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Short lipidated peptide sequences derived from various intracellular loop regions of G protein-coupled receptors (GPCRs) are named pepducins and act as allosteric modulators of a number of GPCRs. Recently, a pepducin selectively targeting the C-X-C chemokine receptor type 4 (CXCR4) was found to be an allosteric agonist, active in both cell-based assays and in vivo. However, the precise mechanism of action of this class of ligands remains poorly understood. In particular, given the diversity of signaling effectors that can be engaged by a given receptor, it is not clear whether pepducins can show biased signaling leading to functional selectivity. To explore the ligand-biased potential of pepducins, we assessed the effect of the CXCR4 selective pepducin, ATI-2341, on the ability of the receptor to engage the inhibitory G proteins (Gi1, Gi2 and Gi3), G13, and β-arrestins. Using bioluminescence resonance energy transfer-based biosensors, we found that, in contrast to the natural CXCR4 ligand, stromal cell-derived factor-1α, which promotes the engagement of the three Gi subtypes, G13 and the two β-arrestins, ATI-2341 leads to the engagement of the Gi subtypes but not G13 or the β-arrestins. Calculation of the transduction ratio for each pathway revealed a strong negative bias of ATI-2341 toward G13 and β-arrestins, revealing functional selectivity for the Gi pathways. The negative bias toward β-arrestins results from the reduced ability of the pepducin to promote GPCR kinase-mediated phosphorylation of the receptor. In addition to revealing ligand-biased signaling of pepducins, these findings shed some light on the mechanism of action of a unique class of allosteric regulators.

The CB 1 cannabinoid receptor mediates many of the psychoactive effects of Δ 9 THC, the principal active component of cannabis. However, ample evidence suggests that additional non-CB 1 /CB 2 receptors may contribute to the behavioral, vascular, and immunological actions of Δ 9 THC and endogenous cannabinoids. Here, we provide further evidence that GPR55, a G protein-coupled receptor, is a cannabinoid receptor. GPR55 is highly expressed in large dorsal root ganglion neurons and, upon activation by various cannabinoids (Δ 9 THC, the anandamide analog methanandamide, and JWH015) increases intracellular calcium in these neurons. Examination of its signaling pathway in HEK293 cells transiently expressing GPR55 found the calcium increase to involve G q , G 12 , RhoA, actin, phospholipase C, and calcium release from IP 3 R-gated stores. GPR55 activation also inhibits M current. These results establish GPR55 as a cannabinoid receptor with signaling distinct from CB 1 and CB 2 .

RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Gα12 and Gα13 both singly and in combination, demonstrating the Gα13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gβ2 correlated with a reduced Ca 2+ response to C5a. Insertion of a GFP transgene upstream of the Gβ2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.

The catenin p120 (p120 ctn ) is an armadillo repeat domain protein that binds to cadherins and has been shown to facilitate strong cell–cell adhesion. We have investigated a possible link between heterotrimeric G proteins and p120 ctn , and found that both Gα 12 and Gα 13 can completely and selectively abrogate the p120 ctn -induced branching phenotype in different cell types. Consistent with these observations, the expression of Gα 12 or Gα 13 compensates for the reduction of Rho activity induced by p120 ctn . On the other hand, p120 ctn can be selectively coimmunoprecipitated with Gα 12 , and the coimmunoprecipitation was favored by activation of the G protein. A specific interaction between p120 ctn and Gα 12 Q231L was also observed in in vitro binding experiments. In addition, p120 ctn can be immunoprecipitated along with Gα 12 Q231L in L cells in absence of E-cadherin. Interestingly, the expression of Gα 12 Q231L increases the amount of p120 ctn associated with E-cadherin. These findings demonstrate that Gα 12 and p120 ctn are binding partners, and they also suggest a role for Gα 12 in regulating p120 ctn activity and its interaction with cadherins. We propose that the Gα 12 –p120 ctn interaction acts as a molecular switch, which regulates cadherin-mediated cell–cell adhesion.

Gα12/13-mediated pathways have been shown to be involved in various fundamental cellular functions in mammalian cells such as axonal guidance, apoptosis, and chemotaxis. Here, we identified a homologue of Rho-guanine nucleotide exchange factor (GEF) in Caenorhabditis elegans ( CeRhoGEF ), which functions downstream of gpa-12 , the C. elegans homologue of Gα12/13. CeRhoGEF contains a PSD-95/Dlg/ZO-1 domain and a regulator of G protein signaling (RGS) domain upstream of the Dbl homology–pleckstrin homology region similar to mammalian RhoGEFs with RGS domains, PSD-95/Dlg/ZO-1–RhoGEF and leukemia-associated RhoGEF. It has been shown in mammalian cells that these RhoGEFs interact with activated forms of Gα12 or Gα13 through their RGS domains. We demonstrated by coimmunoprecipitation that the RGS domain of CeRhoGEF interacts with GPA-12 in an \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{AIF}}_{4}^{-}\end{equation*}\end{document} activation-dependent manner and confirmed that the Dbl homology–pleckstrin homology domain of CeRhoGEF was capable of Rho-dependent signaling. These results proved conservation of the Gα12–RhoGEF pathway in C. elegans . Expression of DsRed or GFP under the control of the promoter of CeRhoGEF or gpa-12 revealed an overlap of their expression patterns in ventral cord motor neurons and several neurons in the head. RNA-mediated gene interference for CeRhoGEF and gpa-12 resulted in similar phenotypes such as embryonic lethality and sensory and locomotive defects in adults. Thus, the Gα12/13–RhoGEF pathway is likely to be involved in embryonic development and neuronal function in C. elegans .