We demonstrate a heteromeric D1-D2 dopamine receptor signaling complex in brain that is coupled to Gq/11 and requires agonist binding to both receptors for G protein activation and intracellular calcium release. The D1 agonist SKF83959 was identified as a specific agonist for the heteromer that activated Gq/11 by functioning as a full agonist for the D1 receptor and a high-affinity partial agonist for a pertussis toxin-resistant D2 receptor within the complex. We provide evidence that the D1-D2 signaling complex can be more readily detected in mice that are 8 months in age compared with animals that are 3 months old, suggesting that calcium signaling through the D1-D2 dopamine receptor complex is relevant for function in the postadolescent brain. Activation of Gq/11 through the heteromer increases levels of calcium/calmodulin-dependent protein kinase II␣ in the nucleus accumbens, unlike activation of Gs/olf-coupled D1 receptors, indicating a mechanism by which D1-D2 dopamine receptor complexes may contribute to synaptic plasticity.heterooligomerization ͉ SKF83959 ͉ calcium signaling ͉ calcium/calmodulin-dependent protein kinase II␣ D iverse roles for each of the five dopamine receptors (D1-D5) have been shown to be initiated primarily through stimulation or inhibition of adenylyl cyclase (AC) via G s /olf or G i /o signaling proteins, respectively (1). There have been reports, however, of a D1-like receptor in brain that is coupled to G q /11, stimulating phospholipase C (PLC) and intracellular calcium release (2-5). Activation of this G q /11-coupled D1-like receptor by specific receptor agonists does not correlate with the ability of these same agonists to activate AC (4), suggesting that the G q /11-coupled D1-like receptor is a molecular entity distinct from the G s /olfcoupled D1 receptor.Molecular identification of the G q /11-coupled D1-like receptor has proven elusive because D1 receptor coupling to PLC has not been demonstrated in a variety of cell types in which the D1 receptor was expressed. We had postulated that G q /11 activation by D1 receptor agonists in brain could occur by concurrent activation of the D1 receptor and the D2 receptor (6). We have shown that heterologously coexpressed D1 and D2 dopamine receptors formed heterooligomers (7) and that coactivation of these receptors resulted in a PLC-dependent rise in intracellular calcium (6). We also demonstrated that D1 and D2 receptors could be coimmunoprecipitated from striatal membranes (6). These results suggested the possibility of a unique signaling complex in brain composed of PLC-coupled D1-D2 receptor heterooligomers.In this work we report the presence of such a D1-D2 dopamine receptor signaling complex in striatum that is coupled to rapid G q /11 signaling on activation of both receptors and which can be defined by a unique pharmacology. The complex was more readily detected in older mice and could modulate levels of calcium/calmodulin-dependent protein kinase II␣ (CaMKII␣) in the nucleus accumbens, indicating a potential role for the D...

