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University of Modena, Modena, Italy; National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland; University of Barcelona, Barcelona, Spain; Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
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opioid receptors. Heteromerization was also discovered among distinct types of G protein-coupled receptors with the initial demonstration of somatostatin SSTR5/dopamine D2 and adenosine A1/dopamine D1 heteromeric receptor complexes. The functional meaning of these heteromeric complexes is to achieve direct or indirect (via adapter proteins) intramembrane receptor/receptor interactions in the complex. G protein-coupled receptors also form heteromeric complexes involving direct interactions with ion channel receptors, the best example being the GABAA/dopamine D5 receptor heteromerization, as well as with receptor tyrosine kinases and with receptor activity modulating proteins. As an example, adenosine, dopamine, and glutamate metabotropic receptor/receptor interactions in the striatopallidal GABA neurons are discussed as well as their relevance for Parkinson's disease, schizophrenia, and drug dependence. The heterodimer is only one type of heteromeric complex, and the evidence is equally compatible with the existence of higher order heteromeric complexes, where also adapter proteins such as homer proteins and scaffolding proteins can exist. These complexes may assist in the process of linking G protein-coupled receptors and ion channel receptors together in a receptor mosaic that may have special integrative value and may constitute the molecular basis for some forms of learning and memory. |
I. Experimental Evidence on Protein/Protein Interactions Involving G Protein-Coupled Receptors in the Central Nervous System |
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Emerging evidence shows that G protein-coupled receptors (GPCR2) can form homo- and heteromers (Bouvier, 2001
; Marshall, 2001). It all began in 1979-1980 in search of an explanation of where all the recently discovered neuropeptides in the brain could integrate their messages with those of classical transmitters such as the monoamines. Luigi F. Agnati and Kjell Fuxe postulated that an intramembrane interaction between neuropeptide and monoamine receptors could be involved. The first observations were published in 1980 (Agnati et al., 1980), showing that substance P could modulate the high-affinity serotonin binding sites in spinal cord membrane preparations using biochemical binding techniques (also, see Agnati et al., 1983b). The same year, an interesting paper was published by Maggi et al. (1980) showing that the 
adrenergic receptor agonist isoproterenol could increase 
2 adrenergic receptor binding in cortical slices, supporting the concept of intramembrane receptor/receptor interactions of GPCR, in this case among isoreceptors. Subsequently, in 1981 the existence of cholecystokinin (CCK) receptor/dopamine D2 receptor interactions using biochemical binding techniques was indicated since CCK-8 could modulate the dopamine D2 receptor antagonist and agonist binding sites in striatal membrane preparations (Fuxe et al., 1981, 1983b; Agnati et al., 1983a,b, 1985). Further evidence for receptor/receptor interactions came in 1982 from Lundberg, Bartfai, and colleagues (Lundberg et al., 1982) and from Zarbin and colleagues (Zarbin et al., 1982). Using the same type of approach, a large number of papers were published in 1983 that suggested the existence of intramembrane receptor/receptor interactions between different GPCR (Fuxe et al., 1983b; Agnati et al., 1984; Fuxe and Agnati, 1985). Those included neurotensin (NT receptor)/D2 (Agnati et al., 1983c; Nemeroff, 1986; Von Euler and Fuxe, 1987; Von Euler, 1991), CCKB/serotonin 5-HT2 (Agnati et al., 1983a, 1985), vasoactive intestinal peptide (VIP) receptor/serotonin 5-HT1 (Rostene et al., 1983a,b), neuropeptide Y (NPY) receptor/2 adrenergic (Agnati et al., 1983d) and neurokinin NK1/5-HT1 receptor/receptor interactions (Agnati et al., 1983e). Subsequently in the 1980s, indications for glutamate receptor/D2 receptor interactions (Fuxe et al., 1984) were obtained in striatal membrane preparations after earlier observations had indicated the existence of interactions at the membrane level among glutamate receptor subtypes (Fuxe et al., 1983c). This early research led to the following postulation in the opening address of Fuxe and Agnati at the International Wenner-Gren Symposium on receptor/receptor interactions in 1986 (Fuxe and Agnati, 1987) "... we will find out that some sophisticated elaborations are performed at the membrane level, via interactions within and among different classes of macromolecules (such as receptors, ion pumps, ion channels)" (also, see Agnati et al., 1988). In 1988, evidence for galanin (Gal) receptor/serotonin 5-HT1A receptor interactions in limbic cortical membranes (Fuxe et al., 1988a), as well as for angiotensin II receptor (AT1)/2 adrenergic receptor interactions (Fuxe et al., 1988b) in the medulla oblongata membrane preparations were obtained. In the early 1990s, adenosine A2A receptor/D2 receptor interactions (Ferré et al., 1991d, 1993; Ferré and Fuxe, 1992) were demonstrated in striatal membrane preparations.
Thus, not only neuropeptide and monoamine receptors were involved in intramembrane receptor/receptor interactions but also certain types of glutamate and adenosine receptors (Agnati et al., 1986, 1990, 1993; Härfstrand et al., 1988; Tanganelli et al., 1989, 1990, 1993; Von Euler et al., 1989; Fuxe et al., 1990a-c, 1991, 1992a-c; Ferré et al., 1992, 1993b; Fior et al., 1993; Yang et al., 1994b). These results were all obtained at the recognition site of the receptors, using saturation and competition binding experiments. The modulation of binding could be shown as changes in KD and Bmax values (saturation analysis) and as KL, KH, and RH values (competition analysis) allowing a determination of modulation of the high- versus the low-affinity states of the receptor. An indication of an effect on the G protein-coupling and thus on the efficacy of the modulated receptor could be obtained by studying how, e.g., the modulator could control the GTP-induced disappearance of the high-affinity state of the receptor (reduction of the RH values). This would imply a G protein activation with formation of G-GTP and 
dimers associated with a cross-regulation of the GPCR with a disappearance of the high-affinity state of the receptor.
