Expression and characterization of human D4 dopamine receptors in baculovirus‐infected insect cells

Abstract

The human D4 dopamine receptor has been genetically engineered for expression in insect cells using the baculovirus system. A D4 cDNA gene fusion construct [(1991) Nature 350, 610-6141 was synthetically modified to remove two introns from the coding region, and expressed in S frugiperda (St9) cells as a fusion with a short sequence from the polyhedrin protein. Binding assays with [3H]spiperone indicated high levels of D4 receptor binding 90 h after infection and a pharmacological profile identical to that reported for D4 receptors expressed in COS-7 cells using the cDNA gene hybrid. We also show that the agonist binding affinity of D4 receptors expressed in Sf9 cells can be shifted by GTP-y-S, indicating coupling to G-proteins.

Effect of Dopamine D3 Antagonists on PPI in DBA/2J Mice or PPI Deficit Induced by Neonatal Ventral Hippocampal Lesions in Rats

“…The discovery of the existence of dopamine subtypes, D 2 , D 3 and D 4 receptor in the D 2 -like receptor family (Giros et al, 1990;Mills et al, 1993), has evoked great interest in developing new antipsychotic agents specifically targeting D 3 and D 4 receptor subtypes with the desire to limit the presumed D 2 receptor-mediated side effects. The clinical significance of D 4 antagonists has come into question after clinical trials showed the lack of antipsychotic activity with D 4 receptor antagonists (Corrigan et al, 2004;Kramer et al, 1997).…”

Expression and Purification of the Saccharomyces cerevisiae α-Factor Receptor (Ste2p), a 7-Transmembrane-segment G Protein-coupled Receptor

“…Several GPCRs have been expressed in a functional form using the baculovirus system, including the D 4 and D 2 dopamine receptors (15,16), the M1 and M2 muscarinic acetylcholine receptors (17,18), and the ␤ 2 -adrenergic receptor (17). The D 2S dopamine receptor has been functionally expressed in both S. cerevisiae and Schizosaccharomyces pombe (19,20), and an E. coli expression system has also been successfully used for expression of GPCRs (21,22).…”

Expression and functional characterisation of a synthetic version of the human D4 dopamine receptor in a stable human cell line

“…These conditions are different to those employed by others in previous hD4.2 studies [1,2,6,16]. However, neither the change in assay buffer nor the change in temperature had The Ki'S obtained are shown in Table 1.…”

“…4 ing the human dopamine D, receptor, has been demonstrated in this cell line [8-lo]. The transfected HER293 cells expressed high levels of a receptor with pharmacology similar to that described for the hD4.2 isoform [1,2,6]. The receptor expressed from the synthetic gene was also shown, in functional assays, to be negatively coupled to adenylate cyclase.…”

“…Variants of the hD4 receptor, containing a variable number of 48 bp imperfect repeat units in intracellular loop three, were later reported [3-51. Expression of the hybrid hD4.2 (hD4 receptor with two repeat units) receptor clone was reported in baculovirus [6]. Expression was achieved by deleting the first two introns of the D4 genomi=DNA hybrid and replacing them with a synthetic oligonucleotide with a reduced G/C content.…”