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Commentary 4215 G--coupled heteromer dynamics

Jean-Pierre Vilardaga1,2,*, Luigi F. Agnati3, Kjell Fuxe4 and Francisco Ciruela5 1Laboratory for GPCR , Department of Pharmacology and Chemical Biology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261, USA 2Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA 3IRCCS, San Camillo, Lido Venezia, Italy 4Department of Neuroscience, Karolinska Institute, Stockholm SE-17177, Sweden 5Pharmacology Unit, Department of Pathology and Experimental Therapy, Faculty of Medicine, University of Barcelona, 08907 Barcelona, Spain *Author for correspondence ([email protected])

Journal of Science 123, 000-000 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jcs.063354

Summary G-protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors, and have evolved to detect and transmit a large palette of extracellular chemical and sensory signals into cells. Activated receptors catalyze the activation of heterotrimeric G , which modulate the propagation of second messenger molecules and the activity of ion channels. Classically thought to signal as monomers, different GPCRs often pair up with each other as homo- and heterodimers, which have been shown to modulate signaling to G proteins. Here, we discuss recent advances in GPCR heteromer systems involving the kinetics of the early steps in GPCR , the dynamic property of receptor–receptor interactions, and how the formation of receptor heteromers modulate the kinetics of G-protein signaling.

Key words: G-protein-coupled receptors, Heterodimers, Signaling

Introduction the signaling and trafficking mechanisms of GPCRs is thus central G-protein-coupled receptors (GPCRs) constitute the main family for the development of new and safer therapies for many of cell surface receptors for a large variety of chemical stimuli physiological and psychological disorders. (, , chemoattractants, calcium ions and GPCRs have traditionally been considered to exist and function pain killers, among others molecules) and sensory stimuli (light, as cell-surface-receptor monomers (Chabre and Le Maire, 2005; odorants and taste molecules). GPCRs are expressed in virtually Chabre et al., 2009). Consistent with this view, recent studies every cell and consist of seven membrane-spanning a-helical characterized in reconstituted phospholipid bilayer systems structures with the N- and C-termini exposed to the extracellular demonstrated that purified receptors such as , b2-adrenergic

Journal of Cell Science and intracellular environment, respectively (Hanson and Stevens, and -opioid receptors can activate heterotrimeric G proteins as 2009). Signal transduction begins when an extracellular single GPCR monomers (Whorton et al., 2007; Whorton et al., ‘’ binds and switches the receptor from an inactive state to 2008; Kuszak, 2008). These studies indicate that a single GPCR an active state conformation. Activated receptors then catalyze the protomer is capable of activating G proteins, and that receptor exchange of GDP for GTP on the a-subunit of heterotrimeric G oligomerization is not necessary to mediate G-protein activation. proteins (Gabg), which in turn engages conformational and/or Several GPCRs can, however, assemble and signal in both homo- dissociation events between the Ga and dimeric Gbg subunits that oligomer and hetero-oligomer complexes at the surface membrane are associated with G-protein activation (Hofmann et al., 2009). of native and cultured cells (Prinster et al., 2005; Guo et al., 2008; Both the GTP-bound Ga subunit and the Gbg dimer can then Milligan, 2009) (Box 1). Two recent studies provided strong initiate or suppress the activity of effector (e.g. adenylyl experimental evidence of the in vivo homodimerization of GPCRs. cyclases, phosphodiesterases, phospholipases) and ion channels. The first showed dimerization of the mouse luteinizing These ion channels in turn modulate the flow of secondary receptor (LHR) by using a transgenetic approach to express binding messengers such as cAMP, cGMP, diacylglycerol or inositol and signaling variants of LHR together (Rivero-Muller et al., 2010), trisphosphate, which are involved in the regulation of multiple whereas the second study demonstrated the existence of native intracellular signaling pathways, and which modulate cell functions dimers by using a fluorescence resonance energy in body systems as diverse as the skeletal, endocrine, cardiovascular transfer (FRET) approach (Albizu et al., 2010). In this Commentary, and nervous systems, among others (Fig. 1). Activated GPCRs can we discuss the kinetics of the early steps involved in GPCR signaling also interact with cytosolic (b--1 and b-arrestin- (receptor activation, the receptor–G-protein interaction, G-protein 2), which coordinate receptor–G-protein uncoupling in space and activation), the stability of the receptor–receptor interaction, and time, promote receptor internalization and modulate various distal how this interaction modulates the kinetics of G-protein signaling. signaling responses such the MAP cascades (Pierce et al., 2002). Malfunction of GPCRs are involved in many human Initial steps in the GPCR signaling system diseases (Schöneberg et al., 2004), for instance, diabetes insipidus Biophysical and biochemical approaches have revealed that GPCR and mellitus, hypercalcemia, obesity, hypertension, cancer, signaling systems involve a succession of events that initially take hypothyroidism, retinitis pigmentosa and psychotic disorders, and place at the and modulate the production and are targets of most of the available clinical drugs used in humans, propagation of second-messenger molecules inside the cell (Fig. 2) such as b-blockers, antipsychotics and analgesics. Understanding (Lamb, 1996; Ferrandon et al., 2009). These events are initiated by 4216 Journal of Cell Science 123 (24)

