The Wnt/Frizzled GPCR Signaling Pathway
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ISSN 00062979, Biochemistry (Moscow), 2010, Vol. 75, No. 12, pp. 14281434. © Pleiades Publishing, Ltd., 2010. Published in Russian in Biokhimiya, 2010, Vol. 75, No. 12, pp. 16421650. REVIEW The Wnt/Frizzled GPCR Signaling Pathway V. L. Katanaev1,2 1Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia 2Department of Biology, University of Konstanz, Universitatsstrasse 10, Box 643, Konstanz 78457, Germany; fax: (0049) 7531884944; Email: [email protected] Received May 24, 2010 Revision received July 27, 2010 Abstract—G proteincoupled receptors (GPCRs) represent the biggest transmembrane receptor family. The Frizzled group of GPCRs is evolutionarily conserved and serves to transduce signals from the Wnttype lipoglycoprotein growth factors. The Wnt/Frizzled signaling cascades are repeatedly used during animal development and are mostly silent in the adult. Improper activation of these cascades, e.g. through somatic mutation, underlies cancer development in various tissues. Our research over the past years has identified the trimeric G proteins as crucial transducers of the Wnt/Frizzled cascades in insect and mammalian cells. The current minireview summarizes our findings on the role of G proteins in Wnt/Frizzled signaling, as well as on identification of other signaling intermediates in this physiologically and pathologically important type of intracellular signal transduction. DOI: 10.1134/S0006297910120023 Key words: Wnt, Frizzled, G proteincoupled receptor, trimeric G proteins, development, carcinogenesis, morphogen INTRODUCTION INTO GPCR SIGNALING nating at various GPCRinduced cellular responses, e.g. transcription regulation, cell shape change or motility, G proteincoupled receptors (GPCRs) represent the proliferation, or death. biggest receptor family in the animal kingdom [1]. More The main intracellular GPCR effector molecules are than one thousand GPCR genes are encoded by the the heterotrimeric G proteins, which provide the name human genome, and more than half of all marketed drugs for this receptor group. A heterotrimeric G protein con target GPCRs and their signaling pathways [2]. The evo sists of three subunits, α, β, and γ, of which the αsubunit lutionary success of GPCRs has allowed diversification of is responsible for binding to guanine nucleotides. Four their sequences, building structures competent to recog main subgroups of Gαsubunits can be identified: Gαs, nize and transduce across the cell membrane a wide vari Gαq, Gαi/o, and Gα12/13 [7]. ety of signals, from quanta of light, ions, small organic In the GDPbound state, the G protein can exist as molecules, to large macromolecules [3]. Yet the principal the heterotrimer and is competent to interact with the organization of GPCRs is stable: they have the extracel cognate GPCR. The activated receptor acts as a GEF lular N and the intracellular Cterminus, and seven (guanine nucleotide exchange factor), catalyzing the transmembrane helixes connected by three extracellular exchange of GDP for GTP on the Gα. In most cases, this and three intracellular loops [1, 3]. event is followed by dissociation of the G protein trimer Ligand binding is mediated by receptor regions dis into GαGTP and the βγheterodimer; both also typical tributed throughout the extracellular and transmembrane ly lose their contact with the GPCR. The two active domains and causes conformational changes in the halves of the G protein bind and activate various down receptor, most noticeable of which is the outward move stream transducer proteins [8]. With time, GTP on Gα is ment of transmembrane helix VI [4, 5]. The consequence hydrolyzed back to GDP; this reaction is strongly stimu of these changes is the release of intracellular protein lated by the group of GAP (GTPase activating proteins) recognition sites, hidden in the inactive receptor state and regulators, most of which belong to the RGS (regulator of for different GPCRs located on different intracellular Gprotein signaling) family of proteins [9]. In the con parts of the receptor [5, 6]. Interaction with and resultant ventional view, GTP hydrolysis is immediately followed activation of the intracellular effector molecules is the by the reassociation of the inactive Gαβγ trimer, which first step in the series of signal transduction events culmi is ready for the new cycle of activation by the GPCR. 1428 Wnt/FRIZZLED GPCR SIGNALING PATHWAY 1429 KINETIC DIVERSITY IN GPCR SIGNALING response modes, as well as existence of multiple steady states with different flux rates) are the result of applica Thousands of GPCRs are encoded by animal tion of basic Michaelis–Mentenlike enzyme–substrate genomes, yet the number of Gprotein subunits is much analysis. In contrast, oscillations are predicted to occur more limited. For example, six Gα genes are present in upon inclusion of the positive and the negative feedback Drosophila melanogaster and 16 in human [10]. As the βγ loops in our analysis [13], or of the negative feedback with identity of the trimeric G protein complex is at large irrel timedelay (not shown). Such regulations are well known evant for the GPCR–G protein coupling [11], this mere to exist in the GPCR signaling [14]. However, the direct comparison of numbers poses the issue of how signal demonstration of oscillations in the component concen specificity is achieved in the GPCR signaling. This issue trations of the GPCR signaling is still missing. One of our is further aggravated by the GPCR promiscuity, when any goals is to test experimentally whether such oscillations given receptor can efficiently signal through multiple indeed exist. To this end, we had to develop an efficient Gαproteins [12]. So, how does the cell know which readout system using a selected GPCR. Many of our GPCR has just been activated and which response pro experiments in this direction utilize the somewhat atypi gram to choose, if, simply stated, numerous GPCRs cal GPCR subfamily—the Frizzled family of receptor expressed on its surface all signal through the same G proteins. proteins? Our analysis has provided a possible answer to this question. We performed mathematical modeling of the THE Wnt/FRIZZLED SIGNALING PATHWAY early events in GPCR signal transduction, relying on pre vious rigorous experimental measurements of GPCR, G Frizzled (Fz) proteins serve as receptors to the Wnt protein, and RGS intracellular concentrations, as well as family of secreted lipoglycoproteins [15]. The human of their kinetic interaction constants [13]. We came across genome contains 19 genes for the Wnt ligands and 10 a number of unexpected predictions concerning the genes for Fz receptors. The Wnt/Fz signaling cascades are kinetics of GPCR signaling. First of all, we found that the well conserved in animal evolution, with the basic design conventional view of the G protein activation, sketched principles of this pathway present already in sponges [16]. above, is just one of the several predicted signaling modes. In the absence of pathway activation, the cytoplasmic Indeed, high concentrations of free GTPloaded Gα and protein Axin organizes the socalled destruction complex Gβγ are predicted to result from GPCR activation in one of proteins which additionally includes the adenomatosis subset of experimentally measured concentrations and polyposis coli protein (APC), glycogen synthase 3 kinase constants. However, in another subset, the predicted (GSK3), and casein kinase [15]. The function of this mode of system response is the production of high con destruction complex is to bind and phosphorylate cyto centrations of GDPloaded but monomeric Gα (plus free plasmic βcatenin, which is followed by ubiquitination Gβγ), while concentration of GαGTP is low in this and proteasomal degradation of the latter. Binding of the regime. Interestingly, this response mode is predicted to Wnt ligand to Fz and the singlepass transmembrane occur in case of Gαomediated signaling [13], which has coreceptor LRP5/6 leads to reorganization of the Axin multiple implications for the rest of this minireview. One based destruction complex. An important role in this of such implications is the prediction that GαoGDP process is played by the cytoplasmic protein Dishevelled should be at least as effective in its interactions with the [17]. As a result of reorganization, the complex can no effector molecules as GαoGTP. This prediction is con longer bind and phosphorylate βcatenin. Thus, cytoplas firmed by the subsequent experimental analysis (see mic βcatenin levels rise, and it can diffuse into the below). nucleus and bind a number of cofactors to trigger tran Detailed modeling also showed the possibility of scription of a set of Wnt target genes [18]. Among these oscillations in the GPCR–G protein–RGS signaling genes are cell cycle regulators [19, 20]; in general, the system [13]. The frequency and the amplitude of these Wnt signaling is responsible for the transcriptional activa oscillations depend on the exact parameters of the par tion of cell proliferation. ticipating components, most importantly – of the Such cellular effect of the Wnt/Fz signaling on cell GPCR involved. Thus, we predicted that the signal proliferation underlies the main function of the pathway: specificity in GPCR signaling is mediated not by the it is repeatedly used in organism development, being exact identity of the G protein transducer, but by the responsive for multiple developmental programs from kinetic “signatures” of the signaling elicited by the given early embryonic