Kegg Pathway: Hedgehog signaling pathway

J Am Soc Nephrol. 2009 Nov 19;
Lye CM, Fasano L, Woolf AS

After the basic shape of the mammalian ureter is established, its epithelia mature and a coat of smooth muscle cells differentiate around nascent urothelia. The ureter actively propels tubular fluid from the renal pelvis to the bladder, and this peristalsis, which starts in the fetal period, requires coordinated smooth muscle contraction. Teashirt-3 (Tshz3) is expressed in smooth muscle cell precursors that form the wall of the forming mammalian ureter. The Teashirt gene family was first identified in Drosophila where Teashirt (Tsh) protein acts as a transcription factor directing embryonic anterior-posterior patterning and leg and eye development. In fly embryonic renal tubules, Tsh is expressed in mesodermally derived stellate cells intercalating between principal cells, and a paralogue, tiptop, is expressed in forming tubules. Teashirt is a component of several gene networks in flies and it is notable that similar networks control mammalian renal tract development. Null mutation of Tshz3 in mice leads to failure of functional muscularization in the top of the ureter and this is followed by congenital hydronephrosis. A signaling pathway can be envisaged, starting with sonic Hedgehog secreted by the nascent ureteric urothelium and ending with ureteric smooth muscle cell differentiation, with Tshz3 downstream of bone morphogenetic protein 4 and upstream of myocardin and smooth muscle cell contractile protein synthesis. The phenotype of Tshz3 mutant mice resembles that of human congenital pelviureteric junction obstruction, and we suggest these individuals may have mutations of genes encoding molecules in the differentiation pathway mediated by Tshz3.

Pancreas. 2009 Nov 16;
Hui H, Tang YG, Zhu L, Khoury N, Hui Z, Wang KY, Perfetti R, Go VL

OBJECTIVES:: That glucagonlike peptide-1 (GLP-1) induces differentiation of primate embryonic stem (ES) cells into insulin-producing cells has been reported by several groups and also confirmed with our observations. METHODS:: To further elucidate the process in detail and the signaling pathways involved in this differentiation, we induced human ES cells HUES1 differentiated into insulin secretion cells by GLP-1 treatment. RESULTS:: A time-dependent pattern of down expression of the stem cell markers (human telomerase reverse transcriptase and octamer-4), and the appearance of multiple beta-cell-specific proteins (insulin, glucokinase, glucose transporter, type 2, and islet duodenal homeobox 1) and Hedgehog signal molecules (Indian Hedgehog, sonic Hedgehog, and Hedgehog receptor, patched) have been identified. Cotreatment with Hedgehog signal inhibitor cytopamine was able to block this differentiation, providing evidence of the involvement of the Hedgehog signaling pathway in GLP-1-induced differentiation. We also observed increased transcripts of the transcription factors of activator protein 1, serum response element-1, DNA-binding transcription factors, and cAMP response element in GLP-1-induced ES cell differentiation. Inhibition profile by its specific inhibitors indicated that the cyclic adenosine monophosphate and phosphatidylinositol-3-kinase pathways, but not the mitogen-activated protein kinase pathway, were required for the induced differentiation of ES cells. CONCLUSIONS:: These data support that GLP-1 directs human ES cell differentiation into insulin-producing cells via Hedgehog, cyclic adenosine monophosphate, and phosphatidylinositol-3-kinase pathways.

Cell Cycle. 2009 Dec 21; 8(24):
Varas A, Sacedón R, Hidalgo L, Martínez VG, Valencia J, Cejalvo T, Zapata A, Hernández-López C, Vicente A