particularly helices 3 and 6 (Bourne, 1997). Activation of the b2- Box 1. Receptor heterodimer and receptor mosaic (b2-AR), for example, proceeds through the concepts release of an ‘ionic lock’ that holds together the cytoplasmic sides In the 1980s, Agnati and Fuxe introduced the concept of of helix 3 and helix 6 in the inactive conformation of the receptor, intermembrane receptor–receptor interactions between different and a rotamer toggle switch mechanism that modulates the types of GPCRs, based on the effects of on the binding characteristics of monoamine receptors in plasma conformation of helix 6 (Yao et al., 2006). These conformational membrane preparations from discrete regions of the brain (Agnati changes are considered to expose the domains et al., 1980; Fuxe et al., 1981; Fuxe et al., 1983; Fuxe and Agnati, at the cytosolic side, which interact and activate G proteins. Our 1985). These results were in line with earlier findings (Limbird et FRET approach (for a review, see Vilardaga et al., 2009; Lohse al., 1975) showing negative cooperativity in b-ARs, which could et al., 2008) revealed that intramolecular rearrangements associated be explained by the existence of receptor homodimers leading to with receptor activation in live cells proceed with fast kinetics site–site interactions. Additional in vivo studies, which examined (Vilardaga et al., 2003). The activation of a receptor by a small the interactions of clonidine (a partial agonist for a -ARs) and 2A such as the , a2A- and Y (NPY; a 36 amino acid neurotransmitter) on b1-adrenergic receptors, and muscarinic receptors takes place with sleep–wakefulness cycles and arterial blood pressure control in a time constant tϷ40 mseconds, whereas activation of the receptor both normal and spontaneously hypertensive rats (Fuxe et al., Ϸ 1989; Fuxe, 1990; Yang et al., 1994), further supported the for the larger parathyroid hormone proceeds within t 1 second existence of receptor–receptor interactions and their potential (Vilardaga et al., 2003; Vilardaga, 2010; Maier-Peuschel et al., functional relevance. As a logical consequence for the indications 2010). One of the mechanistic reasons for this 25-fold difference of direct physical interactions between neuropeptide and in receptor activation kinetics is thought to depend on distinct monoamine receptors, the term heteromerization was introduced modes of binding between hormones and smaller molecules to describe a specific interaction between different types of (Castro et al., 2005; Vilardaga, 2010). GPCRs (Zoli et al., 1993). The concept of the GPCR heterodimer Activated GPCRs then interact with heterotrimeric G proteins was later confirmed by studies reporting that two non-functional with kinetics that, in the context of the low level of G proteins, are GPCR monomers, GABAB1 and GABAB2, can assemble in not determined by the time course of receptor activation, but rather signaling heterodimers at the cell surface to transmit the by a diffusion-limited collision process. At a high level of G- inhibitory effect of the g-aminobutyric acid (GABA) neurotransmitter (Marshall et al., 1999): when expressed alone, protein expression, however, a receptor–G-protein interaction can be as fast as receptor activation itself, with a time constant ranging the GABAB1 receptor is retained in the endoplasmic reticulum, and is able to reach the cell surface and bind GABA only in the from 40 mseconds to 1 second, depending on the nature of the presence of its partner, the GABAB2 receptor. receptor (Hein, 2005; Ferrandon et al., 2009). The step following In 1982, Agnati and colleagues suggested that clusters of the rapid interaction between an activated receptor and a more than two receptors, representing higher-order oligomers, triggers the GDP–GTP exchange on the Ga subunit through termed receptor mosaics (RMs), in which each receptor conformational rearrangements or dissociation between the Ga represents a single tessera within the mosaic, could exist and and Gbg subunits that occur with much slower kinetics, displaying interact with each other (i.e. through allosteric interactions) in the a half-time (t1/2) of ~0.5–1 seconds for adrenergic and adenosine plasma membrane (Agnati et al., 1982; Zoli et al., 1993). It was Ϸ Journal of Cell Science receptors, and t 1–2 seconds for the parathyroid hormone proposed that an RM functions as an integrated unit with unique 1/2 signaling properties that are modulated by receptor–receptor receptor (Bünemann et al., 2003; Nikolaev et al., 2006; Hein et al., allosteric interactions. The integrative action of an RM is likely to 2006; Ferrandon et al., 2009). This step, as opposed to receptor be affected not only by its stoichiometry, but also by the activation and receptor–G-protein interactions, is thus the rate- topological arrangement of its individual receptors (Agnati et al., limiting step for G-protein activation. 2005; Agnati et al., 2007). A recent study by Fung and colleagues supports the RM concept by demonstrating that highly purified GPCR oligomers: static or dynamic b2AR protomers reconstituted into phospholipid vesicles can interactions? assemble together to form a tetramer complex that is stabilized The family C GPCRs, which include the metabotropic glutamate by the binding of inverse (Fung et al., 2009). This study receptors (mGluR1–mGluR5), the calcium-sensing receptor (CaSR) shows not only that a GPCR can indeed exhibit cooperativity by and the -aminobutyric acid receptors (GABA R), among others, forming higher-order oligomers beyond just homodimers, but also g B that GPCRs can assemble together through direct receptor– differentiate themselves from the other GPCR families by large receptor interactions. Indeed, most studies of GPCR bilobed N-terminal extracellular domains known as the venus oligomerization being performed in live cells with both optical and flytrap domains (Pin et al., 2003; Pin et al., 2005). These receptors biochemical approaches [e.g. FRET and bioluminescence associate with themselves and function as constitutive dimers. In resonance energy transfer (BRET)-based techniques, co- many cases, receptor dimers are stabilized by disulfide bridges immunoprecipitation] to determine the formation and modulation between Cys residues located in the venus flytrap domains (Romano of receptor–receptor complexes do not rule out the possibility that et al., 1996; Ray et al., 1999; Ray and Hauschild, 2000). Most receptor oligomers are held together by scaffold proteins, rather oliogomers of class A GPCRs, however, are formed through non- than engaging in direct receptor–receptor interactions. The highly covalent interactions, and recent fluorescence recovery after purified nature of the study by Fung and co-workers convincingly photobleaching (FRAP) studies support the existence of a dynamic demonstrated that the formation of a higher-order b2AR complex takes place without other proteins. equilibrium between monomer and homodimer states of class A GPCRs, such as the D2 receptor (D2R) and the b1-AR (Dorsch et al., 2009; Fonseca and Lambert, 2009). The dynamics ligand binding to an inactive receptor that switches to an active of a class A GPCR dimer has been further detailed by total internal conformation. This receptor switch is driven by intramolecular reflection fluorescence (TIRF) microscopy, revealing that ~30% of rearrangement between the receptor’s transmembrane helices, recombinant muscarinic M1 receptor (M1R) expressed GPCR oligomers 4217