In this period, the above work was extended to show multiple receptor/receptor interactions. Thus, evidence was obtained for a dopamine D1 receptor involvement in the CCKB receptor/D2 receptor interaction. Coactivation of D1 and D2 receptors led to an enhancement of the affinity of D2 receptor agonist sites by CCK-8, instead of a reduced affinity of D2 receptor agonist sites observed without D1 receptor stimulation (Li et al., 1994a). These results are in line with the findings of Seeman et al. (1989) suggesting reciprocal interactions between D1 and D2 receptors in striatal homogenates. Thus, there may exist striatal nerve cell populations where intramembrane multiple CCKB receptor/D1 receptor/D2 receptor interactions can take place (Agnati et al., 1982).
In the early 1990s, evidence was also obtained that striatal NT receptors involved in the G protein-independent antagonistic regulation of striatal D2 receptors (Von Euler et al., 1991) may represent a novel type of a high-affinity NT receptor. This was suggested in view of the rank order of potency found among COOH-terminal NT fragments, neuromedin N, and NT in this response versus the rank order of potency found at the cloned high-affinity NT receptors (NT1 receptors) (Li et al., 1993a,b). These effects were stronger in striatal sections (Li et al., 1994b), and recently, the NT-induced reduction of D2 receptor affinity in striatal sections has been found to be blocked by a NT1-like antagonist (Diaz-Cabiale et al., 2002a).
In 1993 (Zoli et al., 1993), the hypothesis was introduced that the molecular mechanism for these large numbers of intramembrane receptor/receptor interactions among GPCR could be the formation of heteromeric complexes, the simplest being a heterodimer. This concept was based on the indication at the time that dimerization upon agonist activation may be a general phenomenon essential for receptor activation (Hollenberg, 1991), the best example being the dimerization of tyrosine kinase receptors (Schlessinger 1988, 2000; Helldin 1995). Thus, it was assumed that GPCR exist mainly as homodimers that interact with other types of homodimers to form heterodimers. The relative proportions of homo- and heterodimers would be determined by the concentrations of the two transmitters, the density of the two receptors and their distribution patterns, and the unique features of each receptor/receptor interaction (Zoli et al., 1993). In fact, early evidence obtained on purified GPCR by, e.g., Venter and Fraser (1983) and Conn et al. (1982) indicated that the functional GPCR were in a dimeric state. The same year that our review article appeared, the first evidence was published that GPCR can exist as dimers (Ng et al., 1993). Thus, the 5-HT1B receptor in Sf9 cells was in the immunoblot analysis shown to exist as dimers and monomers. Finally, it should be mentioned that in 1982 we had introduced the receptor mosaic hypothesis of learning and memory based on the formation of membrane receptor clusters and thus of high order oligomeric receptor complexes (Agnati et al., 2002). It was postulated (Agnati et al., 1982; Zoli et al., 1993) that the formation and/or stabilization of the heteromeric complexes of GPCR could be enhanced by associated (adapter) proteins especially in the synaptic membranes. It must be noted that most of the GPCR are located in extrasynaptic membranes and therefore the potential heteromeric complexes discussed above may also be reached by volume transmission (VT) signals (Agnati and Fuxe, 2000).
Recent experimental data exist confirming this hypothesis, and they are described in this review (see Table 1). Despite all this novel experimental evidence, there are many questions regarding the molecular mechanism of receptor heteromerization and among them the mapping of the residues involved in the interaction in the case of direct interactions and the identification of scaffolding and adapter proteins in the case of indirect interactions. It should be noted that both types of interaction, direct and indirect, are likely to occur.
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B. G Protein-Coupled Receptors Homo- and Heteromerization
1. Homomerization of G Protein-Coupled Receptors. A number of new approaches made it possible to obtain convincing evidence for the existence of homomers of many types of GPCR. Those include complementary chimeras, coimmunoprecipitation with differentially epitope-tagged receptors, the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), often in combination with covalent cross-linking, and finally biophysical methods, namely bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET).
In the mid 1980s, evidence for dimerization was obtained with experiments with photo-affinity labeling, radiation inactivation, cross-linking, and hydrodynamic analysis (Fraser and Venter, 1982; Avissar et al., 1983; Herberg et al., 1984; Peterson et al., 1986). However, it was the work of Maggio, Wess, and colleagues (Maggio et al., 1993) that offered strong indications for the existence of dimers, with the transcomplementation results obtained using cholinergic M3 muscarinic receptor/2 adrenergic receptor chimeras. In fact, when a chimera consisting of 5TM regions of one receptor and two of the other was expressed, there was a lack of ligand binding and of function that was recovered after coexpression of the two types of chimeras. This finding had a major impact, and the interpretation that the functional transcomplementation was caused by intermolecular interactions leading to the formation of a dimeric complex was early on accepted (Monnot et al., 1996). The early work in the mid-1970s demonstrating negative cooperativity in -adrenergic receptors, opening up the possibility of dimer formation should also be mentioned (Limbird et al., 1975; Limbird and Lefkowitz, 1976). More recently, structural implications for V2 vasopressin receptor oligomerization have been given by Schultz et al. (2000) from functional reconstitution studies.
The SDS-PAGE approach provided one of the first demonstrations of GPCR dimers, namely of 5-HT1B, D1, and D2 receptor homomers (Ng et al., 1993, 1994a,b, 1996; George et al., 1998; Zawarynski et al., 1998). These observations are based on the fact that several homomers are resistant to the denaturation properties of SDS. Thus, it was possible early on for George, O'Dowd, and colleagues to observe upon SDS-PAGE that the 5-HT1B, D1, and D2 receptors expressed in cell lines formed molecular species not only corresponding to monomers but also to dimers (Ng et al., 1993, 1994a,b; Lee et al., 2000). These dimeric and even higher order oligomeric complexes were not caused by glycosylation of monomers nor to the presence of G proteins (Lee et al., 2000). Treatment with covalent cross-linkers before solubilization increased the proportion of dimeric complexes and facilitated the demonstration of dimers in immunoblots (Hebert et al., 1996; Romano et al., 1996). It should be considered that monomers at least in some cases could represent the disruption of dimers or higher oligomeric complexes (Lee et al., 2000). Also by using the SDS-PAGE strategy, the existence of D2 and A1 receptors homomers was demonstrated, for the first time, in brain tissue, showing their existence in situ and not only in cell lines where artificially high levels of receptors are expressed (Ciruela et al., 1995; Ng et al., 1996).
The coimmunoprecipitation approach was first described in the article by Bouvier and colleagues in 1996 (Hebert et al., 1996) on 2 adrenergic receptors with coexpression of differentially tagged 2 adrenergic receptors. This coimmunoprecipitation was taken as evidence for the existence of a 2 adrenergic receptor dimer. Peptides corresponding to TM6 were found to disrupt the dimerization as well as receptor activation indicating a participation of the hydrophobic forces in the TM6 region in the dimerization interface (Hebert et al., 1996; Ng et al., 1996). The focus on the TM6 came from the work of Engelman et al. (Lemmon et al., 1992; Lemmon and Engelman, 1994), showing that dimerization is driven by specific interactions among TM -helices. A number of GPCR homomers have been demonstrated with this approach, e.g., the metabotropic glutamate receptor (mGlu5) homomer, where the disulfide bridges between the large extracellular NH2-terminal domains play a role in the formation of the homomer (Romano et al., 1996; Bouvier, 2001).
The techniques of BRET and FRET imply as close distances as 5 to 10 nM between donor and acceptor for energy transfer. In FRET both the donor and acceptor are fluorescent molecules, whereas for BRET the donor is bioluminescent and the acceptors fluorescent. These techniques have been very valuable in detecting dimers in living cells without the risk of solubilization artifacts (for details on methodology, see Bouvier, 2001). In 2000, the BRET approach could for the first time demonstrate homomerization of 2 adrenergic receptors in living cells (Angers et al., 2000) independently, and at the same time, the FRET procedure revealed the homomerization of the yeast mating factor in living cells (Overton and Blumer, 2000). In 2001, using the FRET and BRET technologies, constitutive homo-oligomerization could be demonstrated for opioid receptors in intact cells (McVey et al., 2001). The same was also found to be true for the thyrotropin-releasing hormone (TRH) receptors using the BRET technique (Kroeger et al., 2001). With FRET technique, the somatostatin receptor subtypes have been shown to assemble, e.g., as homomers (Rocheville et al., 2000b) using differential epitope tagging and fluorescently labeled antibodies against the epitopes.
Together with results from coimmunoprecipitation experiments these results indicate that several, if not many, GPCR undergo constitutive homomerization, i.e., the basal state of the GPCR may be the dimer. It remains to be shown if the existence of constitutive dimers can help explain the constitutive, agonist independent, activity of several GPCR. We have, e.g., recently observed that D2 receptor antagonists do not affect or even reduce the D2 receptor clustering (increased by D2 receptor agonists) in the basal state of CHO cell lines expressing human D2L receptors on their surface. Such an effect of the D2 receptor clustering by a D2 receptor antagonist in the absence of a dopamine receptor agonist could at least in part explain an inverse D2 agonist activity of the D2 antagonist (L. F. Agnati, S. Ferré, R. Franco, and K. Fuxe, unpublished data). Therefore, the action of the agonist at GPCR may sometimes be to produce a conformational change in the basal homomer leading to the development of the active state. Again it should be emphasized that the basal state instead sometimes may be represented by a monomeric or higher oligomeric form of the GPCR.
2. Heteromeric Complexes Involving G Protein-Coupled Receptors.
The existence of heteromers of GPCR was postulated in the 1993 Molecular Neurobiology review article (Zoli et al., 1993) to give a molecular basis for the large amount of evidence we had obtained on the existence of receptor/receptor interactions among GPCR (see above). It was therefore inspiring when the first evidence of the existence of a GPCR heteromer, namely the GABAB heteromer, came in 1998/99 (Jones et al., 1998; Kaupman et al., 1998; White et al., 1998; Kuner et al., 1999; Marshall et al., 1999; Ng et al., 1999). The field of heteromerization has been excellently summarized by Marshall (2001) and by Bouvier (2001). Here some key examples will be discussed together with the evidence suggesting that the heterodimerizaton and hetero-oligomerization may be the main molecular basis for the previously observed receptor/receptor interactions among GPCR (Fuxe and Agnati, 1985, 1987; Agnati et al., 1993; Zoli et al., 1993; Fuxe et al., 1995, 1996, 1998; Ferré et al., 1997). The functional relevance, the pathological implications and the relevance for new drug development (see Agnati et al., 1986, 1990; Fuxe et al., 1989, 1998) of certain selected receptor heteromers are also discussed.
