Efficient functional coupling of the human D3 dopamine receptor to G(o) subtype of G proteins in SH-SY5Y cells.
1 The D3 dopamine receptor presumably activates Gi/Go subtypes of G-proteins, like the structurally analogous D2 receptor, but its signalling targets have not been clearly established due to weak functional signals from cloned receptors as heterologously expressed in mostly non-neuronal cell lines. 2 In this study, recombinant human D3 receptors expressed in a human neuroblastoma cell line, SH-SY5Y, produced much greater signals than those expressed in a human embryonic kidney cell line, HEK293. Quinpirole, a prototypic agonist, markedly inhibited forskolin-stimulated cyclic AMP production and Ca2+-channel (N-type) currents in SH-SY5Y cells, and enhanced GTPgamma35S binding in isolated membranes, nearly ten times greater than that observed in HEK293 cell membranes. 3 GTPgamma35S-bound Galpha subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Galpha-specific antibodies. Galphao subunits (not Galphai) were highly labelled with GTPgamma35S in SH-SY5Y, but not in HEK293 cell membranes, despite their abundance in the both cell types, as shown with reverse transcription-polymerase chain reaction and Western blots. N-type Ca2+ channels and adenylyl cyclase V (D3-specific effector), on the other hand, exist only in SH-SY5Y cells. 4 More efficient coupling of the D3 receptor to Go subtypes in SH-SY5Y than HEK293 cells may be attributed, at least in part, to the two D3 neuronal effectors only present in SH-SY5Y cells (N-type Ca2+-channels and adenylyl cyclase V). The abundance of Go subtypes in the both cell lines seems to indicate their availability not a limiting factor.
Zaworski
P G
PG
Cell & Molecular Biology, Pharmacia and Upjohn, Inc., 301 Henrietta Street, Kalamazoo, Michigan, MI 49007, USA.
Alberts
G L
GL
Pregenzer
J F
JF
Im
W B
WB
Slightom
J L
JL
Gill
G S
GS
D000262:Adenylyl Cyclases / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D018491:Dopamine Agonists / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D019257:Quinpirole / Q000378:metabolism / Q000494:pharmacology; D012313:RNA / Q000235:genetics / Q000378:metabolism; D011869:Radioligand Assay; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D050637:Receptors, Dopamine D3; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013462:Sulfur Radioisotopes; D014316:Tritium; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; C493593:DRD3 protein, human; D018491:Dopamine Agonists; D017448:Receptors, Dopamine D2; D050637:Receptors, Dopamine D3; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D014316:Tritium; D005576:Colforsin; D019257:Quinpirole; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012313:RNA; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases