The existence of neurotransmitter receptor heteromers is becoming broadly accepted and their functional significance is being revealed. Heteromerization of neurotransmitter receptors produces functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer (1-3). The occurrence of receptor heteromers with different pharmacological and signaling properties opens a complete new field to search for novel drug targets useful against a variety of neuropsychiatric disorders with potentially less side effects 1-3). We have recently hypothesized that neurotransmitter receptor heteromers can function as processors of computation that modulate cell signaling (1), and some neurotransmitter heteromers can exert an effect as processors of computations that directly modulate pre- or postsynaptic neurotransmission (1).We have also hypothesized that some neurotransmitter heteromers play a key integrative role in local modules (4), which we have defined as a minimal portion of one or more neurons and/or one or more glial cells that operates as an independent integrative unit (4). Our recent work is related to the identification and study of the neurotransmitter receptor heteromers that modulate the function of the striatal spine modules, which are the most common local module in the striatum (4,5). The striatal spine modules are comprised of the dendritic spine of the medium spiny neuron, its glutamatergic, dopaminergic and/or GABAergic nerve terminals and astroglial processes (4,5). We believe that understanding the integrated function of the striatal spine modules will have important implications for the study of basal ganglia function and disfunction, including drug abuse.? ? The main neurotransmitters influencing the striatal spine modules are dopamine, glutamate, adenosine, GABA, acetylcholine, endocannabinoids, serotonin and histamine. In previous studies we discovered the existence of heteromerization between the following receptors localized in different elements of the striatal spine module: adenosine A2A and dopamine D2, adenosine A1 and dopamine D1, A1 and A2A, A2A and metabotropic glutamate mGlu5, D2 and nicotinic acetylcholine (alpha4-beta2) and A2A and cannabinoid CB1 (reviewed in 4-6). During the last year we demonstrated the existence of D2-histamine H3 and D1-dopamine D3 receptor heteromers (cannabinoid CB1 receptor (7,8). Our results demonstrate that an important part of the previously unknown function of the enigmatic H3 and D3 receptors depends on their ability to modulate D2 and D1 receptor function, respectively, by means of their ability to form D2-H3 and D1-D3 receptor heteromers (7,8). In addition, by using a new biophysical method we called sequential resonance energy transfer (SRET, 9), a method that combines bioluminescence and fluorescence resonance energy transfer techniques (BRET and FRET, respectively, 10), we have identified complexes of A2A, D2 and CB1 receptors in living transfected cells (9). Although not yet demonstrated, it is likely that functional A2A-CB1-D2 receptor heteromers are found where these receptors are highly co-expressed, in the GABAergic enkephalinergic striatal neurons, and that they play an important role in the behavioral effects of cannabinoids (5). ? ? The introduction of the two concepts local module and receptor heteromer not only facilitates the understanding of the functional role of interactions between different neurotransmitters in the brain, but also the understanding of the mechanisms of action of central acting drugs. For instance, we have also been studying for some years the mechanisms of action behind the central effects of caffeine, a non-selective A1-A2A receptor antagonist. We now know that a critical aspect of the mechanisms of the psychostimulant effects of caffeine is its ability to release the pre- and postsynaptic brakes that adenosine imposes on dopaminergic transmission by acting on different adenosine receptor heteromers localized in different elements of the striatal spine modules (11,12). Thanks to this approach we are beginning to understand some of the unsolved issues about caffeines pharmacological properties, such as the tolerance to its psychostimulant and discriminative stimulus effects upon caffeine chronic administration and (11-12). ? ? We are also interested in studying some molecular and dynamic properties of receptor heteromers. We have previously found some epitopes in A2A and D2 receptors that are determinant in their ability to heteromerize. Those are an Arg-rich domain of the third intracellular loop of the D2 receptor and a phosphorylated Ser in the c-terminus of the A2A receptor (6). Previous studies indicate that calmodulin (CaM) can also bind to the Arg-rich epitope of the D2 receptor localized in the N-terminal segment of the third intracellular loop of the dopamine D2 receptor. A mass spectrometric analysis indicated that an electrostatic interaction involving the Arg-rich epitope of the D2 receptor and several CaM acidic epitopes are mainly responsible for D2 receptor-CaM binding (13). CaM disrupted the electrostatic interactions between the D2 receptor epitope and the A2A receptor epitope, indicating a possible regulatory role for CaM in receptor heteromer formation (13). Finally, by combining biomolecular fluorescence complementation and fluorescence correlation spectroscopy we measured the membrane diffusional characteristics of A1 and A2A homo- and heteromers in transfected cells (14). The results demonstrated, for the first time, differences in the plasma membrane diffusion between homo- and heteromers, with the A1-A2A receptor heteromer exhibiting a significantly faster membrane diffusion coefficient compared to A1 receptor homomers (14).
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