One question that arises out of the recent reports is how to understand the architecture of cross-talk among heteromeric complexes. It is interesting to ask, for instance, how the strongly antagonistic A2A/D2 intramembrane receptor/receptor interaction through its heteromeric complex (Hillion et al., 2002) becomes integrated with the strongly facilitatory SSTR5/D2 intramembrane receptor/receptor interaction through its heteromeric complex (Rocheville et al., 2000a). Do these heteromeric complexes directly interact? Are they part of the same molecular circuits of the same striatal nerve cell or are they independently located in different membrane domains of the same cell or even in different striatal nerve cell populations? These will be important studies to perform in order to understand the integration of transmitter signals in the striatum and in general in the brain. How do the various heteromeric complexes of GPCR interact at membrane level and downstream at intracytoplasmatic level? How are these two levels of interactions integrated? These are fundamental questions to be answered and a possible heuristic frame to tackle these questions is discussed below (Section II.C.).
a. The GABAB Receptor Heterodimer.
The first evidence that two subtypes of GABAB receptors, GABABR1 and GABABR2, undergo heterodimerization and that this process is essential for the cell surface expression of the functional receptor was given in three studies published simultaneously in December 1998 (Kaupmann et al., 1998; Jones et al., 1998; White et al., 1998). This was also demonstrated by Kuner et al. and Ng et al. in January 1999 (see also Gordon et al., 1999; Mitrovic et al., 2000). The physiological relevance of these findings is supported by the demonstration of coimmunoprecipitation in cerebral cortex membranes of GABABR1 (a or b) with GABABR2 proteins, of their colocalization in dendritic spines (Kaupmann et al., 1998) and of substantial degree of coexpression of GABABR1 and GABABR2 mRNA levels in many nerve cell populations (Kuner et al., 1999). Changes also occurred at the recognition site level as a result of the heterodimerization, since the potency of agonists and partial agonists became increased (Kaupmann et al., 1988).
The most dramatic change is, however, that the formed GABAB receptor heteromer, unlike its monomeric components, can become functional and couple to the G protein leading to regulation of the inwardly rectifying K+ channels, the Ca2+ channels, and adenylyl cyclase (Alger and Nicoll, 1979; Bettler et al., 1998). Thus, it seems as if the predominant native GABAB receptor is the GABAB receptor heteromer. Evidence has been presented that the COOH-terminal domain is involved in the formation of this heterodimer by a coiled-coil interaction (Kammerer et al., 1999; Kuner et al., 1999).
In 2000, the important observation was made that the coiled-coil interaction at the COOH-terminal domain blocks a retention motif for the endoplasmic reticulum of the GABABR1 receptor (Margeta-Mitrovic et al., 2000). The masking of this motif allows the heterodimers to travel to the cell surface. However, even if in the mutant GABABR1 the retention motif had been removed and this mutant receptor could be expressed on the cell surface, it still remained functionally inactive underlining a probable crucial role of GABAB receptor heterodimerization in signaling.
These important findings give a clear example of the functional relevance of intramembrane receptor/receptor interactions through heteromerization, namely in receptor trafficking, including receptor maturation and receptor cell surface expression, and in receptor signaling, i.e., in G protein coupling and in increased binding potency of agonists and partial agonists, in line with previous work on receptor/receptor interactions (see Fuxe and Agnati, 1985, 1987; Zoli et al., 1993).
It is well known that GABAB receptors play a distinct role in modulating the neuronal networks, and agonist drugs acting on these receptors appear to have inter alia anticonvulsive and anxiolytic properties. It is therefore of substantial interest that the anticonvulsive compound gabapentin is a selective agonist at the GABABR1a/GABABR2 heteromer (Ng et al., 2001). This is an example of how the molecular composition of the heteromer determines its pharmacological profile and gives rise to a novel GABAB receptor agonist selective for a certain type of GABAB receptor heteromer dependent on the splice variant involved. In this study, these results were correlated with a selective ability to increase postsynaptic GABAB receptor signaling (opening of inwardly rectifying K+ channels) without altering GABA transmission at the presynaptic level (Ng et al., 2001). This novel type of pharmacological selectivity based on unique heteromers may therefore have considerable potential for drug development. It serves to show the pharmacological relevance of intramembrane receptor/receptor interactions that may give rise to novel receptor subtypes with a unique pharmacology based on the composition of the heteromer formed changing the biochemical characteristics of the binding pocket of the receptor.
b. Heteromerization of and Opioid Receptors and of µ and Opioid Receptors.
After the discovery of the GABAB receptor heterodimers the discovery of the / opioid receptor dimer came next (Jordan and Devi 1999; Jordan et al., 2000) followed by another interesting paper on µ/ opioid receptor heteromerization by George et al., (2000). In this case, however, the two receptors of the heterodimer were functional on their own and could reach the cell surface without heteromerization with another opioid receptor subtype.
These discoveries were in a way expected, since early work had given indications for the existence of a µ/ opioid receptor complex (Rothmann et al., 1988; Schoffelmeer et al., 1990). Furthermore, the opiate receptor field had for some time discussed the possibility that heterodimerization among the cloned µ, , and opioid receptors could explain the existence of more than three opioid receptor subtypes as characterized pharmacologically (Kieffer, 1999). The / opioid heteromer (Jordan and Devi, 1999; Jordan et al., 2000) was shown to have a unique pharmacology with high affinity for rather unselective ligands but very little affinity for - and -selective compounds. Nevertheless, the selective and agonists, when given at the same time, bound synergistically to the heterodimers associated with a synergistic activation of the mitogen-activated protein kinase (MAPK). Thus, a novel subtype of opioid receptor binding pocket may have appeared through this receptor/receptor interaction via / opioid receptor heterodimerization. Also, the and heteromer had consequences for the agonist-induced internalization of the opioid receptor, which became reduced. Thus, another functional role of this intramembrane receptor/receptor interaction through heteromerization may be the control of receptor internalization.
