Growth differentiation factor 15 (GDF15), a distant member of the transforming growth factor (TGF)-β family, is a secreted protein that circulates as a 25-kDa dimer. In humans, elevated GDF15 correlates with weight loss, and the administration of GDF15 to mice with obesity reduces body weight, at least in part, by decreasing food intake. The mechanisms through which GDF15 reduces body weight remain poorly understood, because the cognate receptor for GDF15 is unknown. Here we show that recombinant GDF15 induces weight loss in mice fed a high-fat diet and in nonhuman primates with spontaneous obesity. Furthermore, we find that GDF15 binds with high affinity to GDNF family receptor α-like (GFRAL), a distant relative of receptors for a distinct class of the TGF-β superfamily ligands. Gfral is expressed in neurons of the area postrema and nucleus of the solitary tract in mice and humans, and genetic deletion of the receptor abrogates the ability of GDF15 to decrease food intake and body weight in mice. In addition, diet-induced obesity and insulin resistance are exacerbated in GFRAL-deficient mice, suggesting a homeostatic role for this receptor in metabolism. Finally, we demonstrate that GDF15-induced cell signaling requires the interaction of GFRAL with the coreceptor RET. Our data identify GFRAL as a new regulator of body weight and as the bona fide receptor mediating the metabolic effects of GDF15, enabling a more comprehensive assessment of GDF15 as a potential pharmacotherapy for the treatment of obesity.

The folding of K K-helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable K K-helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure. ß

We present an approach that allows rapid determination of the topology of Escherichia coli inner-membrane proteins by a combination of topology prediction and limited fusion-protein analysis. We derive new topology models for 12 inner-membrane proteins: MarC, PstA, TatC, YaeL, YcbM, YddQ, YdgE, YedZ, YgjV, YiaB, YigG, and YnfA. We estimate that our approach should make it possible to arrive at highly reliable topology models for roughly 10% of the Ϸ800 inner-membrane proteins thought to exist in E. coli.bioinformatics ͉ fusion protein A n important first step in the characterization of an integral membrane protein of the helix bundle class (1) is to determine its membrane topology-i.e., the number of transmembrane ␣-helices and the overall in͞out orientation of the protein relative to the membrane. In Escherichia coli, this step is usually accomplished by using reporter enzymes such as PhoA or LacZ fused to different portions of the membrane protein (2). In general, the number of fusions that need to be made and analyzed for a complete topology determination is equal to or larger than the number of transmembrane helices in the protein, thus requiring a significant experimental effort.In the absence of experimental information, one can use various topology prediction methods to gain an idea of a protein's topology. The best current methods predict the correct topology with a success rate of 65-70% (3, 4) and thus provide a reasonable guide to minimizing the number of fusion proteins that have to be made for a given membrane protein (5). Recently, we have shown that the reliability of a given topology prediction can be estimated by comparing the predictions from a number of different prediction programs (6): when all methods agree, the topology is virtually certain to be correct, whereas the fraction of correct predictions drops with increasing levels of disagreement between the different methods.Here, we suggest that the amount of experimental work needed to establish a topology should be inversely related to the reliability of the theoretical topology prediction, and we provide data that allows the topology for 12 E. coli inner-membrane proteins to be deduced from a combination of topology predictions and single C-terminal reporter-protein fusions. Given that there are only Ϸ60 experimentally determined topologies for E. coli inner-membrane proteins available in the literature (6, 7), our 12 additional topologies represent a substantial increase in topology information. From topology predictions for the whole complement of E. coli inner-membrane proteins (Ϸ800 proteins), we estimate that the topology for an additional Ϸ75 proteins can be rapidly mapped by using our approach. (10) DNA Techniques. All plasmid constructs were confirmed by DNA sequencing using T7 DNA polymerase. The genes encoding the E. coli MarC, PstA, TatC, YaeL, YcbM, YddQ, YdgE, YedZ, YgjV, YiaB, YigG, and YnfA proteins were amplified from E. coli JM109 by using Taq polymerase. The genes were cloned by using primer-introduced sites 5Ј XhoI and...

The human cannabinoid receptor 1 (CB1) belongs to the G protein-coupled receptor (GPCR) family. Among the members of GPCR family, it has an exceptionally long extracellular Nterminal domain (N-tail) of 116 amino acids but has no typical signal sequence. This poses questions of how the long N-tail affects the biosynthesis of the receptor and of how it is inserted into the endoplasmic reticulum (ER) membrane. Here we have examined the process of membrane assembly of CB1 in the ER membrane and the maturation of the receptor from the ER to the plasma membrane. We find that the long N-tail cannot be efficiently translocated across the ER membrane, causing the rapid degradation of CB1 by proteasomes; this leads to a low level of expression of the receptor at the plasma membrane. The addition of a signal peptide at the N terminus of CB1 or shortening of the long N-tail greatly enhances the stability and cell surface expression of the receptor without affecting receptor binding to a cannabinoid ligand, CP-55,940. We propose that the N-tail translocation is a crucial early step in biosynthesis of the receptor and may play a role in regulating the stability and surface expression of CB1.