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Wnt Signaling, Ca2+, and Cyclic GMP: Visualizing Frizzled Functions

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Science  06 Jun 2003:
Vol. 300, Issue 5625, pp. 1529-1530
DOI: 10.1126/science.1085259


Wnts control the specification of cell fate, cell adhesion, migration, polarity, and proliferation. Their roles in development have been probed in fruit flies, nematodes, zebrafish, frogs, and mice. Some Wnts inhibit the degradation of β-catenin, which can regulate transcription of specific genes. Other Wnts exert their influences in other ways, such as increasing intracellular concentrations of Ca2+ and decreasing intracellular concentrations of cyclic guanosine monophosphate (cGMP). Heterotrimeric guanine nucleotide–binding proteins (G proteins) and RGS proteins have been implicated in Wnt signaling. Wnt regulation of intracellular Ca2+ and cGMP levels requires the G protein transducin and a cGMP-specific phosphodiesterase, which are major elements in signaling of the visual pathway.

Wnt proteins are secreted signaling molecules orchestrating many aspects of early development and are well studied in the fly, worm, fish, frog, mouse, and human (1). Although there is broad agreement that Wnts exert these influences on embryonic cells through cell surface receptors named Frizzleds (2), the most proximal steps of the signaling cascade beyond Frizzleds are less well understood. The overall downstream targets of Frizzled actions can be segregated into three pathways: the canonical Wnt pathway, which acts through the protein β-catenin to regulate transcription; the planar cell polarity (PCP) pathway, which regulates Drosophila development independently of β-catenin; and the Ca2+ pathway. In the Wnt-Ca2+ pathway (3, 4), Frizzled appears to act through heterotrimeric guanine nucleotide–binding proteins (G proteins) (58) and to activate phospholipase C (PLC) (5) and phosphodiesterase (PDE) (7), which lead to increased concentrations of free intracellular calcium {[Ca2+]i} (9) and to decreased intracellular concentrations of cyclic guanosine monophosphate (cGMP) (7), respectively (Fig. 1). This newly discovered role of cGMP in Wnt signaling is perhaps the least well understood. Frizzled signaling seems to have co-opted a signaling paradigm from the visual pathway, including the G protein α subunit Gαt (known in the visual system as transducin) and the Gαt effector PDE6. Interrupting signaling in these networks with pertussis toxin (which inactivates members of the Gi class of G protein α subunits) or with chemical inhibitors of protein kinases or PDEs leads to disruptions in Wnt signaling and interferes with normal development in zebrafish (7). Further confirmation of Wnt signaling through Ca2+ and cGMP will be important to establish this conservation of signaling paradigms between divergent and temporally distinct signaling pathways. Effectors sensing intracellular concentrations of cGMP include cyclic nucleotide–gated ion channels, guanylylcyclases, and protein kinase G. To what extent these effectors play a role in Wnt-Frizzled signaling remains to be established.

Fig. 1.

Wnt-Ca2+–cGMP signaling via Frizzleds. The binding of Wnt5A activates the rat Frizzled-2 (Rfz2), leading to an activation of G proteins (composed of Gα and Gβ/γ subunits). Gαt2 then activates PDE (PDE → PDE6*), which stimulates a decline in the intracellular concentration of cGMP. The Gβ/γ subunits activate PLC Cβ (PLCβ), which hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 catalyzes the release of stored intracellular Ca2+, activating Ca2+-calmodulin–sensitive CamKII and the Ca2+-sensitive protein phosphatase calcineurin (Calcn). DAG activates PKC directly. Both PKC and Calcn directly and indirectly influence the activity of various TFs; for example, using the NF-AT TF to direct gene expression.

Wnts are secreted glycoprotein molecules that bind to Frizzleds: cell membrane–localized receptors with seven transmembrane-spanning segments (2), which are characteristic of receptors that couple to G proteins (GPCRs). Defining whether Frizzled proteins are actually GPCRs is of major import for developmental biologists, in order to understand the immediate downstream elements involved in Wnt signaling to planar polarity, β-catenin, and Ca2+ signaling. Several sorts of evidence support the notion that Frizzleds may function as GPCRs: (i) Frizzled proteins have heptihelical structure conserved in all members of the superfamily of GPCRs (>1500 members). (ii) Wnts show chemical similarity to other secreted glycoprotein ligands (such as gonadotropins) that act through GPCRs. (iii) Wnt signaling is inhibited by pertussis toxin or by depletion of specific G protein α or β subunits (6, 7, 10). (iv) Functional chimeras of Frizzleds can be made with exofacial and transmembrane portions of other GPCRs (6). (v) Wnt signaling is sensitive to inhibitors of enzymes integral to GPCR signaling. (vi) Finally, members of the regulators of G protein signaling family of proteins have been shown to participate in development (11).

