Efficient functional coupling of the human D3 dopamine receptor to G_o subtype of G proteins in SH‐SY5Y cells

Abstract

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 Ca 2+ -channel (N-type) currents in SH-SY5Y cells, and enhanced GTPg 35 S binding in isolated membranes, nearly ten times greater than that observed in HEK293 cell membranes. 3 GTPg 35 S-bound Ga subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Ga-speci®c antibodies. Ga o subunits (not Ga i ) were highly labelled with GTPg 35 S 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 Ca 2+ channels and adenylyl cyclase V (D3-speci®c e ector), on the other hand, exist only in SH-SY5Y cells. 4 More e cient coupling of the D3 receptor to G o subtypes in SH-SY5Y than HEK293 cells may be attributed, at least in part, to the two D3 neuronal e ectors only present in SH-SY5Y cells (Ntype Ca 2+ -channels and adenylyl cyclase V). The abundance of G o subtypes in the both cell lines seems to indicate their availability not a limiting factor.

The RGSZ2 Protein Exists in a Complex with μ-Opioid Receptors and Regulates the Desensitizing Capacity of Gz Proteins

“…It is possible that in these mice, the effects that remained after administering doses of 10 nmol morphine and above are produced by the activity of Gai/o subunits on effectors other than those acted upon by the Gaz subunits. Indeed, there are some reports detailing the preferences of different classes of G proteins in the regulation of certain effectors, for example, Gao and adenylyl cyclase V (Zaworski et al, 1999), Gaz, and adenylyl cyclases II and VI (Ho et al, 2000).…”

G Protein Coupling and Ligand Selectivity of the D2Land D3Dopamine Receptors

“…At the end of assay, an immunoprecipitation step was incorporated using antisera raised against the C-terminal decapeptide of G␣ i1/2 (i and ii), G␣ i3 (iii), or G␣ o1 (iv). The significance of differences between basal and dopaminestimulated [ to G␣ o1 (Zaworski et al, 1999;Beom et al, 2004), although Zaworski et al postulated that efficient coupling to G␣ o1 required the expression of adenyl cyclase subtype V. However, this study is the first to assess coupling to each of the four G␣ i/o subunits directly and, in parallel, to explore pharmacological consequences of attempting to expand the G protein coupling profile of the D 3 receptor. Key to this process was maintaining a clearly defined receptor to G protein expression stoichiometry, and, despite using both a series of transient cotransfection studies and establishing cells lines stably expressing dopamine receptors that allowed regulated expression of a G protein, this was only possible by using GPCR-G protein fusion proteins.…”

“…Initial reports indicated that the D 3 receptor, when expressed in a variety of cell systems, did not exhibit a decrease in affinity for agonists in the presence of guanine nucleotides and did not modulate adenylate cyclase activity (Sokoloff et al, 1990;Freedman et al, 1994;Tang et al, 1994). However, more recently, it has been shown that dopaminergic agonists dose-dependently stimulate guanosine 5Ј-O-(thiotriphosphate) ([ 35 S]GTP␥S) binding in Sf-9 insect cells (Alberts et al, 2000), Chinese hamster ovary cells (Malmberg et al, 1998;Newman-Tancredi et al, 1999;Vanhauwe et al, 1999;Vanhauwe et al, 2000), and SH-SY5Y human neuroblastoma cells (Zaworski et al, 1999) in which the D 3 receptor was expressed. Because these effects were abolished by pertussis toxin treatment, they must reflect activation of one, or more, members of the "G i " G protein family.…”

Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery

“…There are a number of phenotypic drug responses associated with cell types that are not specifically ascribed to mechanisms but are nevertheless noted in the literature (i.e., levomedetomidine for ␣ 2A -adrenoceptors (Jansson et al, 1998;Kukkonen et al, 2001), isoprostane 8-iso-prostaglandin F for thromboxane A 2 receptors (Weber and Markillie, 2003), and quinpirole for dopamine D 3 and D 2 receptors (Zaworski et al, 1999;Alberts et al, 2000). In addition, cells can adjust their signaling capability according to their needs through control of reagents required for receptor phosphorylation [i.e., through GRKs (Lohse, 1993;Zamah et al, 2002;Ribas et al, 2007;Tobin et al, 2008) or protein kinase A (Daaka et al, 1997;Tobin, 2008)] and internalization.…”