The µ/ opioid receptor heteromer, demonstrated by George et al. (2000), also showed changes in the pharmacological properties at the recognition site with reduced affinity for selective agonists and increased affinity for certain enkephalin peptides. Can in fact the µ/ heteromer be the target of distinct enkephalin peptides? Of special interest was the demonstration that the µ/ heterodimer, unlike the µ and receptors when expressed alone, could become coupled to G proteins resistent to pertussis toxin, like GZ. Thus, the G protein coupling has become markedly altered in the heteromer. In fact, the major function of this intramembrane receptor/receptor interaction based on µ/ heteromerization may be a change in the selection of G protein coupling involving a conformational change in the G protein interface of the µ/ receptor heteromer. The other functional change is altered binding properties of the recognition site seen as a novel pattern of ligand binding based on affinity changes; a novel binding pocket seems to have appeared (see Levac et al., 2002).
c. The Serotonin 5-HT1D/5-HT1B Receptor Heteromer.
This is an interesting demonstration of how two receptor subtypes of the type A receptor family (rhodopsin-like GPCR) when coexpressed preferentially form heteromers (Xie et al., 1999) without homomers. In contrast, when the two receptor subtypes were expressed alone homomers were formed. It is of substantial interest that the two receptor subtypes when coexpressed prefer the heteromer, since it indicates that at least in some cases there is a markedly displaced equilibrium between homomers and heteromers, since the heteromer is so clearly preferred. In this example the formation of the heteromer was not associated with a change in the binding pocket, and the functional relevance of this receptor/receptor interaction still remains to be determined. Based on their expression patterns in the brain it seems as if they form different types of complexes in the brain, namely 5-HT1B receptor and 5-HT1D receptor homomers and 5-HT1B/5-HT1D receptor heteromers.
d. The Dopamine D2/D3 Receptor Heteromer.
The D2 and D3 receptors are known to exist as monomers and homomers (Nimchinsky et al., 1997; see Lee et al., 2000). In 2001, the evidence also came that D2 and D3 receptors can form heteromers with unique functional properties (Scarselli et al., 2001). Coimmunoprecipitation experiments using differentially tagged D2 and D3 receptors showed that D2 and D3 receptors in HEK-293 cells can form a heteromeric complex. Furthermore, they were able to demonstrate that heterologous cotransfected dopamine receptor fragments [D2 trunc (TM1-5)/D3 tail (TM6-7); D3 trunc (TM1-5)/D2 tail (TM6-7)] could form functional dopamine receptors that bound dopamine agonists and antagonists with a different pharmacological profile compared with native D2 and D3 receptors, with the highest affinity of all being found with the D3 trunc/D2 tail fragment combination. Thus, split D2/D3 heteromers may be formed through the domain-swapping mechanism as proposed by Gouldson, Reynolds, and colleagues (Gouldson et al., 1998, 2000) based inter alia on demonstrations of functional complementation between chimeras of 2 adrenergic and M3 muscarinic receptors (Maggio et al., 1993).
In agreement with the formation of D2/D3 heteromers in cells, these types of D2 and D3 receptor fragments, when coexpressed with native D2 and D3 receptors, reduced the expression of native dopamine receptors indicating fragment/native receptor complex formation. However, not only are D2 trunc/D3 tail and D3 trunc/D2 tail receptors able to bind ligands but they can also couple in an inhibitory way to adenylyl cyclase and to the same extent as the native D2 receptor. It is also of substantial interest that the D3 receptor under conditions in which it cannot inhibit adenylate cyclase VI (Robinsson and Caron, 1997) can develop such a coupling by cotransfection with D2 receptors. It is therefore possible that in the D2/D3 heteromeric receptor complex formed, the D2 receptor can make possible the G protein coupling of the D3 receptor to adenylyl cyclase VI. Alternatively, the D3 receptor binding pocket upon activation by D3 agonists can, through conformational changes, transfer the D2 pocket of the heteromer into an activated state, leading to Gi activation and adenylyl cyclase VI inhibition. Finally, in the case of adenylyl cyclase V activity, the coexpression of D2 and D3 receptors even resulted in an increased potency of the D3 receptor agonist to inhibit this adenylyl cyclase compared with wild-type D2 receptors when activated by D2 agonists. One function of the D2/D3 heteromeric receptor complex may therefore be to allow a stronger inhibitory coupling of the D3 receptors to adenylyl cyclase.
Colocalization of D2 and D3 receptors has been demonstrated in nerve cells of the basal ganglia (Le Moine and Bloch, 1996; Gurevich and Joyce, 1999) showing that there is the potential to form functional D2/D3 heteromers also in vivo.
e. The Somatostatin SSTR5/SSTR1 Receptor Heteromer.
The study by Rocheville et al. (2000b) gives a fine illustration of intramembrane receptor/receptor interactions and their functional relevance and of the relationship of monomers, homomers, and heteromers among five somatostatin receptor subtypes. Using FRET analysis, the human somatostatin receptor subtype SSTR5 was shown to exist as a monomer in the basal state, which upon agonist activation was converted into a homomer. The data suggested that the agonist-induced dimerization of SSTR5 receptors was essential for signaling. Agonist-induced heteromerization of SSTR5 and SSTR1 receptors could also be demonstrated, which appeared to be subtype specific.
