Wnt Signaling

Fzs bind and synergize with Wnts to activate two signaling pathways in vertebrates referred to as the Wnt/p-catenin and Wnt/calcium pathways [2,7]. The canonical or Wnt/p-catenin pathway (Fig. 1A) promotes the interaction between P-catenin and the Lef/Tcf family of transcription factors [1] to regulate cell proliferation and cell fate determination. Upon binding Wnt, Fz signals to Dishevelled (Dsh), which inhibits the "destruction complex" [2]. The destruction complex is composed of a large assembly of proteins, including Axin, APC, PP2A, and GSK-3 that continually promotes the ubiquitination and proteosomal degradation of P-catenin in the absence of active Wnt signaling. Once Dsh has inactivated this complex, P-catenin accumulates and interacts with the Lef/Tcf family of transcription factors to activate transcription of Wnt-responsive genes. In Xenopus and mammalian cells, strong evidence shows that Fz signaling to Lef/Tcf occurs via G-protein subunits (discussed below). A lack of biochemical data showing how G proteins might then regulate the function of Dsh and ultimately Lef/Tcf transcription factors represents a significant gap in our knowledge of Wnt/p-catenin signaling. (Detailed maps of this pathway can be found at http://www.ana.ed.ac.uk/ rnusse/pathways/cell2.html and http://stke.sciencemag.org/ cgi/cm/CMP_5533.)

Although the net effect of activation of the Wnt/Ca++ pathway (Fig. 1B) is poorly understood, at a minimum it regulates cell behavior and some cell fates [7]. Activation of this pathway has also been reported to oppose the effects of Wnt/P-catenin pathway activation. Fz stimulates G proteins, which activate phopholipase C to turn on Ca++ signaling. Wnts and Fzs increase the release of Ca++ from intracellular stores and activation of the Ca++-sensitive

Figure 1 The two vertebrate Wnt signaling pathways are discussed in detail in the text. Abbreviations: cysteine-rich domain (CRD); Dishevelled (Dsh), phospholipase C (PLC), phosphatidylinositol 4,5-bisphosphate (PIP2), diacylglycerol (DAG), inositol 1,4,5-triphosphate (IP3), protein kinase C (PKC), and calcium-/calmodulin-dependent protein kinase II (CaMKII).

Figure 1 The two vertebrate Wnt signaling pathways are discussed in detail in the text. Abbreviations: cysteine-rich domain (CRD); Dishevelled (Dsh), phospholipase C (PLC), phosphatidylinositol 4,5-bisphosphate (PIP2), diacylglycerol (DAG), inositol 1,4,5-triphosphate (IP3), protein kinase C (PKC), and calcium-/calmodulin-dependent protein kinase II (CaMKII).

protein kinase C (PKC) and calcium-/calmodulin-dependent kinase II (CaMKII).

Evidence for Frizzleds as G-Protein-Coupled Receptors

Structural comparison to GPCRs and interesting experimental findings argue that Frizzleds signal through het-erotrimeric G proteins. The seven hydrophobic domains of the predicted Fz protein, the predicted NH2-terminal signal sequence, and the potential signal peptidase cleavage site suggest topological homology to all known GPCRs [8,9]. Phylogenetically, fz is most closely related to smoothened (smo) [5], which was recently reported to signal through G proteins [10]. Some reports have noted that fz has no amino acid sequence similarity to the rhodopsin superfamily of GPCRs [4,5,11,12]. However, reevaluation of Fz predicted protein sequences reveals that Fzs share more characteristics with established GPCR families than was previously thought (Table 1) [4,11,13]. Because Fzs are phylogenetically linked to a known GPCR, Smo, and Fzs contain several GPCR sequence motifs, Fzs might also share with GPCRs a mechanism of conformational change that can activate G proteins.

Fzs not only resemble GPCRs but experimental evidence also argues that Fzs rely upon G proteins for signaling. Recent work examined Fzs as GPCRs using rat Fz-2 signaling in the Wnt/Ca++ pathway and rat Fz-1 signaling in the Wnt/ P-catenin pathway. The first report showing a requirement for G proteins by Fz came from the analysis of intracellular calcium in zebrafish [14]. An increase in the frequency of intra-cellular calcium transients was measured in zebrafish embryos over-expressing Wnt-5A or rat Fz-2. Whether G proteins were required for this phenomenon was tested by treating embryos expressing rat Fz-2 with several G-protein inhibitors.

The elevation of Ca++ stimulated by rat Fz-2 was blocked by the G-protein inhibitors GDP^S, which prevents G-protein activation; pertussis toxin, which adenosine diphosphate (ADP) ribosylates and which specifically inhibits guanosine diphosphate (GDP)-guanosine triphosphate (GTP) exchange on Gai, Gao, and Gat; and a-transducin, which sequesters PY subunits. Subsequent studies in Xenopus embryos found that rat Fz-2 requires G proteins to activate two Ca++-sensitive enzymes, PKC [15] and CaMKII [16]. Activation of both these enzymes by rat Fz-2 was also inhibited by pertussis toxin and a-transducin, confirming that Wnt/Ca++ signaling by rat Fz-2 is mediated, directly or indirectly, by G proteins.

