Smoothened Activates Gαi-mediated Signaling in Frog Melanophores

Abstract

Smoothened (Smo),1 a protein with homology to G-proteincoupled receptors, was first identified in Drosophila as a necessary component for Hedgehog signaling and shown to be involved in pattern formation and morphogenesis (1-4). Subsequent studies revealed that Smo is the signaling component of the vertebrate Sonic hedgehog (Shh) receptor complex (5) and, along with Patched (Ptc), contributes to the causation of basal cell carcinoma (6 -10). Smo is known to be constitutively active in the absence of Ptc, and its third intracellular loop and seventh transmembrane region are required for function (11). However, Smo has yet to be shown to interact with a G-protein, and the identity of the pathway used for downstream communication is unclear (12, 13).Because detection of a G-protein-coupled response from Smo in transfected mammalian cells has been elusive, we were prompted to examine its effect using a frog melanophore-based assay. Melanophores provide a sensitive, quantitative bioassay for the activity of G-protein-coupled receptors (14). The state of pigment distribution within a cell reflects intracellular levels of cyclic adenosine monophosphate (cAMP) or diacylglycerol. Elevation of either cAMP or diacylglycerol above baseline induces melanosome dispersion throughout the cytoplasm, whereas inhibition of cAMP production (in the absence of additional diacylglycerol production) is associated with melanosome aggregation into a single, centrally located sphere. The melanophore assay is particularly useful as a means of identifying G␣ icoupled receptors, such as the human D 3 receptor, whose ability to lower intracellular cAMP levels is barely noticeable when heterologously expressed (15).Here, it is demonstrated that Xenopus melanophores expressing Smo display persistent aggregation, a hallmark of G␣ i activity. Either pertussis toxin (PTX) or a dominant negative G␣ i , but not a dominant negative G␣ o , can reverse the phenotype. The results show that Smoothened can signal via activation of G␣ i . EXPERIMENTAL PROCEDURESPlasmid DNA Constructs-The human cannabinoid receptor 2 (CB2) was the kind gift of Merck Frosst. Plasmid pRK7 containing the human Smo, Ptc, and mouse Shh genes and purified, recombinant N-Shh (5) were obtained from Genentech, Inc. Clones for rat wild type G␣ i1 , G␣ oa , and the G␣ i1 S47N mutant (16) were a gift from Dr. Alfred Gilman and G␣ i1 Q204H was obtained from Dr. Stephen Sprang, both at the University of Texas Southwestern Medical Center. The coding sequences were subcloned into either pCR3.1 Uni (Smo) or pcDNA3.1/V5/His-TOPO (G␣ i and G␣ o ) vectors from Invitrogen for expression in melanophores. G␣ o Q205L and S47C were constructed using the Transformer Mutagenesis kit from Promega.Transfections-The ␤ 2 -7 cell line of Xenopus melanophores, obtained from Bunsen Rush Laboratories, Inc., was propagated as described in fibroblast-conditioned medium (CFM) (14). Cells were transfected by electroporation using an Electro Cell Manipulator 600 (BTX) as described with the following changes (16). ...

Sonic hedgehog signaling is decoded by calcium spike activity in the developing spinal cord

“…In vertebrates, Shh binds to its receptor, Patched, allowing the recruitment and activation of Smoothened (Smo) in the primary cilium that triggers the regulation of targeted gene expression (23,24). Smo is a seven-transmembrane receptor, and although its interaction with GTP-binding protein α-i (Gαi) has been demonstrated in vivo in Drosophila (25) and in vitro (25,26), the functional relevance of this coupling in vertebrates has remained elusive (27)(28)(29). Activation of G protein-coupled receptors often engages second messengers such as Ca 2+ , and cilia are structures especially suitable for coordinating second-messenger dynamics (30,31).…”

Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8

“…Culture of Xenopus laevis melanophores and DNA transfections by electroporation were done as described (9). For purifying NPB, the acid͞acetone extraction of frozen bovine hypothalami was performed as described (10,11).…”

Altered localization ofDrosophilaSmoothened protein activates Hedgehog signal transduction

“…Two experiments in frog melanophores and zebrafish embryos have suggested possible G␣(i) involvement in Hh signal transduction (Hammerschmidt and McMahon 1998;DeCamp et al 2000). As both studies involved overexpression of proteins, it is unclear as yet whether Smo acts through G protein signal transduction.…”