The suggestion was made that the reported high level of basal homomer expression of GPCR could be due to receptor overexpression. The intramembrane SSTR5 and SSTR1 receptor/receptor interactions through heteromerization was shown to have important functional consequences for the participating receptors. Besides the changes in agonist affinity that usually develop upon changes in the oligomeric state, marked alterations in agonist-dependent internalization and an up-regulation of SSTR1 receptors occurred through formation of a heteromer with SSTR5 receptors. Thus, the SSTR1 receptor only underwent agonist-induced internalization as a heterodimer with the SSTR5 receptor. Furthermore, the heteromerization allowed a somatostatin receptor agonist (not binding to SSTR1) to induce up-regulation of agonist binding at the SSTR1 receptor. SSTR5 receptor signaling via its G protein is probably not involved in this response of the heteromer, since the COOH-terminal tail of the SSTR5 receptor had been deleted abolishing adenylyl cyclase regulation. These results are of substantial interest since in this way the desensitization of activated somatostatin receptor subtypes can be compensated for by an up-regulation of the nonactivated somatostatin receptor subtypes, such as SSTR1, made possible through the heteromerization. Thus, another functional meaning of intramembrane receptor/receptor interactions via heteromerization may be the sensitization of one isoreceptor as the other isoreceptor of the heteromer undergoes desensitization.
f. The Somatostatin SSTR5 and Dopamine D2 Heteromeric Receptor Complex.
The discovery of this intramembrane receptor/receptor interaction through hetero-oligomerization gave a novel way to understand the well known somatostatin/dopamine interactions in the brain involved, e.g., in the control of motor activity (Cohn and Cohn, 1975; Havlicek et al., 1976; Kastin et al., 1978; Chneiweiss et al., 1985; Glowinski and Premont, 1985; Martin-Iverson et al., 1986; Leblanc et al., 1988; Izquierdo-Claros et al., 1997; Rodriguez-Sanchez et al., 1997). In this case the hetero-oligomerization involved distinct GPCR and not isoreceptors having the same or similar endogenous ligands. It was by means of photobleaching FRET microscopy that the direct SSTR5/D2 receptor/receptor interaction could be determined, and oligomerization was hardly observed in the basal state but only after treatment with either agonist. Simultaneous treatment with the two types of agonists together had no further action (Rocheville et al., 2000a). It is known that D2 receptor homomers exist in the basal state (Lee et al., 2000), and somatostatin homomers are induced by somatostatin receptor agonists (see above). It remains to be determined whether heterodimers are formed or larger oligomeric complexes in the case of the SSTR5/D2 heteromerization.
The functional meaning of this direct intramembrane receptor/receptor interaction appeared to be severalfold (Rocheville et al., 2000a). First, the binding pocket of SSTR5 receptor was markedly altered, since a 30-fold increase in affinity was found upon D2 receptor agonist activation, whereas D2 receptor antagonists reduced the affinity of SSTR5 receptors for the somatostatin agonist SST-14. Thus, different conformational states (agonist-antagonist states) of the D2 receptor have a substantial modulatory action on the binding pocket of the SSTR5. The interaction at the recognition site level was also reciprocal since the somatostatin agonist enhanced the affinity of the D2 receptor for antagonists. Second, the G protein coupling of the SSTR5 receptor was enhanced by the D2 receptor activation, since the reduction of SSTR5 receptor agonist binding by GTPS was enhanced by the D2 agonist. Furthermore, the inhibitory responses on cAMP accumulation were significantly enhanced by simultaneous agonist treatments, emphasizing the enhancement of the functional activity through the hetero-oligomer formed and the associated conformational changes induced by agonists in this complex. In fact, these functional changes in the intramembrane receptor/receptor interaction induced by SSTR5 and D2 receptor agonists may explain the increased somatostatin- and D2 receptor-mediated neurotransmission found in vivo after somatostatin or dopamine agonist treatments.
In this study, Rocheville et al. (2000a) also used the mutant 
318-SSTR5 receptor, with a COOH-terminal tail deletion. This mutant SSTR5 receptor had been previously shown to bind somatostatin agonists with unchanged affinity, but different than the wild-type SSTR5 receptor, it is not able to produce inhibition of forskolin-induced cAMP accumulation (Hukovic et al., 1998). The interesting finding was that cotransfection with D2 receptors and mutant 318-SSTR5 receptors could restore the somatostatin agonist signaling to the adenylate cyclase provided the D2 recognition site was not blocked by a D2 antagonist. These observations can be explained by the formation of a hetero-oligomer in the CHO-k cells used, in which the SSTR5 binding pocket when activated by agonists can signal via a conformational change in the dopamine D2 binding pocket. This would lead to a coupling of the D2 recognition site to the Gi protein followed by its activation and subsequent inhibition of adenylate cyclase. This conformational change cannot occur when the D2 binding pocket is in an antagonistic binding state. It indicates in fact that the SSTR5 receptor can signal via a conformational change in the D2 receptor similar to that produced by the D2 agonist. In other words, a cross-activation of the D2 receptor can occur in the absence of dopamine by a direct receptor/receptor interaction in the receptor interfaces of the hetero-oligomer. Thus, the activated SST receptor cannot only modulate the activated D2 receptor/Gi protein coupling to adenylate cyclase but also produce a constitutive activity of the D2 receptor when it is not locked into an antagonistic state. From another perspective, it represents an example of how a mutant GPCR can rescue its signaling by activating another receptor coupled to the same type of G protein.
g. The Adenosine A1 and Dopamine D1 Heteromeric Receptor Complex.
The article on A1/D1 heteromers came out a couple of months after the appearance of the SST5/D2 receptor oligomer article and gives another example of heteromerization between distinct GPCR (Gines et al., 2000). A number of morphological and neurochemical observations indicate that adenosine A1 and dopamine D1 receptor/receptor interactions exist in the basal ganglia (Ferré et al., 1994b, 1996a,b, 1997; Fuxe et al., 1998, 2002; Franco et al., 2001) and colocalization of A1 and D1 receptors exists in primary cortical cultures (Gines et al., 2000). The article by Ginés et al. (2000) gives the first evidence that this receptor/receptor interaction can involve A1/D1 heteromeric receptor complexes since such complexes could be demonstrated in cotransfected A1/D1 fibroblast Ltk- cells by means of coimmunoprecipitation. Thus, the previously found A1 receptor-induced uncoupling of the D1 receptor, demonstrated as the A1 receptor-induced disappearance of the high-affinity D1 receptor agonist binding sites in membrane preparations (Ferré et al., 1994b, 1998; Fuxe et al., 1998), could be the result of a physical interaction of the A1 receptor with the D1 receptor in this heteromeric complex, leading to an uncoupling of the D1 receptor to its Gs-like protein in this functionally interacting heteromeric complex. The coimmunoprecipitation analysis demonstrates its existence already in the basal state and the specificity by the failure to show A1/D2 receptor heteromerization in A1/D2 receptor cotransfected fibroblast cells.