Bone morphogenetic proteins (BMPs) play a pivotal role during vertebrate embryogenesis and organogenesis, and have also been described to function in regulating cell fate and determination in self-renewing tissues in adults. Recent results have demonstrated that the different components of the BMP2/4 signaling pathway are expressed in the human thymus. In this study, we provide evidence that BMP4 and IL-7 interplay is important in the maintenance of the human thymic progenitor population. Intrathymic CD34(+) cells express BMP receptors (BMPRIA, BMPRIB, ActRIA, BMPRII), signal transduction molecules (Smad1, 5, 8 and 4), and produce BMP4. Neutralization of endogenous BMP4 by treatment with the antagonist Noggin reduces thymic precursor cell survival, and the addition of exogenous BMP4 decreases their proliferation. The treatment of chimeric human-mouse fetal thymus organ cultures with BMP4 inhibits cell expansion, arrests thymocyte differentiation, and leads to the accumulation of human CD34(+) precursor cells. This effect is mainly attributed to the ability of BMP4 to counteract the IL-7-induced proliferation and differentiation of CD34(+) cells. BMP4 downregulates in the precursor cell population the expression of CD127 and inhibits the IL-7-dependent STAT5 phosphorylation. In addition, BMP signaling is promoted by IL-7. Our results also demonstrate that in thymic progenitors BMPs act downstream of Sonic Hedgehog, previously described to function as a maintenance factor for human intrathymic CD34(+) precursor cells.

Zhonghua Wei Chang Wai Ke Za Zhi. 2009 Nov; 12(6): 603-606
Li XW, Li JF, Qu Y, Cai Q, Ji J, Nie H, Chen XH, Zhu ZG, Liu BY

OBJECTIVE: To investigate the effect of Hedgehog(HH) pathway on proliferation and in vitro tumorigenicity of gastric cancer cell lines. METHODS: The expression of SHH, PTCH, SMO, SUFU and GLI1 in seven cell lines were tested by RT-PCR. siRNA targeting GLI1 mRNA was transfected into MKN28 cells. Cell proliferation and in vitro tumorigenicity were examined by CCK8 and soft agar colony formation test. RESULTS: SHH in six gastric cancer cell lines was up-regulated. Expression of PTCH in KATOIII( cell lines and expression of SUFU in MKN28 and KATOIII( were reduced. GLI1 siRNA significantly inhibited the expression of GLI1 in MKN28 cell line. Growth rate and colony formation rate of MKN28 cells treated with GLI1 siRNA were significantly lower than those of control cells(all P <0.001). CONCLUSIONS: HH signaling pathway is widely activated in gastric cancer cell lines. The activation of HH signaling pathway promotes the growth of MKN28 cells.

Biochem Biophys Res Commun. 2009 Nov 12;
Tanese K, Fukuma M, Ishiko A, Sakamoto M

Vasoactive peptide endothelins are a group of small peptides with diverse paracrine/autocrine actions and are reported to be involved in the pathogenesis of many human malignancies. Basal cell carcinoma (BCC) is a common malignant skin tumor that frequently has aberrant activation of the Hedgehog (HH) signaling pathway. We show here that endothelin-2 (ET-2) is overexpressed in BCC under the control of HH signaling. By real-time quantitative RT-PCR analysis, significant expression of ET-2 mRNA was observed in 19 of 20 cases (95%) compared to normal skin. In addition, inhibition of the HH signaling pathway in a mouse BCC cell line downregulated endogenous ET-2, and activation of HH signaling in mouse embryonic fibroblast upregulated endogenous ET-2. Moreover, the 3' promoter region of ET-2 gene contains the GLI-binding site and a 0.8 kb downstream fragment containing GLI-binding sites activates transcription in a reporter assay. These data indicate that ET-2 is a direct target gene of HH signaling in BCC.

J Neurosci Res. 2009 Nov 11;
Hu X, Huang J, Feng L, Fukudome S, Hamajima Y, Lin J

Sonic Hedgehog (SHH) is essential for the development of the cochlear duct that harbors the organ of Corti. However, little is known about the molecular signaling pathway through which SHH promotes the development of the organ of Corti, especially cochlear sensory epithelial cells. In this study, we demonstrated that SHH contributes to the differentiation of cochlear neural progenitors (CNPs), which are derived from the postnatal day 1 organ of Corti in mice. Addition of SHH to CNPs increased the formation of epithelial cell islands, simultaneously activated the expression of Math1 that is a transcription factor for the initial differentiation of auditory hair cells. The increased expression of Math1 then regulated the promoter activity of Brn3.1, another transcription factor that controls the further differentiation and survival of auditory hair cells. Taken together, our data suggest that SHH plays an important role in the promotion of auditory hair cell differentiation via the Math1-Brn3.1 signaling pathway. (c) 2009 Wiley-Liss, Inc.

