A clone encoding a human D2 dopamine receptor was isolated from a pituitary cDNA library and sequenced. The deduced protein sequence is 96% identical with that of the cloned rat receptor with one major difference: the human receptor contains an additional 29 amino acids in its putative third cytoplasmic loop. Southern blotting demonstrated the presence of only one human D2 receptor gene. Two overlapping phage containing the gene were isolated and characterized. DNA sequence analysis of these clones showed that the coding sequence is interrupted by six introns and that the additional amino acids present in the human pituitary receptor are encoded by a single exon of 87 base pairs. The involvement of this sequence in alternative splicing and its biological significance are discussed. Dopamine neurons in the vertebrate central nervous system are involved in the initiation and execution of movement, the maintenance -of emotional stability, and the regulation of pituitary function. Several human neurological diseases, including Parkinson disease (1) and schizophrenia (2), are thought to be manifestations of imbalances between dopamine receptors and dopamine. The receptors that mediate dopamine's effects have been divided into D1 and D2 subtypes, which are distinguished by their guanine nucleotidebinding protein (G-protein) coupling (3, 4), ligand specificities, anatomical distribution, and physiological effects (5). The D2 dopamine receptors have been of particular clinical interest due to their regulation of prolactin secretion (6) and their affinity for antipsychotic drugs (7, 8).The D2 dopamine receptors belong to the family of Gprotein-coupled receptors. The sequence similarity shared by members of this family enabled Bunzow et al. (9) to clone a rat brain D2 dopamine receptor cDNA. We have used that clone to isolate the human pituitary D2 dopamine receptor cDNA described here. § We have found that the deduced amino acid sequences of these two receptors are very similar, with one notable difference. The human receptor contains an additional 29 amino acids in its putative third cytoplasmic loop that are encoded by one of the gene's exons. This genomic organization suggests that the existence of two D2 receptor mRNAs is the result of an alternative splicing event. Expression and Pharmacology. The 2.5-kb human pituitary cDNA (hPitD2) was cloned into pZem3 (a gift from E. Mulvihill, Zymogenetics, Seattle) and cotransfected with pRSVneo into thymidine kinase-deficient mouse cells (Ltkcells) by calcium phosphate precipitation (10). A stable transfectant (L-hPitD2Zem) was selected and maintained in medium containing G418 sulfate (Geneticin; GIBCO) at 750 ,ug/ml. Twenty hours prior to the harvesting of membranes, these cells were incubated with 70 ,uM zinc sulfate. Membranes were prepared from L-HPitD2Zem, from the Ltk-cell line expressing the cloned rat D2 dopamine receptor (LRGB2Zem-1), and from freshly dissected rat striata (Taconic Farm, Germantown, NY) as described (11,12). For the binding assays, membrane p...

Cyclic AMP regulates a variety of cellular responses through activation of the catalytic subunit of cAMP-dependent protein kinase. The cDNAs for two protein isoforms of the catalytic subunit, C alpha and C beta, were placed into expression vectors, and their ability to stimulate cAMP-dependent transcription of the human enkephalin promoter was examined in transiently transfected CV-1 cells. Expression vectors for C alpha and C beta that were directed by the human cytomegalovirus promoter produced up to 350- and 200-fold increases in chloramphenicol acetyltransferase activity, respectively, when cotransfected with the ENKAT-12 reporter plasmid. Transcriptional activation was shown to be dependent upon functional kinase activity by point mutations in catalytic subunit vectors which eliminated activation. Transcriptional activation by C alpha and C beta was eliminated when the cAMP response elements (CREs) were deleted from the native enkephalin promoter, but activation was recovered when this region was replaced with an oligonucleotide containing two copies of the somatostatin CRE consensus TGACGTCA. C alpha expression vectors were found to produce 2-fold greater transcriptional activation than C beta expression vectors. These results were most likely due to the cellular kinase activity produced by the catalytic subunit expression vectors and did not appear to be dependent on CRE motif or substrate specificity. In vitro mutagenesis indicates that neither C alpha nor C beta requires N-terminal myristylation for transcriptional activation, but threonine-197 is critical to subunit function.