In zebrafish embryos and mouse F9 embryonic teratocarcinoma cells, Wnt5A stimulates increases in [Ca2+]i, a response that is sensitive to pertussis toxin (5, 7). Such G protein–linked phosphatidylinositol signaling in the Wnt-Ca2+ pathway (5, 8) is likely to be mediated by PLC (Fig. 1). Several Ca2+-sensitive targets—protein kinase C (PKC) (12), Ca2+-calmodulin–dependent protein kinase II (CamKII) (13), and the Ca2+-calmodulin–sensitive protein phosphatase calcineurin (14)— have been identified downstream of the Wnt-Ca2+ pathway in vertebrates. Ca2+ signaling appears to drive dorsoventral axis patterning of vertebrates, whereas such noncanonical Wnt signaling (that is, signaling not mediated by β-catenin) regulates focal adhesion kinase activity and cell motility in Drosophila (15). Details about substrates of PKC (16), CamKII, and calcineurin specifically involved in the downstream events of Wnt-Ca2+ signaling are few. The transcription factor (TF) NF-AT is both a substrate for calcineurin and a regulator of ventral cell fate (14), providing one carefully detailed example. Targets of the Wnt-Ca2+ pathway appear to cross-talk to the Wnt–β-catenin pathway at multiple points (17). The fact that PKC appears to participate not only in the Wnt-Ca2+ pathway but also in the Wnt–β-catenin (17) and perhaps in the Wnt-PCP pathways suggests that cellular context and temporal and spatial considerations must be highlighted in our quest to fully understand Wnt-Ca2+ signaling.

We have implicated the G protein transducin 2 (Gt2), cGMP PDE, and intracellular cGMP in Wnt-Ca2+ signaling (7). Each of these signaling elements is well known in the visual excitation pathway of both invertebrates and vertebrates (18). Disruption of the Gt2 → PDE → cGMP pathway in response to Wnt activation blocks primitive endoderm formation in mouse F9 cells and normal early development in zebrafish embryos (7). There may be cross-talk among the Wnt signaling pathways mediated by cGMP. Inhibitors of cGMP PDEs are reported to inhibit the Wnt–β-catenin pathway by increasing intracellular concentrations of cGMP, activating cGMP-dependent protein kinase (which phosphorylates β-catenin), reducing β-catenin concentrations, and promoting apoptosis (19). The PDE inhibitor Exisulind has been proposed as a lead compound for anticancer therapies (20).

Understanding the Wnt-Ca2+–cGMP pathway and its full biological role may yield rich dividends in several areas. Genes regulated by the Wnt–β-catenin pathway have been described (21), whereas gene profiling for the Wnt-Ca2++-cyclic GMP pathway has not been reported. Several early lines of evidence suggest that the Wnt-Ca2+–cGMP pathway may antagonize the Wnt–β-catenin pathway (17), which has many linkages to human cancer. Unknown is the extent to which the Wnt-Ca2+–cGMP pathway may participate in the regulation of the PCP function of Wnts. Because Wnts can interact with more than one Frizzled, detailed analysis of individual Frizzleds may yield new insights into Frizzled-specific pathways of signaling and gene expression. The role of low-density lipoprotein receptor–related proteins 5 and 6 (LRP5 and LRP6) as coreceptors with Frizzled in the Wnt–β-catenin pathway raises the question of a possible similar role for LRP proteins in Wnt-Ca2+ signaling. Finally, the macromolecular complexes involved in Wnt-Frizzled signaling are tantalizing targets for analysis. Scaffolds such as Axin, as well as A kinase anchor proteins (22), will likely provide explanations for the exquisite temporal and spatial responses we find for many GPCR-mediated pathways, including Wnt signaling. Given the apparent function of primary elements of the visual pathway in the regulation of embryonic development, we can also imagine other roles for Wnts and Frizzleds outside of their critical roles in early development.

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