Cold Spring Harb Symp Quant Biol. 2009 Nov 10;
Richards GS, Degnan BM

Intercellular signaling underpins metazoan development by mediating the induction, organization, and cooperation of cells, tissues, and organs. Herein, the origins of the four major signaling pathways used during animal development and differentiation-Wnt, Notch, transforming growth factor-beta (TGF-beta), and Hedgehog-are assessed by comparative analysis of genomes from bilaterians, early branching metazoan phyla (poriferans, placozoans, and cnidarians), and the holozoan sister clade to the animal kingdom, the choanoflagellates. On the basis of the incidence and domain architectures of core pathway ligands, receptors, signal transducers, and transcription factors in representative species of these lineages, it appears that the Notch, Wnt, and TGF-beta pathways are metazoan synapomorphies, whereas the Hedgehog pathway arose in the protoeumetazoan lineage, after its divergence from poriferan and placozoan lineages. Examination of the binding domains and motifs present in signaling pathway components of nonbilaterians reveals cases in which signaling interactions are unlikely to be operating in accordance with bilaterian canons. Overall, this study highlights the stability and antiquity of the core cytosolic components of each pathway, juxtaposed with the more variable and recently evolved molecular interactions taking place at the cell surface.

Cancer Biol Ther. 2009 Sep; 8(18): 1691-8
Yao Z, Mishra L

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, with a median survival of 6-16 m. Factors responsible for the poor prognosis include late onset diagnosis, underlying cirrhosis and resistance to chemotherapy; 40% of HCCs are clonal and therefore potentially arise from progenitor/stem cells. New insights are provided from several signaling pathways, such as STAT3, NOTCH, Hedgehog and transforming growth factor-beta (TGFbeta), which are involved in stem cell renewal, differentiation, survival, and are commonly deregulated in HCC. Control of stem cell proliferation by the TGFbeta, Notch, Wnt and Hedgehog pathways to suppress hepatocellular cancer and to form the endoderm suggest a dual role for this pathway in tumor suppression as well as progression of differentiation from a stem or progenitor stage. This review provides a rationale for detecting and analyzing tumor stem cells as one of the most effective ways to treat cancers such as hepatocellular cancer.

Curr Eye Res. 2009 Aug; 34(8): 623-35
Qian YS, Chu RY, Hu M, Hoffman MR

PURPOSE: To investigate whether sonic Hedgehog (Shh) plays a role in postnatal eye development and the development of experimentally induced myopia. METHODS: Expression of Shh, Patched-1 (Ptc-1), and Gli3 was evaluated in the eyes of 13- to 14-day-old C57B/L6 mice with form-deprivation myopia (FDM) (n = 100) and controls (n = 100) using real-time PCR and Western blot analysis. In a second experiment, 336 mice were divided into two groups: the first wore a unilateral translucent diffuser to induce myopia and the second served as a control. Both groups received four intravitreal injections of either Shh-N (Sonic Hedgehog amino-terminal peptide) or cyclopamine (a specific inhibitor of the Shh pathway) every other day. Retinoscopic refraction and axial length measurements were performed on the 11th day of form deprivation. Sections of the eyes were observed using a light microscope. RESULTS: Inducing myopia caused a significant increase in expression of Shh mRNA (7 days: t = 6.09, p = 0.004; 14 days: t = 3.48, p = 0.025) and protein (7 days: t = 4.06, p = 0.015; 14 days: t = 4.25, p = 0.013). Expression of both Gli3 mRNA (t = 7.61, p = 0.002) and protein (t = 2.89, p = 0.045) increased after 7 days of occlusion. Administration of Shh-N stimulated the development of myopia and axial growth in both occluded (refraction: F = 7.49, p = 0.001; axial length: F = 9.89, p < 0.001) and non-occluded eyes (refraction: F = 14.20, p < 0.001; axial length: F = 20.37, p < 0.001). Cyclopamine reduced myopic refractive error and axial elongation in occluded eyes (refraction: F = 27.91, p < 0.001; axial length: F = 15.18, p < 0.001). It also influenced non-occluded eyes, reducing axial growth and shifting the refraction toward hyperopia (refraction: F = 14.81, p < 0.001; axial length: F = 3.99, p = 0.024). No difference in retinal thickness was found between experimental and control eyes. CONCLUSIONS: The Shh signaling pathway may influence both form-deprivation myopia and the postnatal growth of eyes with normal visual input.

Cancer Res. 2009 Nov 15; 69(22): 8572-8
Noubissi FK, Goswami S, Sanek NA, Kawakami K, Minamoto T, Moser A, Grinblat Y, Spiegelman VS

Wnt and Hedgehog signaling pathways play central roles in embryogenesis, stem cell maintenance, and tumorigenesis. However, the mechanisms by which these two pathways interact are not well understood. Here, we identified a novel mechanism by which Wnt signaling pathway stimulates the transcriptional output of Hedgehog signaling. Wnt/beta-catenin signaling induces expression of an RNA-binding protein, CRD-BP, which in turn binds and stabilizes GLI1 mRNA, causing an elevation of GLI1 expression and transcriptional activity. The newly described mode of regulation of GLI1 seems to be important to several functions of Wnt, including survival and proliferation of colorectal cancer cells.

Tissue Eng Part A. 2009 Nov 3;
Dohle E, Fuchs S, Kolbe M, Hofmann A, Schmidt H, Kirkpatrick J

A number of previous studies documented the angiogenic potential of outgrowth endothelial cells (OEC) in vitro and in vivo and provided evidence that therapeutic success could depend on co-culture or co-implantation strategies. Thus, deeper insight into the molecular mechanisms underlying this pro-angiogenic effect of co-cultures might provide new translational options for tissue engineering and regenerative medicine. One promising signaling pathway in bone repair involved in neoangiogenesis and bone formation, is the sonic Hedgehog (Shh) pathway. In this paper we focus on the effect of Shh on the formation of microvessel-like structures and osteoblastic differentiation in co-cultures of primary osteoblasts (pOB) and OEC. Already after 24 hours of treatment, Shh leads to a massive increase in microvessel-like structures compared to untreated co-cultures. Increased formation of angiogenic structures seems to correlate with the upregulation of vascular endothelial growth factor (VEGF) or angiopoietins (Ang-1, Ang-2) studied at both the mRNA and protein-level. In addition, treatment with cyclopamine, an inhibitor of Hedgehog signaling, blocked the formation of microvessel-like structures in the co-cultures. However, exogenous Shh also resulted in the upregulation of several osteogenic differentiation markers in real time PCR, as well as in an increased mineralization and alkaline phosphatase (ALP) activity. The present data highlight the central role of the Shh pathway in bone regeneration and vascularization. Furthermore sonic Hedgehog might have the potential to improve both angiogenesis and osteogenesis in clinical applications in the future.

Curr Biol. 2009 Oct 28;
Tsiairis CD, McMahon AP

Breaking bilateral symmetry is critical for vertebrate morphogenesis. In the mouse, directional looping of the heart and rotation of the embryo, the first overt evidence of left/right asymmetry (L/R), are observed at early somite stages ( approximately E8.5) [1, 2]. Activation of a Nodal-Pitx2 regulatory pathway specifically within the left lateral plate mesoderm (LPM) is critical for these events [3-10]. Asymmetric expression of Nodal is thought to be triggered by left-oriented, cilia-generated flow within the ventral, midline node [11, 12]. Genetic removal of Hedgehog (Hh) signaling in the mouse demonstrates a requirement for Hedgehog signals in the symmetry-breaking process [13], and analysis of node trafficking has suggested a mechanism of directional transport in the node that might relate to symmetry breaking in the LPM [14]. Here we provide evidence that Hedgehog signaling in the node is not essential for breaking bilateral symmetry. In contrast, direct Hh signaling in the LPM is critical. Evidence is presented that Sonic and Indian Hedgehog signals act together, through a Foxf1/Bmp4 pathway, to enable the initiation and propagation of Nodal signaling within the LPM, regulating the competence of that tissue to respond to the Nodal pathway.

Exp Cell Res. 2009 Oct 21;
Maloverjan A, Piirsoo M, Michelson P, Kogerman P, Osterlund T

The Hedgehog (Hh) signaling pathway plays crucial roles in embryonic development and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Aberrant activation of Hh signaling is associated with various developmental abnormalities and several types of cancer. Genetic and biochemical studies ascertain serine/threonine kinase Fused (Fu) as a protein involved in Hh signaling in Drosophila. However, the role of Fu is not fully conserved in mammals suggesting involvement of other kinases in the mammalian Hh signaling pathway. In search of potential homologues to Drosophila and human Fu, we have cloned human serine/threonine kinase ULK3 and assessed its ability to regulate GLI transcription factors, mediators of SHH signaling. We demonstrate that ULK3 enhances endogenous and over-expressed GLI1 and GLI2 transcriptional activity in cultured cells, as assessed by GLI-luciferase reporter assay. Besides that, ULK3 alters subcellular localization of GLI1, as assessed by immunofluorescent staining and immunoblotting assays. We show that ULK3 is an autophosphorylated kinase and phosphorylates GLI proteins in vitro. We also demonstrate that ULK3 catalytical activity is crucial for its function in SHH pathway. We show that ULK3 is widely expressed and its expression is higher in a number of tissues where Shh signaling is known to be active. Our data suggest that serine/threonine kinase ULK3 is involved in the SHH pathway as a positive regulator of GLI proteins.

Evol Dev. 2009 Nov-Dec; 11(6): 710-8
Lin Y, Cai Z, Huang S, Yang L, Wang C, Liu Z, Cao J, An Y, Zhang H

The Hedgehog (Hh) signaling pathway regulates many developmental processes both in vertebrates and in invertebrates. However, little is known about this pathway in the cephalochordate amphioxus. In this paper, we focus on the Ptc, Smo, and Sufu homologs in amphioxus, which are the key members of the Hh signaling pathway. Their genomic structures show their comparability with homologs in vertebrates. In situ hybridization reveals that amphioxus Ptc, Smo, and Sufu have similar expression patterns in embryogenesis. They are expressed in the neural plate at early neurula stage, and then down-regulated in dorsal neural ectoderm. During development, their transcripts appear and persist in the notochord, the wall of the head cavity, the epithelium of the pharynx, and the gut. The data show that the expression patterns of these three genes are overlapping with Hh and Gli during the embryonic development in amphioxus. Moreover, injection of amphioxus Hh RNA into zebrafish-fertilized eggs can expand the expression domains of Ptc1 and Nk2.2a, the target genes of the Hh signaling pathway, which is similar to the injection of zebrafish Sonic hh a (zShha) and Sonic hh b (zShhb). Our results suggest that amphioxus may possess a conserved and functional Hh signaling pathway similar to that of vertebrates.

Dev Dyn. 2009 Oct 29; 238(12): 3035-3042
Heydeck W, Zeng H, Liu A

Precise planar cell polarity (PCP) is critical for the development of multiple organ systems in animals. A group of core-PCP proteins are recognized to play crucial roles in convergent extension and other PCP-related processes in mammals. However, the functions of another group of PCP-regulating proteins, the PCP-effector proteins, are yet to be fully studied. In this study, the generation and characterization of a mouse mutant for the PCP effector gene Fuzzy (Fuz) is reported. Fuz homozygous mutants are embryonically lethal, with multiple defects including neural tube defects, abnormal dorsal/ventral patterning of the spinal cord, and defective anterior/posterior patterning of the limb buds. Fuz mutants also exhibit abnormal Hedgehog (Hh) signaling and inefficient proteolytic processing of Gli3. Finally, a significant decrease in cilia was found in Fuz homozygous mutants. In conclusion, Fuz plays an important role in cilia formation, Hh signal transduction, and embryonic development in mammals. Developmental Dynamics 238:3035-3042, 2009. (c) 2009 Wiley-Liss, Inc.