ABLigands Sensory stimuli (light, odor, taste), ions, neurotransmitters, , hormones, prescribed drugs Effectors Adenylyl cyclases (Ff) F GPCR Phospholipase C (F) GIRK ( ) F N K+, Cl–, Na+ channels (F) PI-3 kinase ( ) GPCR VSCC(F) extracellular GDP membrane AC on intracellular

C off GDPDP GTPTP Gα Gγ β G proteins G + G , G , G , G cAMP K current s q i/o 12/13 PI-3 kinase IP3, DAG

Fig. 1. General principle of the GPCR signaling system. (A)Molecular representation of a GPCR in complex with a Gabg, based on crystal structures of rhodopsin (red; coordinates from PDB code 1GZM) and the inactive heterotrimeric Gi protein (from PDB 1GG2). (B)Following ligand binding, the receptor undergoes conformational changes, which promote the coupling with heterotrimeric G proteins (Gabg), and catalyzes the exchange of GDP for GTP on the a- subunit. This event triggers conformational and/or dissociation events between the a-subunit and bg-subunit. GaS activates adenylyl cyclases, leading to cAMP synthesis, which in turn activates A (PKA). Gaq activates phospholipase C, which cleaves (4,5)-bisphosphate (PIP2) into 2+ diacylglycerol (DAG) and inositol (1,4,5)-trisphosphate (IP3). IP3 then diffuses through the cytosol and activates IP3-gated Ca channels in the membranes of the endoplasmic reticulum, causing the release of stored Ca2+ into the cytosol. The increase of cytosolic Ca2+ promotes PKC translocation to the plasma membrane, and then activation by DAG. Activation of Gi blocks adenylyl-cyclase-mediated cAMP synthesis by its a-subunits, whereas Gbg-mediated signaling processes such as activation of G-protein-regulated inwardly rectifying potassium (GIRK) channels. VSCC, voltage-sensitive Ca2+ channel.

in CHO cells form transient homodimers with a life-time as short T1R1–T1R3 heterodimer mediates the glutamate umami taste, as 0.5 seconds (Hern et al., 2010). These studies suggest that at whereas the T1R2–T1R3 heterodimer mediates the sweet taste least some class A GPCRs, such as M1R, b1-AR and D2R, can (Nelson et al., 2001; Zhao et al., 2003; Mueller et al., 2005). transiently associate with each other, with half-lives presumably Receptor heterodimers between the adenosine A2A and dopamine long enough to permit G-protein activation. D2 receptors is another attractive example of how receptor hetero- dimerization can modulate receptor function. This receptor dimer

Journal of Cell Science Modulation of by fast inter-conformational is expressed in striatopallidal GABAergic that are present switches in the basal ganglia, a region of the brain that is involved in During the past decade, studies recently reviewed by Rozenfeld sensory–motor integration (Canals et al., 2003). Adenosine inhibits and Devi (Rozenfeld and Devi, 2010) revealed that receptor dopamine-induced locomotor activity, and by exerting opposite heterodimerization modulates pharmacological, signaling and effects in the basal ganglia, the A2A–D2 receptor heteromer trafficking properties of individual parent receptors. Examples of contributes to the fine-tuning of neural activity. At the biochemical such fascinating aspects of receptor heteromerization are found in level, the activation of A2A receptors by adenosine or adenosine the different combinations between the taste receptors T1R1, TIR2 analogs affects the pharmacology of D2 receptors by modulating and TIR3, which modulate the sensitivity of taste molecules – the the binding characteristics of dopamine agonists, and counteracts

AB C D

Fig. 2. Kinetics of early reactions in the signaling cascade of GPCRs. Time constants (t) of (A) ligand (L) association, (B) receptor (R) activation (R*), (C) receptor–G-protein association, and (D) G protein (G) activation (G*) are shown for two distinct GPCR systems: the parathyroid (PTHR) in 2+ response to the major endocrine regulator of Ca homeostasis, PTH, and a2A-AR in response to the principal neurotransmitter of sympathetic nerves, norepinephrine. 4218 Journal of Cell Science 123 (24)

Norepinephrine Morphine

Fig. 3. Modulation of G-protein signaling by receptor heterodimers. Representation of the a2A- AR (red)–MOR (blue) heterodimer complex based t1/2=400 ms on the crystal structure of rhodopsin (coordinates Crosstalk from PDB code 1GZM), and chemical structures of norepinephrine and morphine. G-protein signaling mediated by the receptor heterodimer in response to norepinephrine or morphine is modulated by the simultaneous action of both signaling ligands. This X modulation proceeds through conformational changes (arrows) that propagate from one receptor G-protein signaling G-protein signaling G-protein signaling to the other with a half-life of 400 mseconds.

