Evidence for G Protein Coupling of FZDs

There is accumulating evidence that FZDs indeed signal through the recruitment of heterotrimeric G proteins as recently reviewed by Egger-Adam and Katanaev [17]. The strongest evidence is based on genetic analysis in Drosophila [70], inhibition of signaling by the blockade of G proteins, and the use of chimeric FZDs [10] - Here, I would like to briefly summarize the facts from the literature in order to show that the evidence for G protein coupling of FZDs is rather indirect, being based on loss- or gain-of-function experiments rather than on a direct proof of a WNT- i nduced and FZD-mediated activation of guanine nucleotide exchange at a FZD-interacting G protein [9]. The reason we are forced to rely on circumstantial evidence is again the lack of tools such as purified WNTs, lack of information (e.g., pharmacological profiles of WNT-FZD interaction), as well as the peculiar characteristics of FZDs, which possibly represent an evolutionarily more modern form of seven transmembrane spanning receptors. As recently proposed by Egger-Adam and Katanaev [17], FZDs have strong responsibility during embryonic development that might have resulted in an evolutionary transformation of signaling modes adapting them from predominantly G protein-dependent to alternative signaling mechanisms. The striking difference between FZDs and "classical" GPCRs was recently emphasized by the separate classification of FZDs (and the related Smoothened) as a novel family of GPCRs by the International Union of Clinical and Experimental Pharmacology [12].

The first indication that FZDs could be GPCRs was based on structural analysis of the receptor by hydropathy plots revealing the presence of putative 7 transmembrane helices [ 4], Further structural analysis indicated that the receptors might differentially couple to G proteins [71] and the first strong experimental indications resulted from experiments in zebrafish embryos overexpressing rat FZD- [72] , Coexpression of Xenopus WNT-5A induced pertussis toxin-sensitive Ca2+ transients, which could also be inhibited by Py-sequestering transducin expression as well as GDP-P-S injection. The results implied a connection between Frizzleds, Py release from Gyo family proteins and phospholipase C signaling. Subsequently, several reports showed that inhibition or downregulation of heterotrimeric G proteins affected both P -catenin-independent as well as P - catenin - dependent signaling pathways. Especially, work by Wang and collaborators led to the elucidation of a novel FZD - dependent Ca2+ pathway through cGMP- selective phosphodiesterases [73-76] [ However, these reports have in common that the conclusions are based on downregulation/blockade of the G proteins rather than on a direct measure of WNT[[nduced and FZD^ediated activation of G proteins, for example, by a GTPyS binding assay. Thus, one explanatory model could still include the pure requirement of active G proteins for successful WNT signaling, which does not necessarily require direct FZD-G protein interaction. Thus far, the most compelling experimental evidence for a WNT-induced dissociation of a receptor-G protein complex was presented by Endo et al. [77]. They reported a rapid and prolonged WNT-3A-induced rearrangement of a FZD-DVL-G protein complex, which could be the consequence of a WNT-induced and FZD-mediated activation of a heterotrimeric G protein.

In addition, chimeric receptors consisting of FZD-intracellular loops and transmembrane and extracellular domains from adrenergic receptors [76, 78-81] ; designed with the aim of overcoming the lack of purified WNTs, provided important insight into FZD structure and function relationships as well as some pharmacological aspects. Even though it is difficult to interpret the results obtained with these chimeric receptors, with major parts from classical GPCRs, the experiments represent milestones in the understanding of FZDs as unconventional GPCRs. Most prominently, the agonist affinity shift in the presence or absence of guanine nucleotides, which was obtained in membrane preparation using isoproterenol competition binding to the FZD1/P2- adrenergic [78, 79] and FZD2/P2- adrenergic [76, 79] chimera, strongly suggests that the intracellular loops of FZDs could directly bind to and activate heterotrimeric G proteins.

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