title: Advantages of heterologous expression of human D2long dopamine receptors in human neuroblastoma SH-SY5Y over human embryonic kidney 293 cells.
abstract: The human D2long dopamine receptor when expressed heterologously in a human neuronal cell line, SH-SY5Y, produced more robust functional signals than when expressed in a human embryonic kidney cell line, HEK293. Quinpirole (agonist)-induced GTPgamma(35)S binding and high affinity sites were 3 - 4 fold greater in SH-SY5Y than in HEK293 cells. N-type Ca(2+) channel currents present in SH-SY5Y cells, but not HEK293 cells, were inhibited potently by quinpirole with a half-maximal inhibitory concentration of 0.15+/-0.03 nM. Inhibition of adenylyl cyclases by agonists, on the other hand, was of similar potency and efficacy in the two cell lines. GTPgamma(35)S-Bound Galpha subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Galpha-specific antibodies. Galpha(i) and Galpha(o) subunits were highly labelled with GTPgamma(35)S in SH-SY5Y cells, but only Galpha(i) subunits were labelled in HEK293 cells. The additional G(o) coupling in SH-SY5Y cells could arise, at least in part, from the presence of G(o) coupled-effectors, such as the N-type Ca(2+) channel, and may contribute to robust agonist-induced GTPgamma(35)S binding, which is a reliable means for measuring ligand intrinsic efficacy. It appears that expression of neuronal G protein-coupled receptors in neuronal environments could reveal additional functional characteristics that are absent in non-neuronal cell lines. This appears to be due to, at least in part, to the presence of neuron-specific effectors. These findings underscore the importance of the cellular environment in which drug actions are examined, particularly in the face of intensive efforts to develop drugs for G protein-coupled receptors of various origins.
authors.affiliation: BiologyII/Neurobiology, Pharmacia, 7251-209-512, 301 Henrietta Street, Kalamazoo, Michigan, MI 49007, USA.
authors.forename: G L
authors.initials: GL
authors.lastname: Alberts
authors.forename: J F
authors.initials: JF
authors.lastname: Pregenzer
authors.forename: W B
authors.initials: WB
authors.lastname: Im
mesh_terms: D015220:Calcium Channels / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D018492:Dopamine Antagonists / Q000494:pharmacology; D065127:Dopamine D2 Receptor Antagonists; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D009447:Neuroblastoma; D009474:Neurons / Q000378:metabolism*; D020891:Raclopride / Q000494:pharmacology; D017448:Receptors, Dopamine D2 / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000378:metabolism; D013134:Spiperone / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D015220:Calcium Channels; D018492:Dopamine Antagonists; D065127:Dopamine D2 Receptor Antagonists; D017448:Receptors, Dopamine D2; D011994:Recombinant Proteins; C113776:dopamine D2L receptor; D020891:Raclopride; D013134:Spiperone; D020962:Heterotrimeric GTP-Binding Proteins