ACCELERATED COMMUNICATION D5 Dopamine Receptors are Required for Dopaminergic Activation of Phospholipase C

Dopamine activates phospholipase C in discrete regions of the mammalian brain, and this action is believed to be mediated through a D1-like receptor. Although multiple lines of evidence exclude a role for the D1 subtype of D1-like receptors in the phosphoinositide response, the D5 subtype has not been similarly examined. Here, mice lacking D5 dopamine receptors were tested for dopamine agonist-induced phosphoinositide signaling both in vitro and in vivo. The results show that hippocampal, cortical, and striatal tissues of D5 receptor knockout mice significantly or completely lost the ability to produce inositol phosphate or diacylglycerol messengers after stimulation with dopamine or several selective D1-like receptor agonists. Moreover, endogenous inositol-1,4,5-trisphosphate stimulation by the phospholipase C-selective D1-like agonist 3-methyl-6chloro-7,8-dihydroxy-1-[3methylphenyl]-2,3,4,5-tetrahydro1H-3-benzazepine (SKF83959) was robust in wild-type animals but undetectable in the D5 receptor mutants. Hence, D5 receptors are required for dopamine and selective D1-like agonists to induce phospholipase C-mediated phosphoinositide signaling in the mammalian brain. Multiple subtypes of dopamine receptors expressed in the mammalian brain may be categorized into D1-like and D2like subclasses based on their structural homology, pharmacological selectivity, and functional similarities (Civelli et al., 1993; Lachowicz and Sibley, 1997). Members of the D1-like subclass include the D1 and D5 receptors (Civelli et al., 1993; Lachowicz and Sibley, 1997). Expressed in clonal cell lines, both the D1 and D5 receptors have demonstrated coupling to multiple signaling cascades as assessed through the activation of specific G proteins or the formation of downstream second messengers (Sidhu and Niznik, 2000; Neve et al., 2004). However, promiscuous or inconsistent coupling to multiple or alternate G proteins and signaling cascades might be a frequent feature for many G protein-coupled receptors expressed in artificial cell lines (Sidhu and Niznik, 2000; Hermans, 2003). Thus, experiments aiming to definitively assign a signaling pathway to a given receptor would benefit from the use of physiological tissue preparations and receptor-selective pharmacological agents. Although various chemical entities that are highly selective for D1-like receptors exist, there remains a lack of agonists or antagonists that sufficiently discriminate between the D1 and D5 subtypes. Moreover, typical agonists of D1-like receptors generally activate both the D1 and D5 subtypes and elicit multiple signaling responses in mammalian tissues (Neve et al., 2004). Such responses include cascades mediated by adenylyl cyclase (Monsma et al., 1990), mitogenactivated protein kinase (Zhen et al., 1998), intracellular calcium mobilization (Lezcano and Bergson, 2002; Yasumoto This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grant R01-DA017614] and by the Intramural Research Program of the National Institutes of Health. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.053017. ABBREVIATIONS: PLC, phospholipase C; PI, phosphatidylinositide; D5KO, D5 dopamine receptor knockout; HB, HEPES bicarbonate; CDPdiacylglycerol, cytidine diphosphate diacylglycerol; SKF38393, 1-phenyl-2,3,4,5-tetrahydro-(H1)-3-benzazepine-7,8-diol; SKF83959, 3-methyl-6chloro-7,8-dihydroxy-1-[3methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine; ANOVA, analysis of variance; kb, kilobase. 0026-895X/09/7503-447–453 MOLECULAR PHARMACOLOGY Vol. 75, No. 3 U.S. Government work not protected by U.S. copyright 53017/3436733 Mol Pharmacol 75:447–453, 2009 Printed in U.S.A. 447 at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from et al., 2004), and phospholipase C (PLC) (Felder et al., 1989; Undie and Friedman, 1990). The PLC response, which is associated with the hydrolysis of phosphatidylinositides (PIs) to release inositol phosphate and diacylglycerol second messengers, is evident in discrete brain regions, including tissues expressing D1-like receptors that show relatively negligible functional coupling to adenylyl cyclase (Undie and Friedman, 1990; Montague et al., 2001) and persists when D1 receptors have been genetically deleted (Friedman et al., 1997) or chemically inactivated (Rosengarten and Friedhoff, 1998; Undie et al., 2000). Thus, although a D1-like receptor mediates dopaminergic-induced PI signaling in the brain, the structurally defined D1 receptor itself is not the specific subtype that is involved. Given that extensive data mining of mammalian genomes has not revealed the existence of additional dopamine receptors beyond the D1–D5 entities, the foregoing suggests that the D5 subtype might be the D1-like receptor that mediates the PLC response. This hypothesis has not been directly tested in the intact brain. In the present work, we sought to further examine the nature of D5 receptor coupling in native brain tissues. For this, we used mutant mice lacking D5 receptors to test the hypothesis that the D5 receptor is required for dopaminergic activation of PLC in the intact brain. The results demonstrate that dopamine and selective D1-like receptor agonists lose their efficacy to induce inositol phosphate accumulation or diacylglycerol production in brain tissues of mice lacking the D5 subtype of D1-like receptors. Materials and Methods Animals. Dopamine D5 receptor knockout (D5KO) mice were obtained along with their wild-type litter mates from the National Institutes of Health (Bethesda, MD). Produced from a genetic background of 129/SvJ1 and C57BL/6J, the mice are viable, develop normally, and are fertile and capable of reproduction (Hollon et al., 2002). The genotype of each animal was determined by polymerase chain reaction techniques, and confirmed D5KO animals showed complete loss of immunoreactivity for the D5 receptor in the brain (Hollon et al., 2002). Mice used in this study were at least 10 weeks old. Protocols for the care and use of the animals were approved by the Institutional Animal Care and Use Committee and conformed to the National Institutes of Health Guide for the Care and Use of