G Protein bc-Subunit Signaling in Asthma Figure 6. Inhibition of Gbc-signaling prevents upregulation of PDE activity in lungs from OVA-sensitized/challenged rabbits and passively sensitized ASM tissues. Relative to controls, PDE activity is significantly increased in lung tissues isolated from OVA- sensitized+challenged rabbits (A) and in OVA serum-sensitized (B) and IL-13-treated (C) isolated ASM tissues. The upregulated PDE activity is abrogated in lungs isolated from OVA-sensitized rabbits that are treated with inhaled anti-Gbc peptide prior to antigen challenge, as well as in OVA serum- or IL-13-exposed ASM tissues that were pretreated with anti-Gbc peptide or gallein. Data are mean 6 SE values from 3–5 determinations. Comparisons are made using two-tailed Student t-test. **p,0.01. doi:10.1371/journal.pone.0032078.g006 demonstrated that activation of both c-Src and ERK1/2 by IL-13 in HASM cells was suppressed by pretreatment with different inhibitors of Gbc and, based on related co-immunoprecipitation studies, c-Src tyrosine kinase was identified as a direct effector of Gbc signaling (Fig. 7). Collectively, these data strongly implicate Gi-bc signaling leading to c-Src-induced ERK1/2 activation and its upregulation of PDE4 expression as a key signaling mechanism that underlies the changes in airway function observed under the Figure 7. Gbc signaling regulates IL-13-induced c-Src and ERK1/2 activation in HASM cells. (A) Immunoblots depicting IL-13-induced transient phosphorylation of c-SrcTyr416 and ERK1/2 in HASM cells, with peak phosphorylation detected at 10 and 30 min, respectively. (B) Immunoblots depicting that, contrasting the lack of effect of pretreatment with MPS alone, IL-13-induced phosphorylation of c-SrcTyr416 and ERK1/2 is suppressed in HASM cells pretreated with either anti-Gbc peptide (20 mM) or gallein (10 mM). Additionally, in contrast to HASM cells transfected with adeno-LacZ (i.e., negative control), IL-13-induced phosphorylation of c-SrcTyr416 and ERK1/2 is also suppressed in HASM cells wherein Gbc signaling is inhibited by transfection with adeno-bARK-ct. (C) Western blot depicting interaction of Gb and c-SrcTyr416 in HASM cells stimulated with IL-13 in the absence and presence of inhibition of Gbc activation. Following preparation of lysates from untreated and IL-13-treated (50 ng/ml610 min) HASM cells, Gb was immunoprecipitated (IP) with anti-Gb monoclonal antibody, and subsequently immunoblotted (IB) with anti-phospho-c-SrcTyr416 antibody (see Methods). Note, relative to untreated cells, association of Gb and c-SrcTyr416 proteins was significantly increased in IL-13-treated HASM cells; and formation of this protein complex was suppressed in IL-13-exposed cells that were pretreated with either anti-Gbc peptide (20 mM) or gallein (10 mM), and also suppressed in IL-13-exposed HASM cells that were transfected with adeno-bARK-ct. The immunoblots shown in A–C are representative from 3–4 experiments. doi:10.1371/journal.pone.0032078.g007 PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e32078