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).

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.

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.

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.

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.

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.

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.