, 2011). To measure the relative expression of ghsr1a and drd2 mRNA was isolated from different regions of the mouse brain. RT-PCR shows ghsr1a expression is most abundant in hypothalamus compared to striatum and
hippocampus and that drd2 is expressed mainly in the striatum with lesser amounts in the hypothalamus ( Figure 1A). Immunofluorescence on brain slices from Ghsr-IRES-tauGFP mice ( Jiang et al., 2006) show colocalization of DRD2 and GFP in subsets of neurons with the most abundant coexpression in the hypothalamus ( Figure 1B). The specificity of the DRD2 monoclonal antibody used for immunofluorescence studies was rigorously tested ( Figures S1A–S1D available online). Importantly, DRD2 immunofluorescence was observed in brain slices from drd2+/+, but not drd2−/− Forskolin clinical trial CHIR-99021 manufacturer mice. To investigate whether neuronal cells that coexpress GHSR1a and DRD2 are characterized by modification of signal transduction, we selected the SH-SY5Y neuroblastoma cell line that expresses DRD2 endogenously and generated SH-SY5Y cells that stably express GHSR1a (SH-GHSR1a). In SH-SY5Y parental cells, DRD2 activation by the selective DRD2 agonist, quinpirole, causes coupling to Gαi without inducing release of intracellular Ca2+, whereas quinpirole treatment of SH-GHSR1a cells produces dose dependent
rapid transient Ca2+ signals reaching a maximum by 20 s (Figures 2A and 2B EC50 = 32.76 ± 3.4 nM). Attenuation of the Ca2+ signal by the DRD2 antagonist raclopride confirms DRD2 specificity (Figure 2C) and attenuation by the GHSR1a antagonist/inverse agonist L-765,867, Subst P derivative (Holst et al., 2004 and Smith et al., 1996). Since GHSR1a and DRD2 colocalize in the hypothalamus (Figure 1B), we prepared primary cultures of hypothalamic neurons. Treatment of the cultured neurons induces rapid Ca2+ transients (Figure 2D, upper panel). After washing to remove quinporole,
ghrelin treatment produces an immediate Ca2+ response (Figure 2D, lower panel). These results are consistent with coexpression of GHSR1a and DRD2 in hypothalamic neurons. To study GHSR1a and DRD2 interactions in a system where we could control the relative concentrations of GHSR1a and DRD2, we performed Ca2+ mobilization assays in HEK293 cells Phosphoprotein phosphatase stably expressing the bioluminescent calcium sensor aequorin (Button and Brownstein, 1993). When DRD2 is expressed alone dopamine does not induce Ca2+ mobilization, but when GHSR1a is coexpressed dopamine induces dose-dependent rapid Ca2+ transients with a maximal response at 15–20 s (Figures 3A and 3B, EC50 = 41.88 ± 1.12 nM). To determine GHSR1a specificity, the closely related motilin receptor that also couples to Gαq/11 (Feighner et al., 1999) was coexpressed with DRD2; in this context, dopamine treatment does not induce a Ca2+ response (Figure 3B).