In order to determine whether G proteins mediate Fz signaling directly, chimeric receptors were constructed to control the activation state of Fz. The intracellular loops of rat Fz-1 and -2 were substituted for the cognate loops of the

Table I Conserved Sequence Characteristics in the G-Protein-Coupled Receptors of the Rhodopsin (Rho) and Smoothened (Smo) Families and Frizzleds (Fz)

Conserved sequence characteristic

Rho

Smo

Fz

Putative signal peptide

Y

Y

Y

Potential N-linked glycosylation sites

Y

Y

Y

Cysteine-rich domain

N

Y

Y

Seven predicted transmembrane domains

Y

Y

Y

Cysteines in extracellular loops 1 and 2

Y

Y

Y

DRY or ERW motif

Y

N

N

Prolines in transmembrane domains 4-6

Y

4,5 only

Y

Leucine-rich transmembrane domain 5

Y

Y

Y

Lys-X-X-Lys in intracellular loop 3

Y

N

Y

Cysteine in intracellular COOH terminus

Y

Y

N

Table II

G-Protein Subunits Required for Wnt-Fz Signaling

Activator

System

Response

Required3

Not required

Xwnt-8

Xenopus

Axis duplication

Gai, Gao, Gat

rat Fz-1

Xenopus

Gap Gao> Gat

ß2-AR/Fz-1

F9 cells

Topflash activation

Gao Gaq

Gas, Gai, Ga11, ß

rat Fz-2

Zebrafish

Intracellular [Ca++]

Gai, Gao, Gat, ß

rat Fz-2

Xenopus

PKC activation

G«i, Gao, Gat, ßY

ß2-AR/Fz-2

Xenopus

Gai, Gao, Gat

ß2-AR/Fz-2

F9 cells

Primitive endoderm

Gaq, Gai, Ga11

ß2-AR/Fz-2

F9 cells

Ligand affinity shift

Gao, Gat

Gas

aInhibition of these G-protein subunits interfered with Fz signaling.

aInhibition of these G-protein subunits interfered with Fz signaling.

P2-adrenergic receptor (P2-AR), so that Fz signaling domains could be kept in an inactive state using a P2-AR antagonist and quickly activated by a P2-AR agonist [17,18]. Stimulation of the P2-AR/rat Fz-2 chimera activated CaMKII within just10 minutes, and that effect was inhibited by treatment with pertussis toxin [16]. The dependence on G proteins for such a rapid response to Fz signaling indicated that G proteins must be integral to Fz signaling. Actual binding of G proteins to Fz has not been reported but can be inferred from the observation of a shift in agonist affinity of the P2-AR/rat Fz chimeras in the presence of a nonhy-drolyzable GTP analog. The presence of GTP causes a reduction in the affinity of most GPCRs for their agonists; the decrease correlates with dissociation of the GPCR from the G protein. The P2-AR/rat Fz chimeras exhibit this classic affinity shift, suggesting that intracellular residues of rat Fz-1 [17] and rat Fz-2 [19] directly bind G proteins also. Together, these experiments suggest that Fzs interact directly with G proteins to activate cytoplasmic signaling molecules.

Additional work aimed to show that Fzs require G proteins to mediate cellular and physiological processes. First, it was observed that a GPCR known to stimulate Ca++ signaling, 5-HT1c, and Xwnt-5A produce the same overexpression phenotype in Xenopus embryos [20]. It was then shown that Wnts require G proteins to produce the classic duplicated-axis over-expression phenotype. The regulator of G-protein signaling (RGS4), which enhances the intrinsic GTPase activity of Gai and Gaq subunits blocked the ability of Wnt, but not Dsh, to induce duplicated axes in Xenopus embryos [21]. This observation placed G proteins between Fz and Dsh for the first time. In cultured mammalian F9 cells, pertussis toxin and oligonucleotides antisense to specific G proteins inhibited both induction of primitive endo-derm by rat Fz-1 [22] and the P2-AR/rat Fz-2 chimera [18] and activation of a Lef/Tcf specific reporter gene by rat Fz-1. This result was confirmed in Xenopus embryos where pertussis toxin blocked activation of Wnt-responsive genes by rat Fz-1 [17]. Recently it has been shown that activation of Frizzled-2 in mouse totitpotent F9 cells involves activation of cyclic GMP phosphodiesterase and a sharp decline in the intracellular concentration of cyclic GMP [23].

Inhibitors of cyclic GMP phosphodiesterases block aspects of Frizzled-2 signaling in the F9 cells as well as in zebrafish oocytes. Wnt-5A and G-protein signaling are required also for collagen-induced DDR1 receptor activation and normal cell adhesion [24]. Taken together, these studies indicate that Fzs not only require G proteins to activate intracellular signaling enzymes, but also couple to G proteins to regulate physiologically relevant events.

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