implies a close physical interaction between the two receptors. In fact, the pharmacology of D2R is affected by adenosine analogs activating A2AR (Canals et al., 2003). Signaling and/or desensitization may vary depending on whether one or the two receptors in the heteromer are activated and the resulting signal is very often not simply the addition of the signals given by individual activation of the receptors; even there are instances in which heteromer-mediated signaling becomes qualitatively different (see below).
Characterization of GPCRs by radioligand binding needs the use of a model considering that receptors are dimers. This model has been devised in two different laboratories (Durroux, 2005, Albizu et al., 2006, Franco et al., 2005, Franco et al., 2006) and, recently, simple equations useful to fit radioligand biding data to receptors dimers have been released (Casadó et al., 2007). Using these equations one can calculate the dissociation constants (KD values) for the binding of the first and the second molecule to the dimer but also a newly defined dimer cooperative index (DC). These parameters will likely be very useful to quantitatively determine receptor regulation, for instance by allosteric regulators (see below).
A variety of techniques have been used to detect heteromers, among them are co-immunoprecipitation and, more recently, energy transfer biophysical assays such as fluorescence resonance energy transfer (FRET) and bioluminescence energy transfer (BRET) which allow the demonstration of receptor heteromers in the natural environment of the living cell. However, these techniques are not appropriate to be applied in tissues, making more difficult the demonstration of neurotransmitter receptor heteromers in the brain. It is quite important to be able to detect receptor heteromers in natural tissues. For this purpose one has to look for the “dimer fingerprint” (Ferré et al., 2007a) which can be functional or biochemical. Significant examples of identification of dimer fingerprints are given below.
Section snippets
GPCR homo and heterodimers as targets for hormones, neurotransmitters and drugs
Neurotransmitter and hormone GPCRs cannot only be considered as single functional units, but as forming part of multimolecular aggregates localized in the plane of the plasma membrane (also called “horizontal molecular networks”), which can contain other interacting proteins, including receptors for the same or other neurotransmitters (Franco, 2003, Agnati et al., 2003, Agnati et al., 2005). The hypothesis on the existence of intramembrane receptor–receptor interactions was introduced in the
Pharmacology of GPCR heteromers
The easier detection of pharmacological profile modification of receptor heteromers has been achieved in the case of opioid receptors (Waldhoer et al., 2004, Rios et al., 2001) Different strategies have been considered for making opiates such as morphine more effective, while curbing its liability to be abused. In particular, delta opioid receptor ligands have been useful in enhancing morphine's potency. The underlying molecular basis is a physical association between mu and delta opioid
Fitting data to GPCR homo and heteromers
There are reasons for equivocal interpretations of the data fitting to receptor dimers assuming they are monomers. In this regard new models considering receptor dimers have been recently developed (Durroux, 2005, Albizu et al., 2006, Franco et al., 2005, Franco et al., 2006). Until now the approaches for fitting ligand binding data have been based on the existence of receptor monomers. From a recently devised model for receptor dimers, there exists a new approach for fitting data that gives
Quantifying allosteric effects
The possibility to calculate the dissociation constants for the binding of the first agonist/antagonist molecule (KD1) and the second molecule (KD2) and the cooperativity index (DC) for a homodimer allows easy quantitation of the effect of allosteric regulators. These are natural or synthetic molecules that interact with an allosteric center of the receptor altering the binding of agonists to the orthosteric center and subsequently regulating receptor activation. It should be noted that in the
Receptor–heteromer-specific drugs
Heteromerization of opioid receptors has been shown to alter opioid analog effects and affect receptor trafficking in cell culture model systems (Gomes et al., 2004, Waldhoer et al., 2004, Gupta et al., 2006). Waldhoer et al. (2005) demonstrated that 6′-guanidinonaltrindole has the unique property of selectively activating only opioid receptor heteromers but not homomers. The assayed compound induced analgesia in vivo depending on the place of administration. This study constitutes a proof of
Conclusions
Receptors occur as homodimers and/or heterodimers on the cell surface and therefore dimers/oligomers are the real targets for agonists/antagonists and for drugs interacting with these receptors at the orthosteric site. This is a concept that is currently overlooked by pharmaceutical companies, which concentrate in a single receptor whose pharmacological characterization is frequently performed using single transfected cells in which receptor heteromers cannot occur. The physiology of heteromers
Acknowledgments
This research was supported by grants SAF2006–00170 SAF2005–00170 from the Spanish Ministerio de Ciencia y Tecnología, grant 060110 from Fundació La Marató de TV3 and from Intramural Funds of the NIDA, NIH.
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