We provided evidence for the formation of a novel phospholipase C-mediated calcium signal arising from coactivation of D1 and D2 dopamine receptors. In the present study, robust fluorescence resonance energy transfer showed that these receptors exist in close proximity indicative of D1-D2 receptor heterooligomerization. The close proximity of these receptors within the heterooligomer allowed for cross-phosphorylation of the D2 receptor by selective activation of the D1 receptor. D1-D2 receptor heterooligomers were internalized when the receptors were coactivated by dopamine or either receptor was singly activated by the D1-selective agonist (Ϯ)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) or the D2-selective agonist quinpirole. The D2 receptor expressed alone did not internalize after activation by quinpirole except when coexpressed with the D1 receptor. D1-D2 receptor heterooligomerization resulted in an altered level of steady-state cell surface expression compared with D1 and D2 homooligomers, with increased D2 and decreased D1 receptor cell surface density. Together, these results demonstrated that D1 and D2 receptors formed heterooligomeric units with unique cell surface localization, internalization, and transactivation properties that are distinct from that of D1 and D2 receptor homooligomers.The dopamine receptor family is subdivided into two distinct subclasses, based on structural similarities, pharmacological profiles, and signal transduction mechanisms, into D1-and D2-like receptors. Although D1 and D2 receptor subclasses are biochemically distinct in that D1 receptors couple positively and the D2 receptors couple negatively to adenylyl cyclase, many physiological functions are known to be mediated by the coactivation of both receptors. For example, the augmentative effect of cocaine on locomotion and intracranial self-stimulation is mediated by the activation of both D1 and D2 receptors (Kita et al., 1999). Dopamine receptor synergism could occur either at the level of neuronal networks through D1-and D2-like receptors expressed in separate neuronal populations or, on the other hand, within the same neurons through convergent postreceptor mechanisms. The latter mechanism of D1-D2 receptor synergism is possible because dopamine receptor subclasses are colocalized in rat brain, with colocalization of D1-and D2-like receptors in virtually every neuron in the neonatal striatum (Aizman et al., 2000). Furthermore, our own studies have demonstrated robust colocalization of D1 and D2 receptors in a subset of neurons in human caudate nucleus, rat striatum, and cortex . These data suggest, therefore, that functional synergism could occur within individual neurons. In fact, the coactivation of both D1 and D2 receptors has been shown to result in a significant increase in action potential frequency in neurons of the substantia nigra pars reticulata (Waszczak et al., 2002) and a potentiation of arachidonic acid release in Chinese hamster ovary cells coexpre...

There is accumulating evidence that G protein-coupled receptor signaling is regulated by localization in lipid raft microdomains. In this report, we determined that the D1 dopamine receptor (D1R) is localized in caveolae, a subset of lipid rafts, by sucrose gradient fractionation and confocal microscopy. Through coimmunoprecipitation and bioluminescence resonance energy transfer assays, we demonstrated that this localization was mediated by an interaction between caveolin-1 and D1R in COS-7 cells and an isoform-selective interaction between D1R and caveolin-1␣ in rat brain. We determined that the D1R interaction with caveolin-1 required a putative caveolin binding motif identified in transmembrane domain 7. Agonist stimulation of D1R caused translocation of D1R into caveolin-1-enriched sucrose fractions, which was determined to be a result of D1R endocytosis through caveolae. This was found to be protein kinase A-independent and a kinetically slower process than clathrin-mediated endocytosis. Site-directed mutagenesis of the caveolin binding motif at amino acids Phe313 and Trp318 significantly attenuated caveolar endocytosis of D1R. We also found that these caveolin binding mutants had a diminished capacity to stimulate cAMP production, which was determined to be due to constitutive desensitization of these receptors. In contrast, we found that D1Rs had an enhanced ability to maximally generate cAMP in chemically induced caveolae-disrupted cells. Taken together, these data suggest that caveolae has an important role in regulating D1R turnover and signaling in brain.

Aggressive ®bromatosis is a locally invasive soft tissue lesion. Seventy-®ve per cent of cases harbor a somatic mutation in either the APC or b-catenin genes, resulting in b-catenin protein stabilization. Cyclooxygenase-2 (COX-2) is an enzyme involved in prostaglandin synthesis that modulates the formation of colonic neoplasia, especially in cases due to mutations resulting in b-catenin stabilization. Human aggressive ®broma-toses and lesions from the Apc+/Apc1638N mouse (a murine model for Apc-driven ®bromatosis) demonstrated elevated COX-2 levels. COX-2 blockade either by the selective agent DFU or by non-selective COX blocking agents results in reduced proliferation in human tumor cell cultures. Breeding mice with Cox-27/7 mice resulted in no di erence in number of aggressive ®bromatoses formed, but in a smaller tumor size, while there was a decrease in number of GI lesions by 50%. Mice fed various COX blocking agents also showed a decline in tumor size. COX-2 expression was regulated by tcf-dependent transcription in this lesion. COX-2 partially regulates proliferation due to b-catenin stabilization in aggressive ®bromatosis. Although COX blockade alone does not cause tumor regression, this data suggests that it may have a role as an adjuvant therapy to slow tumor growth in this lesion. Oncogene (2001) 20, 451 ± 460.