, 2007), but may not have the ability to support DRD2 signaling. Although WT-GHSR1a and S123A-GHSR1a and M213K-GHSR1a all have identical basal activity, of the three, only WT-GHSR1a coexpression with DRD2 mobilizes Ca2+ in response to dopamine (Figure 4A). By direct contrast, coexpression selleck kinase inhibitor of DRD2

with the basally inactive mutant produces a modest increase in Ca2+ mobilization in response to dopamine (Figure 4A). These results illustrate a lack of correlation between dopamine-induced release of intracellular Ca2+ and GHSR1a basal activity and are consistent with allosteric interactions between GHSR1a and DRD2. Agonist activation of GHSR1a in HEK293 cells results in coupling to Gαq (Smith et al., 1997). Therefore, if modification of DRD2 signaling by GHSR1a is caused by GHSR1a basal activity, ablating Gαq expression should block DRD2-mediated mobilization of Ca2+ by dopamine. When Gαq siRNA is expressed with GHSR1a and DRD2 dopamine-induced Ca2+ signaling is not suppressed (Figure 4B, left panel), whereas ghrelin-induced Ca2+ release is significantly reduced (Figure 4B, right panel). To ensure that ablating Gαq suppresses GHSR1a basal activity, we deliberately overexpressed GHSR1a to produce detectable basal activity as measured

by IP1 production. Coexpression of Gαq siRNA, EGFR inhibitor but not control siRNA, suppresses IP1 production to control levels (Figure S2A). Also, overexpression of Gαq protein does not increase dopamine-induced Ca2+ mobilization (Figure S2B). The PKC inhibitor, BisI, also does not reduce dopamine-induced Ca2+ mobilization (Figure S2C). These data provide evidence that modification of DRD2 signaling by GHSR1a is independent of GHSR1a basal signaling through Gαq and PKC. Functional crosstalk between receptors that depends on basal activity of a Gαq-coupled receptor is indicated when acute preactivation (3 min) with an agonist of the and basally active receptor synergistically increases the agonist response of the protomer partner (Rives et al., 2009). In the case of GHSR1a and DRD2, preincubation with ghrelin for 3 min has no effect on the amount of Ca2+ released in

response to dopamine (Figure S2D). Likewise, if dopamine-induced Ca2+ production is explained by potentiation of GHSR1a activity by DRD2, activation of DRD2 prior to ghrelin treatment would potentiate ghrelin-induced Ca2+ mobilization. However, this is not the case; simultaneous addition of ghrelin and dopamine results in additive Ca2+ accumulation (Figures S2E and S2F). Our collective results argue that dopamine-induced Ca2+ release is independent of GHSR1a basal activity and crosstalk between signal transduction pathways. An alternative mechanism is that GHSR1a-induced modification of canonical dopamine-DRD2 signal transduction is a consequence of allosteric modification of DRD2 signal transduction caused by formation of GHSR1a:DRD2 heteromers.