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The distribution and function of neurons coexpressing the dopamine D1 and D2 receptors in the basal ganglia and mesolimbic system are unknown. We found a subset of medium spiny neurons coexpressing D1 and D2 receptors in varying densities throughout the basal ganglia, with the highest incidence in nucleus accumbens and globus pallidus and the lowest incidence in caudate putamen. These receptors formed D1-D2 receptor heteromers that were localized to cell bodies and presynaptic terminals. In rats, selective activation of D1-D2 heteromers increased grooming behavior and attenuated AMPA receptor GluR1 phosphorylation by calcium/calmodulin kinase IIα in nucleus accumbens, implying a role in reward pathways. D1-D2 heteromer sensitivity and functional activity was up-regulated in rat striatum by chronic amphetamine treatment and in globus pallidus from schizophrenia patients, indicating that the dopamine D1-D2 heteromer may contribute to psychopathologies of drug abuse, schizophrenia, or other disorders involving elevated dopamine transmission.

Opioid receptor pharmacology in vivo has predicted a greater number of receptor subtypes than explained by the profiles of the three cloned opioid receptors, and the functional dependence of the receptors on each other shown in gene-deleted animal models remains unexplained. One mechanism for such findings is the generation of novel signaling complexes by receptor hetero-oligomerization, which we previously showed results in significantly different pharmacology for mu and delta receptor hetero-oligomers compared with the individual receptors. In the present study, we show that deltorphin-II is a fully functional agonist of the mu-delta heteromer, which induced desensitization and inhibited adenylyl cyclase through a pertussis toxin-insensitive G protein. Activation of the mu-delta receptor heteromer resulted in preferential activation of Galpha(z), illustrated by incorporation of GTPgamma(35)S, whereas activation of the individually expressed mu and delta receptors preferentially activated Galpha(i). The unique pharmacology of the mu-delta heteromer was dependent on the reciprocal involvement of the distal carboxyl tails of both receptors, so that truncation of the distal mu receptor carboxyl tail modified the delta-selective ligand-binding pocket, and truncation of the delta receptor distal carboxyl tail modified the mu-selective binding pocket. The distal carboxyl tails of both receptors also had a significant role in receptor interaction, as evidenced by the reduced ability to co-immunoprecipitate when the carboxyl tails were truncated. The interaction between mu and delta receptors occurred constitutively when the receptors were co-expressed, but did not occur when receptor expression was temporally separated, indicating that the hetero-oligomers were generated by a co-translational mechanism.

Although dopamine D1 and D2 receptors belong to distinct subfamilies of dopamine receptors, several lines of evidence indicate that they are functionally linked. However, a mechanism for this linkage has not been elucidated. In this study, we demonstrate that agonist stimulation of co-expressed D1 and D2 receptors resulted in an increase of intracellular calcium levels via a signaling pathway not activated by either receptor alone or when only one of the co-expressed receptors was activated by a selective agonist. Calcium signaling by D1-D2 receptor co-activation was abolished following treatment with a phospholipase C inhibitor but not with pertussis toxin or inhibitors of protein kinase A or protein kinase C, indicating coupling to the G(q) pathway. We also show, by co-immunoprecipitation from rat brain and from cells co-expressing the receptors, that D1 and D2 receptors are part of the same heteromeric protein complex and, by immunohistochemistry, that these receptors are co-expressed and co-localized within neurons of human and rat brain. This demonstration that D1 and D2 receptors have a novel cellular function when co-activated in the same cell represents a significant step toward elucidating the mechanism of the functional link observed between these two receptors in brain.

We have shown in a previous study that desensitization and internalization of the human dopamine D(1) receptor following short-term agonist exposure are mediated by temporally and biochemically distinct mechanisms. In the present study, we have used site-directed mutagenesis to remove potential phosphorylation sites in the third intracellular loop and carboxyl tail of the dopamine D(1) receptor to study these processes. Mutant D(1) receptors were stably transfected into Chinese hamster ovary cells, and kinetic parameters were measured. Mutations of Ser/Thr residues to alanine in the carboxyl tail demonstrated that the single substitution of Thr-360 abolished agonist-induced phosphorylation and desensitization of the receptor. Isolated mutation of the adjacent glutamic acid Glu-359 also abolished agonist-induced phosphorylation and desensitization of the receptor. These data suggest that Thr-360 in conjunction with Glu-359 may comprise a motif necessary for GRK2-mediated phosphorylation and desensitization. Agonist-induced internalization was not affected with mutation of either the Thr-360 or the Glu-359 residues. However, receptors with Ser/Thr residues mutated in the distal carboxyl tail (Thr-446, Thr-439, and Ser-431) failed to internalize in response to agonist activation, but were able to desensitize normally. These results indicate that agonist-induced desensitization and internalization are regulated by separate and distinct serine and threonine residues within the carboxyl tail of the human dopamine D(1) receptor.

The existence of dimers and oligomers for many G protein-coupled receptors has been described by us and others. Since many G protein-coupled receptor subtypes are highly homologous to each other, we examined whether closely related receptors may interact with each other directly and thus have the potential to create novel signaling units. Using mu- and delta-opioid receptors, we show that each receptor expressed individually was pharmacologically distinct and could be visualized following electrophoresis as monomers, homodimers, homotetramers, and higher molecular mass oligomers. When mu- and delta-opioid receptors were coexpressed, the highly selective synthetic agonists for each had reduced potency and altered rank order, whereas endomorphin-1 and Leu-enkephalin had enhanced affinity, suggesting the formation of a novel binding pocket. No heterodimers were visualized in the membranes coexpressing mu- and delta-receptors by the methods available. However, hetero-oligomers were identified by the ability to co-immunoprecipitate mu-receptors with delta-receptors and vice versa using differentially epitope-tagged receptors. In contrast to the individually expressed mu- and delta-receptors, the coexpressed receptors showed insensitivity to pertussis toxin and continued signal transduction, likely due to interaction with a different subtype of G protein. In this study, we provide, for the first time, evidence for the direct interaction of mu- and delta-opioid receptors to form oligomers, with the generation of novel pharmacology and G protein coupling properties.

In this study, we show that a peptide based on the sequence of transmembrane domain 6 of the D1 dopamine receptor (D1DR) specifically inhibited D1DR binding and function, without affecting receptor oligomerization. It has been shown that an analogous peptide from the beta2-adrenergic receptor disrupted dimerization and adenylyl cyclase activation in the beta2-adrenergic receptor (Hebert, T. E., Moffett, S., Morello, J. P., Loisel, T. P., Bichet, D. G., Barret, C., and Bouvier, M. (1996) J. Biol. Chem. 271, 16384-16392). Treatment of D1DR with the D1DR transmembrane 6 peptide resulted in a dose-dependent, irreversible inhibition of D1DR antagonist binding, an effect not seen in D1DR with peptides based on transmembrane domains of other G protein-coupled receptors. Incubation with the D1DR transmembrane 6 peptide also resulted in a dose-dependent attenuation of both dopamine-induced [35S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and receptor-mediated dopamine stimulation of adenylyl cyclase activity. Notably, GTPgammaS binding and cAMP production were reduced to levels below baseline, indicating blockade of ligand-independent, intrinsic receptor activity. Immunoblot analyses of the D1DR revealed the receptor existed as monomers, dimers, and higher order oligomers and that these oligomeric states were unaffected after incubation with the D1DR transmembrane 6 peptide. These findings represent the first demonstration that a peptide based on the transmembrane 6 of the D1DR may represent a novel category of noncompetitive D1DR antagonists.