However, A1/D1 receptor heteromerization in the cotransfected fibroblast cells was strongly reduced by the D1 receptor agonist treatment, showing an agonist dependence, and simultaneous D1 and A1 receptor agonist treatment blocked this disruption of the heteromeric complex. Thus, like the Rocheville et al. (2000a) study, this study shows how agonists alone or simultaneous treatment lead to conformational changes in their respective binding pockets that are transmitted to the heteromeric interface and results in strengthening or disruption of the complex. In this case, the physical interaction is maintained when the A1 and D1 receptor binding pockets are simultaneously activated by agonists allowing the antagonistic intramembrane receptor/receptor interaction to take place, namely the G protein uncoupling with the disappearance of the high-affinity state of the D1 receptor for agonists. One functional meaning of this intramembrane receptor/receptor interaction is therefore uncoupling of the D1 receptor from Gs protein. This is in sharp contrast to the enhanced functional activity of the SSTR5/D2 receptor oligomers, especially after combined agonist treatment (Rocheville et al., 2000a).
The A1/D1 heteromeric receptor complex may therefore give the molecular basis for the well documented antagonistic A1/D1 receptor/receptor interactions found in the neuronal networks of the brain (Ferré et al., 1997; Fuxe et al., 1998, 2002; Franco et al., 2000, 2001). The A1/D1 receptor heteromerization also appears to have an impact on receptor trafficking (Ginés et al., 2000). Thus, an A1 receptor agonist, after 3 h of exposure, produced a coaggregation of A1 and D1 receptors. On the other hand, aD1 receptor agonist after 3 h of exposure only produced an aggregation of D1 receptor immunoreactivity with a lack of coaggregation in agreement with the ability of the D1 receptor agonist to disrupt A1/D1 receptor heteromerization (see above). The D1 receptor signaling remained unaffected by the formation of D1 receptor or A1/D1 receptor clusters, as seen in terms of an unchanged D1 receptor-stimulated cAMP accumulation and thus with no signs of D1 receptor desensitization. In contrast, combined A1 and D1 receptor agonist treatments under the same conditions did not result in the formation of A1/D1 receptor clusters, but the diffuse A1/D1 receptor colocalization was maintained. Furthermore, now signs of D1 receptor desensitization developed as seen from reductions in D1 receptor-induced increases of cAMP levels. Thus, essential features of D1 receptor desensitization may be a maintained heteromerization with no A1/D1 receptor coaggregates formed after prolonged combined exposure to A1 and D1 receptor agonists with no indications of receptor internalization. It seems possible that the D1 receptor desensitization may be mainly caused by a prolonged allosteric change in the D1 receptor brought about by the A1/D1 receptor/receptor interaction within the heteromeric complex, which could be related to subsequent phosphorylation changes and/or association with -arrestin-like molecules (Lefkowitz, 2000; McDonald and Lefkowitz, 2001), leading overall to a reduced D1 receptor/Gs coupling. Thus, it may be suggested that the intramembrane A1/D1 receptor/receptor interaction in this heteromeric complex is relevant not only for acute antagonism of D1 receptor signaling but also for a persistent long-term antagonism of D1 signaling to the Gs protein. The details of the composition and stoichiometry of the A1/D1 heteromeric receptor complex is unknown, and A1 and D1 receptors are known to exist as monomers and homomers (Ciruela et al., 1995; Franco et al., 2000; Lee et al., 2000). It is unknown if heteromers are preferred when A1 and D1 receptors are coexpressed in the same cells.
h. The Metabotropic Glutamate mGluR1 and Adenosine A1 Heteromeric Receptor Complex.
There is evidence that the group I metabotropic glutamate receptor mGluR1 and adenosine A1 receptors colocalize in certain types of cerebellar neurons and that functional interactions occur between adenosine and glutamate receptors in the brain (Ferré et al., 1999a; Ciruela et al., 2001a,b). Coimmunoprecipitation experiments on soluble extracts from the rat cerebellum synaptosomes have shown that mGluR1 receptors can coimmunoprecipitate with anti-A1 receptor antibodies. Thus, mGluR1 and A1 receptors may exist as heteromers in certain cell populations of the cerebellum (Ciruela et al., 2001a). Subsequent coimmunoprecipitation studies on transiently cotransfected HEK-293 cells showed that mGluR1/A1 heteromeric receptor complexes exist also in these cells. The receptor subtype specificity was shown by the failure of the COOH-terminal splice variant, the mGluR1 to immunoprecipitate with the A1 receptor, indicating the involvement of the COOH-terminal tail in the formation of this heteromeric complex. It is unknown how this heteromeric complex relates to mGluR1 receptor dimer, where the interface modulates the glutamate binding site of the extracellular region of the receptor (Kunishima et al., 2000).