Mech Dev. 2009 Oct 25;
Varnat F, Zacchetti G, Ruiz I Altaba A

Several lines of evidence point to the central role of WNT signaling in the initiation of intestinal tumorigenesis, most often due to loss of APC, a negative regulator of the WNT-betaCATENIN/TCF pathway. Modeling human colon cancers in mice through loss of Apc has shown that inappropriate activation of Wnt signaling is sufficient to induce adenoma formation. More recent analyses have also demonstrated a key role for Hedgehog-GLI (HH-GLI) signaling in human colon cancers. However, how the WNT and HH pathways interact during intestinal development, homeostasis and cancer is not clear. Marker analyses suggest predominant paracrine signaling from rare Shh producing cells in the crypt's bottom to adjacent Gli1(+) mesenchymal cells in normal adult mice. Using conditional KO models, we show that inhibition of the function of the critical Hh mediator Smoothened (Smo) rescues the lethality and intestinal phenotypes of loss of Apc. The results uncover an essential role of the Hh pathway in tumors induced by hyperactive Wnt signaling, suggest the action of the Hh pathway in parallel or downstream of Wnt signaling, and validate this model for its use in preclinical work testing Hh pathway antagonists.

Development. 2009 Nov; 136(22): 3779-89
Bastida MF, Sheth R, Ros MA

Normal patterning of tissues and organs requires the tight restriction of signaling molecules to well-defined organizing centers. In the limb bud, one of the main signaling centers is the zone of polarizing activity (ZPA) that controls growth and patterning through the production of sonic Hedgehog (SHH). The appropriate temporal and spatial expression of Shh is crucial for normal limb bud patterning, because modifications, even if subtle, have important phenotypic consequences. However, although there is a lot of information about the factors that activate and maintain Shh expression, much less is known about the mechanisms that restrict its expression to the ZPA. In this study, we show that BMP activity negatively regulates Shh transcription and that a BMP-Shh negative-feedback loop serves to confine Shh expression. BMP-dependent downregulation of Shh is achieved by interfering with the FGF and Wnt signaling activities that maintain Shh expression. We also show that FGF induction of Shh requires protein synthesis and is mediated by the ERK1/2 MAPK transduction pathway. BMP gene expression in the posterior limb bud mesoderm is positively regulated by FGF signaling and finely regulated by an auto-regulatory loop. Our study emphasizes the intricacy of the crosstalk between the major signaling pathways in the posterior limb bud.

Dev Cell. 2009 Oct; 17(4): 443-58
Kovacs JJ, Hara MR, Davenport CL, Kim J, Lefkowitz RJ

Arrestins were identified as mediators of G protein-coupled receptor (GPCR) desensitization and endocytosis. However, it is now clear that they scaffold many intracellular signaling networks to modulate the strength and duration of signaling by diverse types of receptors--including those relevant to the Hedgehog, Wnt, Notch, and TGFbeta pathways--and downstream kinases such as the MAPK and Akt/PI3K cascades. The involvement of arrestins in many discrete developmental signaling events suggests an indispensable role for these multifaceted molecular scaffolds.

Dev Biol. 2009 Oct 21;
Glazer AM, Wilkinson AW, Backer CB, Lapan SW, Gutzman JH, Cheeseman IM, Reddien PW

Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian Schmidtea mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in Hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates.

Dev Biol. 2009 Oct 20;
Cheng S, Maier D, Neubueser D, Hipfner DR

The Hedgehog (Hh) signaling pathway plays a conserved and essential role in regulating development and homeostasis of numerous tissues. Cytoplasmic signaling is initiated by Smoothened (Smo), a G-protein-coupled receptor (GPCR) family member, whose levels and activity are regulated by the Hh receptor Patched (Ptc). In response to Hh binding to Ptc, Ptc-mediated repression of Smo is relieved, leading to Smo activation, surface accumulation, and downstream signaling. We find that downregulation of Drosophila Smo protein in Hh-responding imaginal disc cells is dependent on the activity of G-protein-coupled receptor kinase 2 (Gprk2). By analyzing gain- and null loss-of-function phenotypes, we provide evidence that Gprk2 promotes Smo internalization subsequent to its activation, most likely by direct phosphorylation. Ptc-dependent regulation of Smo accumulation is normal in gprk2 mutants, indicating that Gprk2 and Ptc downregulate Smo by different mechanisms. Finally, we show that both Drosophila G-protein-coupled receptor kinase orthologues, Gprk1 and Gprk2, act in a partially redundant manner to promote Hh signaling. Our results suggest that Smo is regulated by distinct Ptc-dependent and Gprk2-dependent trafficking mechanisms in vivo, analogous to constitutive and activity-dependent regulation of GPCRs. G-protein-coupled receptor kinase activity is also important for efficient downstream signaling.