D2-receptor-mediated intracellular responses (Agnati et al., 2003). only by the action of an A2A agonist. This release might be The binding of ligand to one receptor altering the activation of the accomplished by means of inter-conformational switches between partner receptor in the heterodimer complex led to the hypothesis these two receptors. This hypothesis needs to be tested, but would that conformational changes induced by ligand binding to one indicate that functional selectivity at GPCR heteromers is receptor might be transmitted to the receptor partner to modulate modulated directly at the level of receptors through conformational its function (Zoli et al., 1993). crosstalk. We confirmed this hypothesis with FRET studies in living cells, by showing that allosteric interactions between the a2A-AR and - Perspectives and Conclusions (MOR) are mediated by direct cross-conformational We have recently shown that signal transduction mediated by changes between these receptors (Vilardaga et al., 2008). GPCRs involves a series of reversible reactions (ligand binding, Norepinephrine and morphine, which are the native ligands for the receptor activation and deactivation, receptor–G-protein a2A-AR and MOR, respectively, are well known to stimulate interactions, and G protein activation and deactivation) that initially common inhibitory G protein (Gi)-mediated signaling pathways take place at the plasma membrane (for a review see Vilardaga, through their respective receptors, but the simultaneous action of 2010). These reactions, which are conveyed by interactions between norepinephrine and morphine produces a cellular response different ligands, receptors and G proteins, and conformational changes from the expected additive effects (Jordan et al., 2003). In this associated with the activation–deactivation process of each protein, case, morphine binding to the MOR triggers a rapid conformational can be modulated by dynamic receptor–receptor interactions. The

Journal of Cell Science change in the norepinephrine (NE)-bound a2A-AR that proceeds capacity of GPCR heterodimers to modulate receptor function with a t1/2400 mseconds, which is faster than the kinetics for G- leads to the concept that dysregulation of receptor heterodimer protein activation (Fig. 3). This fast trans-conformational switch expression and function might be implicated in several diseases between receptors decreases both activation of Gi signaling and (Prinster et al., 2005; Dalrymple et al., 2008; Rozenfeld and Devi, stimulation of MAP kinase . A trans-conformational 2010). Recent studies show that A2AR, D2R and mGlu5R form a switch in the reverse direction, from the a2A-AR towards the receptor heterocomplex in the striatal spine module (Cabello et al., MOR, was not directly observed because FRET-based MOR 2009), a site that has a crucial role in controlling motor activity, biosensors are not functional. The inhibition of morphine-mediated motor learning and some forms of associative and visual learning. G-protein activation by NE suggests, however, that such a Imbalance of these activities caused by a gradual and progressive conformational transfer also occurs. Thus, conformational crosstalk loss of dopaminergic neurons, which are implicated in between receptors is bidirectional, and rapidly prevents over- glutamatergic, dopaminergic and adenosinergic , stimulation of signaling pathways by a combination of ligands is thought to be one of the crucial determinants of Parkinson’s acting on a receptor heteromer, by rapidly adjusting the extent of disease. In recent years, the A2AR–D2R–mGlu5R heteromer has G-protein activation. become a principal target for the treatment of Parkinson’s disease, A fundamental property of ligands acting at a given GPCR is because antagonists of both A2AR and mGlu5R enhance D2R their capacity to stabilize different active receptor conformations function (for a review, see Fuxe et al., 2007). The finding that that can each activate a unique signaling pathway. The term adenosine and glutamate ligand binding to their respective receptors ‘functional selectivity’ was introduced as a parameter to express reduces (in the case of agonists) or increases (in the case of the ability of a ligand to produce a selective response (Kenakin and antagonists) dopamine D2R signaling suggests that allosteric Miller, 2010). Functional selectivity in agonism is also linked with interactions within the A2AR–D2R–mGlu5R heteromer regulate the GPCR heterodimer. For example, the Gi-coupled cannabinoid basal ganglia neurotransmission through a presumably fast inter- receptor (CB1R) forms a heterodimer complex with the GS-coupled conformational switch. An extension of this theme implies that the A2AR and signals to Gi only when CB1 and A2A agonists are integrative receptor–receptor interactions through an assembly of combined (Carriba et al., 2007). These findings suggest that the several distinct GPCRs, named higher-order receptor heteromers receptor–receptor interaction constrains the capacity of the CB1R or receptor mosaics (Agnati et al., 1982), increases the complexity to adopt an active conformation that can stimulate Gi activation in by which extracellular stimuli transduce signals into the cell (Box the CB1R–D2R heterodimer, and that this constraint is released 1). Future studies will be needed to better understand this GPCR oligomers 4219

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