The functional role of this heteromerization was especially studied in the HEK-293 cells. After cotransfection of the mGluR1 and A1 receptors, it was found that quisqualic acid substantially enhanced the increase in Ca2+ signaling produced by A1 receptor activation, and the same was true when A1 receptor modulation of mGluR1 receptor function was studied. Thus, it seems as if heteromerization led to the development of synergistic responses in Ca2+ signaling upon simultaneous activation of the receptors within the mGluR1/A1 receptor heteromer. Also in primary cortical cultures, where a high colocalization was observed at dendritic locations, a synergistic interaction was found in terms of a reduction of NMDA-induced neurotoxicity (Ciruela et al., 2001a,b). A reduced hypoxic neuroprotection has been observed in mice lacking A1 receptor (Johansson et al., 2001). It seems possible that the mGluR1/A1 receptor heteromerization can take place indirectly, since the COOH terminus interacts with specific targeting proteins. Thus, the protein Homer-1a with an enabled VASP homology 1-like domain binds to the COOH-terminal of mGluR1 receptor. Homer-1c can link together proteins with a proline-rich motif (PPSPF), since it binds to this motif (Tu et al., 1999). It is of substantial interest that this or a similar motif is found in both the COOH-terminal part of mGluR1 and in the COOH-terminal part of the A1 receptor. Thus, the Homer can be an important part of this heteromeric complex as an adapter protein (Xiao et al., 2000; Ciruela et al., 2000). It should also be noticed that the Homer can also link mGluR1 to Shank, since it also contains a similar motif (PPEEF). Shank is a scaffolding multimeric postsynaptic protein that may bring the mGluR1 to the appropriate location on the cell surface and is part of the NMDA receptor-associated PSD-95 complex (Naisbitt et al., 1999; Tu et al., 1999).
i. The Purinergic P2Y1 and Adenosine A1 Heteromeric Receptor Complex.
In 2001, the fascinating finding was made in coimmunoprecipitation experiments on cotransfected HEK-293 T cells that Gi/o-coupled P1 purinoceptor adenosine A1 receptors can form a heteromeric complex with Gq-coupled P2 purinoceptor ATP P2Y1 receptors (Yoshioka et al., 2001), showing less than a 5% homology with each other (Fredholm et al., 1994). The COOH-terminal part of A1 receptors was shown not to be involved in this type of heteromerization. In agreement immunofluorescence studies with confocal imaging showed a marked colocalization of the A1 and P2Y1 receptors. Furthermore, A1 receptors coimmunoprecipitated with P2Y1 receptors indicating that heteromerization between P1 and P2 receptor subtypes could be a rather widespread mechanism for the immediate cross-talk between, e.g., inhibitory A1 receptors and excitatory P2Y1 receptors.
A marked change in the signaling was found in the heteromeric complex. Thus, it became possible for the P2Y1 receptor agonist ADPS to induce signaling via the Gi/o protein coupled to the A1 receptor, an action blocked by pertussis toxin and the A1 receptor antagonist but not by the P2Y1 receptor antagonist.
These interesting results seemed to be explained by the development of an ability of the ADPS to reduce 3H-labeled (-)-N6-phenylisopropyladenosine binding of the heteromeric complex in the high-affinity range. It was therefore suggested that the A1 receptor ligand pocket of the heteromer had markedly changed so as to bind the P2Y1R agonist associated with activation of the Gi/o protein. Thus, it was correctly suggested that the heteromeric association produces a P2Y1-like A1 receptor. However, it is also possible to propose another molecular mechanism. Thus, there could exist an A1/P2Y1 receptor/receptor interaction at the recognition site level that changes the pharmacology of both the A1 and P2Y1 receptor binding pockets. The P2Y1 agonist-induced conformational change in the P2Y1 receptor binding pocket now not only leads to an activation of Gq proteins but also to a change in the conformation of the A1 receptor, converting it into an agonist state capable of turning on the Gi/o protein. Such a conformational change may no longer occur when the A1 receptor binding pocket is occupied by the A1 receptor antagonist locking it into an antagonist state. The P2Y1 receptor antagonist may not block the action of the P2Y1 receptor agonist since it seems possible that in the heteromeric complex the antagonist used may not have sufficient affinity for the P2Y1 receptor binding pocket. The possible existence of the P2Y1/A1 heteromeric receptor complex in the brain could explain the demonstration of theophylline-sensitive P2Y receptors (Mendoza-Fernandez et al., 2000). This P2Y1/A1 heteromeric receptor complex is of substantial interest, since it allows the excitatory ATP receptor P2Y1 upon activation to immediately activate in parallel the inhibitory A1R mechanism. In this way, the excitation and increased energy expenditure brought about by the ATP P2Y1 receptor activation begins to be counteracted even at a moment when the extracellular ATP has not been broken down to adenosine, the major ligand for the A1 receptor (Fredholm, 1995a,b; Ferré and Fuxe, 2000; Fredholm et al., 2001).
j. The Adenosine A2A and Dopamine D2 Heteromeric Receptor Complex.
In 1991, the antagonistic A2A/D2 receptor/receptor interaction was demonstrated in striatal membrane preparations with A2A receptors reducing the affinity of D2 receptors, especially in the high-affinity state, for agonists (Ferré et al., 1991d). This offered a novel mechanism for the reported antagonistic adenosine/dopamine interactions found in the brain (Ferré, 1992, 1997; Fuxe et al., 1993, 1998; Lepiku et al., 1997). The molecular mechanism was proposed to be one of heteromerization of A2A/D2 receptors (Zoli et al., 1993). The same antagonistic intramembrane modulation of D2 receptor recognition mechanisms by A2A receptor activation was observed in different cell lines stably cotransfected with different species and isoforms of A2A and D2 receptors. These were a native A2A receptor/human D2L receptor neuroblastoma cell line (Salim et al., 2000), a dog A2A receptor/human D2L receptor Ltk- fibroblast cell line (Snaprud e
