Dopamine receptor belong to a large class of neurotransmitter and hormone receptors that are linked to their signal transduction pathways through guanine nucleotide binding regulatory proteins (G proteins). Pharmacological, biochemical and physiological criteria have been used to define two subcategories of dopamine receptors referred to as D1 and D2. D1 receptors activate adenylyl cyclase and are coupled with the Gs regulatory protein. By contrast, activation of D2 receptors results in various responses including inhibition of adenylyl cyclase, inhibition of phosphatidylinositol turnover, increase in K+ channel activity and inhibition of Ca2+ mobilization. The G protein(s) linking the D2 receptors to these responses have not been identified, although D2 receptors have been shown to both copurify and functionally reconstitute with both Gi and Go related proteins. The diversity of responses elicited by D2-receptor activation could reflect the existence of multiple D2 receptor subtypes, the identification of which is facilitated by the recent cloning of a complementary DNA encoding a rat D2 receptor. This receptor exhibits considerable amino-acid homology with other members of the G protein-coupled receptor superfamily. Here we report the identification and cloning of a cDNA encoding an RNA splice variant of the rat D2 receptor cDNA. This cDNA codes for a receptor isoform which is predominantly expressed in the brain and contains an additional 29 amino acids in the third cytoplasmic loop, a region believed to be involved in G protein coupling.

In order to clone the D1 dopamine receptor linked to adenylyl cyclase activation, the polymerase chain reaction was used with highly degenerate primers to selectively amplify a cDNA sequence from NS20Y neuroblastoma cell mRNA. This amplification produced a cDNA fragment exhibiting considerable sequence homology to guanine nucleotidebinding (G)-protein-coupled receptors that have been cloned previously. To characterize this cDNA further, a full-length clone was isolated from a rat striatal library by using the cDNA fragment as a probe. Sequence analysis of this cDNA clone indicated that it is indeed a member of the G-protein-coupled receptor family and exhibits greatest homology with the

Seven different peptides of 14-23 residues in length based on the predicted amino acid sequence of the cloned rat D2 receptor cDNA were used as immunogens to develop antibodies in rabbits. Two of these peptides were derived from the amino terminus and four were from the third cytoplasmic loop, including one to the splice variant insertion sequence and one to the carboxyl terminus of the receptor protein. These peptides were conjugated to bovine thyroglobulin prior to rabbit immunization. Antibody production was monitored by a solid-phase ELISA. With the exception of the carboxylterminal peptide, all of the peptide immunogens produced antiserum of high titer ranging from 1:104 to 1:106 on ELISA. Specificity of the reaction was demonstrated by the absence of a response in the preimmune serum and by the absence of cross-reactivity between the various antisera and the nonimmunization peptides. Moreover, preincubation of the antiserum with the immunization peptide, but not other peptides, blocked the subsequent ELISA reactions. Some of the antisera were additionally characterized by immunodot assays using solubilized rat striatal membranes blotted onto nitrocellulose. Positive reactions with antiserum dilutions of 1:500 were observed that were dependent on the presence and concentration of membrane protein and were not observed using preimmune serum. Additionally, immunofluorescent staining by the D2 receptor antiserum was observed on cells that had been transfected with the D2 receptor cDNA but not on untransfected cells. Immunoprecipitation of the photoaffinity-labeled and solubilized D2 receptor also suggested that the antisera were able to directly recognize the native receptor protein.Immunohistochemical localization of the D2 receptor in slices of fresh frozen and perfusion-fixed rat brain was performed using these antisera. Within the striatum, about 50% of the mediumsized neurons were labeled as well as large, putatively cholinergic interneurons.Two populations of dopamine receptors exist in the central nervous system that are pharmacologically distinct and utilize different transduction mechanisms (1). The cDNA sequence has been reported for the D2 subtype (2), and recently an RNA splice variant has been described for this class of dopaminergic receptor (3-6). The gene products for these cDNA clones have been shown to be functional D2 receptors that effectively inhibit adenylyl cyclase activation (5, 7). The anatomical distribution of both dopamine receptor populations has been described using in vitro autoradiographic methods (8-10) that demonstrate that the highest accumulation of receptor binding sites is in the striatum and mesolimbic regions of the rat central nervous system. Application of in situ hybridization histochemistry has further demonstrated that mRNA for the D2 receptor subtype can be distinguished within these same two rat brain regions with high density (11, 12). We have recently reported the regional as well as the cellular localization of the two dopamine receptor binding sites in...

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