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.

A high-sugar diet produces obesity and insulin resistance in wild-type Drosophila.

Insulin-resistant, 'type 2' diabetes (T2D) results from a complex interplay between genes and environment. In particular, both caloric excess and obesity are strongly associated with T2D across many genetic backgrounds. To gain insights into how dietary excess affects insulin resistance, we studied the simple model organism Drosophila melanogaster. Larvae reared on a high-sugar diet were hyperglycemic, insulin resistant and accumulated fat--hallmarks of T2D--compared with those reared on control diets. Excess dietary sugars, but not fats or proteins, elicited insulin-resistant phenotypes. Expression of genes involved in lipogenesis, gluconeogenesis and ß-oxidation was upregulated in high-sugar-fed larvae, as were FOXO targets, consistent with known mechanisms of insulin resistance in humans. These data establish a novel Drosophila model of diet-induced insulin resistance that bears strong similarity to the pathophysiology of T2D in humans.

E2F8 is a nonreceptor activator of heterotrimeric G proteins.

BACKGROUND: Heterotrimeric G proteins are important for numerous signaling events in eukaryotes, serving primarily to transduce signals that are initiated by G protein-coupled receptors. It has recently become clear that nonreceptor activators can regulate the level of heterotrimeric G protein signaling and, in some cases, drive cycles of receptor-independent G protein activation. In this study, we used a yeast expression cloning strategy to identify novel nonreceptor activators of heterotrimeric G proteins in a human adipocyte cDNA library. RESULTS: The human transcription factor E2F8 was found to activate heterotrimeric G proteins, suggesting a specific biological role for this recently described member of the E2F family. Epistasis studies showed that E2F8 acted at the level of G proteins and was specific for G alpha(i) over Gpa1. E2F8 augmented receptor-driven signaling, but also activated G proteins in the absence of a receptor. The GTPase-activating protein RGS4 antagonized the effect of E2F8, showing that E2F8's effect on G alpha involved nucleotide turnover. The entire E2F8 protein was required for full activity, but the majority of the signaling activity appeared to reside in the first 200 residues. CONCLUSION: In yeast, E2F8 is a guanine nucleotide exchange factor (GEF) for the alpha subunit of heterotrimeric G proteins. The molecular mechanism and biological significance of this effect remain to be determined.

A comprehensive structure-function map of the intracellular surface of the human C5a receptor. I. Identification of critical residues.

G protein-coupled receptors are one of the largest protein families in nature; however, the mechanisms by which they activate G proteins are still poorly understood. To identify residues on the intracellular face of the human C5a receptor that are involved in G protein activation, we performed a genetic analysis of each of the three intracellular loops and the carboxyl-terminal tail of the receptor. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The third intracellular loop contains the largest number of preserved residues (positions resistant to amino acid substitutions), followed by the second loop, the first loop, and lastly the carboxyl terminus. Surprisingly, complete removal of the carboxyl-terminal tail did not impair C5a receptor signaling. When mapped onto a three-dimensional structural model of the inactive state of the C5a receptor, the preserved residues reside on one half of the intracellular surface of the receptor, creating a potential activation face. Together these data provide one of the most comprehensive functional maps of the intracellular surface of any G protein-coupled receptor to date.

A comprehensive structure-function map of the intracellular surface of the human C5a receptor. II. Elucidation of G protein specificity determinants.

Within any given cell many G protein-coupled receptors are expressed in the presence of multiple G proteins, yet most receptors couple to a specific subset of G proteins to elicit their programmed response. Numerous studies demonstrate that the carboxyl-terminal five amino acids of the Galpha subunits are a major determinant of specificity, however the receptor determinants of specificity are less clear. We have used a collection of 133 functional mutants of the C5a receptor obtained in a mutagenesis screen targeting the intracellular loops and the carboxyl terminus (Matsumoto, M. L., Narzinski, K., Kiser, P. D., Nikiforovich, G. V., and Baranski, T. J. (2007) J. Biol. Chem. 282, 3105-3121) to investigate how specificity is encoded. Each mutant, originally selected for its ability to signal through a nearly full-length Galpha(i) in yeast, was tested to see whether it could activate three versions of chimeric Galpha subunits consisting of Gpa1 fused to the carboxyl-terminal five amino acids of Galpha(i), Galpha(q), or Galpha(s) in yeast. Surprisingly the carboxyl-terminal tail of the C5a receptor is the most important specificity determinant in that nearly all mutants in this region showed a gain in coupling to Galpha(q) and/or Galpha(s). More than half of the receptors mutated in the second intracellular loop also demonstrated broadened G protein coupling. Given a lack of selective advantage for this broadened signaling in the initial screen, we propose a model in which the carboxyl-terminal tail acts together with the intracellular loops to generate a specificity filter for receptor-G protein interactions that functions primarily to restrict access of incorrect G proteins to the receptor.

Random mutagenesis of the complement factor 5a (C5a) receptor N terminus provides a structural constraint for C5a docking.

The N terminus of G protein-coupled receptors has been implicated in binding to peptide hormones. We have used random saturation mutagenesis to identify essential residues in the N terminus of the human complement factor 5a receptor (C5aR). In a library of N-terminal mutant C5aR molecules screened for activation by C5a, residues 24-30 of the C5aR showed a marked propensity to mutate to cysteine, most likely indicating that sulfhydryl groups at these positions are appropriately situated to form disulfide interactions with the unpaired Cys(27) of human C5a. This presumptive spatial constraint allowed the ligand to be computationally docked to the receptor to form a model of the C5a/C5aR interaction. When the N-terminal mutant C5aR library was rescreened with C5a C27R, a ligand incapable of disulfide interactions, no individual position in the N terminus was essential for receptor signaling. However, the region 19-29 was relatively highly conserved in the functional mutants, further demonstrating that this region of the C5aR makes a productive physiologic interaction with the C5a ligand.

Essential role for the second extracellular loop in C5a receptor activation.

More than 90% of G protein-coupled receptors (GPCRs) contain a disulfide bridge that tethers the second extracellular loop (EC2) to the third transmembrane helix. To determine the importance of EC2 and its disulfide bridge in receptor activation, we subjected this region of the complement factor 5a receptor (C5aR) to random saturation mutagenesis and screened for functional receptors in yeast. The cysteine forming the disulfide bridge was the only conserved residue in the EC2-mutated receptors. Notably, approximately 80% of the functional receptors exhibited potent constitutive activity. These results demonstrate an unexpected role for EC2 as a negative regulator of C5a receptor activation. We propose that in other GPCRs, EC2 might serve a similar role by stabilizing the inactive state of the receptor.