Frizzled receptors signal through G proteins.

Frizzled receptors have long been thought to couple to G proteins but biochemical evidence supporting such an interaction has been lacking. Here we expressed mammalian Wnt-Frizzled fusion proteins in Saccharomyces cerevisiae and tested the receptors' ability to activate the yeast mitogen-activated protein kinase (MAPK) pathway via heterotrimeric G proteins. Our results show that Frizzled receptors can interact with Gαi, Gαq, and Gαs proteins, thus confirming that Frizzled functions as a G protein coupled receptor (GPCR). However, the activity level of Frizzled-mediated G protein signaling was much lower than that of a typical GPCR and, surprisingly, was highest when coupled to Gαs. The Frizzled/Gαs interaction was further established in vivo as Drosophila expressing a loss-of-function Gαs allele rescued the photoreceptor differentiation phenotype of Frizzled mutant flies. Together, these data point to an important role for Frizzled as a nontraditional GPCR that preferentially couples to Gαs heterotrimeric G proteins.

Beta2-chimaerin in cancer signaling: connecting cell adhesion and MAP kinase activation.

The chimaerins are Rac GTPase-activating proteins that bind diacylglycerol. Emerging evidence implicates beta2-chimaerin in tumor progression. Here, we discuss our recent work in Drosophila melanogaster in the context of previous studies performed in human cancer cell lines that together lend new mechanistic insight into the role of chimaerins in cancer.

Chimaerin and Rac regulate cell number, adherens junctions, and ERK MAP kinase signaling in the Drosophila eye.

The chimaerin family of Rac GTPase-activating proteins (GAPs) has been implicated in neural development and tumor progression, although the cellular mechanisms of their effects are poorly understood. To study their physiologic function, we used the Drosophila retina as a model system. Reduced expression of the fly chimaerin ortholog RhoGAP5a in the pupal eye led to an excess of interommatidial pigment cells, aberrant cell contacts, and an increase in activated ERK that localized specifically to the plasma membrane. Reducing RhoGAP5A levels suppressed the effects of disrupted EGF receptor signaling. Perturbation of Rac activity led to similar phenotypes, whereas coexpression of Rac and RhoGAP5A-dsRNAi resulted in the elimination of adherens junctions between interommatidial cells. Our results reveal a role for chimaerin in the regulation of ERK signaling and cell-cell adhesion and have implications for its participation in epithelial development and tumor progression.

C5a receptor oligomerization. II. Fluorescence resonance energy transfer studies of a human G protein-coupled receptor expressed in yeast.

Recent studies demonstrate that members of the superfamily of G protein-coupled receptors (GPCRs) form oligomers both in vitro and in vivo. The mechanisms by which GPCRs oligomerize and the roles of accessory proteins in this process are not well understood. We used disulfide-trapping experiments to show that C5a receptors, expressed in mammalian cells, reside in membranes as oligomers (Klco, J. M., Lassere, T. B., and Baranski, T. J. (2003) J. Biol. Chem. 278, 35345-35353). To begin to address how C5a receptors form oligomers, we now use fluorescence resonance energy transfer experiments on human C5a receptors expressed in the lower eukaryote Saccharomyces cerevisiae. C5a receptors tagged with variants of the green fluorescent protein display energy transfer in intact yeast, demonstrating that mammalian accessory proteins are not required for C5a receptor oligomerization. In both intact yeast cells and membrane preparations, agonist does not affect FRET efficiency, and little energy transfer is observed between the C5a receptor and a co-expressed yeast pheromone receptor (encoded by STE2), indicating that C5a receptor oligomerization is both receptor-specific and constitutive. FRET studies performed on fractionated membranes demonstrate similar levels of energy transfer between tagged C5a receptors in endoplasmic reticulum compared with plasma membrane, and urea washing of membranes has little effect on the extent of energy transfer. The oligomerization of C5a receptors expressed in yeast displays characteristics similar to those observed for other GPCRs studied in mammalian cells. This model system should prove useful for further studies to define mechanisms of oligomerization of mammalian GPCRs.