Molecular cloning and expression of the rhesus macaque D1 dopamine receptor gene

Curtis Machida, Robert Searles, Valerie Nipper, Julie A. Brown, Laura B. Kozell, Kim Neve

Research output: Contribution to journalArticle

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Abstract

Using homologous probes for the cloning of related genes within the family of guanine nucleotide-binding protein-coupled receptors, we have cloned the gene for the rhesus macaque D1 dopamine receptor. By using the rat D1 receptor coding sequence as a probe under high stringency conditions, the rhesus D1 receptor gene was isolated from a λ EMBL3 rhesus genomic DNA library. The rhesus D1 dopamine receptor gene is intronless and encodes a 446-amino acid protein that contains two consensus sites for asparagine-linked glycosylation (Asn-5 and Asn-176) and two consensus sites for cAMP-dependent protein kinase phosphorylation (Thr-136 and Thr-268). The primary amino acid sequence of the rhesus D1 dopamine receptor shows an extremely high degree of similarity (99.6%) to the human D1 receptor. Genomic DNA analyses conducted with high and reduced stringency hybridizations indicate that the rhesus macaque D1 receptor is a member of a large multigene family. Like the human D1 receptor mRNA, the rhesus D1 receptor mRNA is approximately 4 kilobases in size and is localized predominantly in the caudate, with lesser amounts in the hippocampus and cortex. The rhesus D1 receptor coding region was inserted into the cytomegalovirus promoter-driven expression vector pcDNA-1, and the recombinant (pcDNA-D1) was cotransfected with the selectable marker pRSVneo, conferring G418 resistance, into D1 receptor-deficient C6 glioma cells. Analyses of the selected transfectant demonstrate the expression of a high affinity, functional D1 dopamine receptor. The D1 receptor radioligand [3H]SCH 23390 bound transfectant membranes with an affinity (Kd), of 0.3 nM; the D2-selective ligand spiperone, the dopamine receptor ligand clozapine, and the serotonin receptor antagonist ketanserin bound with considerably lower affinities (102, 80, and 95 nM, respectively). Both dopamine and the D1-selective agonist SKF 38393 inhibited the binding of [3H]SCH 23390 to transfectant cell membranes; the binding of these agonists was sensitive to GTP. Dopamine potently stimulated the accumulation of cAMP in transfected C6 cells, whereas SKF 38393 was a partial agonist in these cells. Also, the density of recombinant D1 receptors on the transfectant cells was decreased 40% upon treatment with 10 μM dopamine, indicating that occupation of recombinant D1 receptors by agonists alters surface expression of the receptors.

Original languageEnglish (US)
Pages (from-to)652-659
Number of pages8
JournalMolecular Pharmacology
Volume41
Issue number4
StatePublished - Apr 1992

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Dopamine D1 Receptors
Molecular Cloning
Macaca mulatta
2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine
Dopamine
Genes
Ligands
Ketanserin
Messenger RNA
Serotonin Antagonists
Guanine Nucleotides
Genomic Library
Clozapine
Asparagine
Dopamine Receptors
Multigene Family
Cyclic AMP-Dependent Protein Kinases
Guanosine Triphosphate
Cytomegalovirus
Gene Library

ASJC Scopus subject areas

  • Pharmacology

Cite this

Molecular cloning and expression of the rhesus macaque D1 dopamine receptor gene. / Machida, Curtis; Searles, Robert; Nipper, Valerie; Brown, Julie A.; Kozell, Laura B.; Neve, Kim.

In: Molecular Pharmacology, Vol. 41, No. 4, 04.1992, p. 652-659.

Research output: Contribution to journalArticle

Machida, Curtis ; Searles, Robert ; Nipper, Valerie ; Brown, Julie A. ; Kozell, Laura B. ; Neve, Kim. / Molecular cloning and expression of the rhesus macaque D1 dopamine receptor gene. In: Molecular Pharmacology. 1992 ; Vol. 41, No. 4. pp. 652-659.
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abstract = "Using homologous probes for the cloning of related genes within the family of guanine nucleotide-binding protein-coupled receptors, we have cloned the gene for the rhesus macaque D1 dopamine receptor. By using the rat D1 receptor coding sequence as a probe under high stringency conditions, the rhesus D1 receptor gene was isolated from a λ EMBL3 rhesus genomic DNA library. The rhesus D1 dopamine receptor gene is intronless and encodes a 446-amino acid protein that contains two consensus sites for asparagine-linked glycosylation (Asn-5 and Asn-176) and two consensus sites for cAMP-dependent protein kinase phosphorylation (Thr-136 and Thr-268). The primary amino acid sequence of the rhesus D1 dopamine receptor shows an extremely high degree of similarity (99.6{\%}) to the human D1 receptor. Genomic DNA analyses conducted with high and reduced stringency hybridizations indicate that the rhesus macaque D1 receptor is a member of a large multigene family. Like the human D1 receptor mRNA, the rhesus D1 receptor mRNA is approximately 4 kilobases in size and is localized predominantly in the caudate, with lesser amounts in the hippocampus and cortex. The rhesus D1 receptor coding region was inserted into the cytomegalovirus promoter-driven expression vector pcDNA-1, and the recombinant (pcDNA-D1) was cotransfected with the selectable marker pRSVneo, conferring G418 resistance, into D1 receptor-deficient C6 glioma cells. Analyses of the selected transfectant demonstrate the expression of a high affinity, functional D1 dopamine receptor. The D1 receptor radioligand [3H]SCH 23390 bound transfectant membranes with an affinity (Kd), of 0.3 nM; the D2-selective ligand spiperone, the dopamine receptor ligand clozapine, and the serotonin receptor antagonist ketanserin bound with considerably lower affinities (102, 80, and 95 nM, respectively). Both dopamine and the D1-selective agonist SKF 38393 inhibited the binding of [3H]SCH 23390 to transfectant cell membranes; the binding of these agonists was sensitive to GTP. Dopamine potently stimulated the accumulation of cAMP in transfected C6 cells, whereas SKF 38393 was a partial agonist in these cells. Also, the density of recombinant D1 receptors on the transfectant cells was decreased 40{\%} upon treatment with 10 μM dopamine, indicating that occupation of recombinant D1 receptors by agonists alters surface expression of the receptors.",
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