Signal transmission by many cell surface receptors results in the activation of phosphoinositide (PI) 3-kinases that phosphorylate the 3' position of polyphosphoinositides. From a screen for mouse proteins that bind phosphoinositides, the protein GRP1was identified. GRP1 binds phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4, 5)P3] through a pleckstrin homology (PH) domain and displays a region of high sequence similarity to the yeast Sec7 protein. The PH domain of the closely related protein cytohesin-1, which, through its Sec7 homology domain, regulates integrin beta2 and catalyzes guanine nucleotide exchange of the small guanine nucleotide-binding protein ARF1, was also found to specifically bind PtdIns(3,4,5)P3. GRP1 and cytohesin-1 appear to connect receptor-activated PI 3-kinase signaling pathways with proteins that mediate biological responses such as cell adhesion and membrane trafficking.

adipocyte ͉ Cide ͉ diabetes ͉ fat droplet ͉ fat metabolism

Fat-specific protein (FSP)27/Cidec is most highly expressed in white and brown adipose tissues and increases in abundance by over 50-fold during adipogenesis. However, its function in adipocytes has remained elusive since its discovery over 15 years ago. Here we demonstrate that FSP27/Cidec localizes to lipid droplets in cultured adipocytes and functions to promote lipid accumulation. Ectopically expressed FSP27-GFP surrounds lipid droplets in 3T3-L1 adipocytes and colocalizes with the known lipid droplet protein perilipin. Immunostaining of endogenous FSP27 in 3T3-L1 adipocytes also confirmed its presence on lipid droplets. FSP27-GFP expression also markedly increases lipid droplet size and enhances accumulation of total neutral lipids in 3T3-L1 preadipocytes as well as other cell types such as COS cells. Conversely, RNA interference-based FSP27/Cidec depletion in mature adipocytes significantly stimulates lipolysis and reduces the size of lipid droplets. These data reveal FSP27/Cidec as a novel adipocyte lipid droplet protein that negatively regulates lipolysis and promotes triglyceride accumulation.Adipose tissue is a major determinant of whole body glucose homeostasis and insulin sensitivity, as evidenced by its ability to secrete bioactive peptides and control lipid storage (1-6). The nuclear receptor peroxisome proliferator-activated receptor-␥ promotes adipogenesis and enhances these functions, acting in mice and humans to increase insulin signaling and glucose tolerance (7-12). Several proteins that are highly and selectively expressed in adipocytes, such as the secreted proteins adiponectin and leptin as well as adipsin, are under the control of peroxisome proliferator-activated receptor-␥ (3-5, 13, 14). These and other adipocyte proteins are highly up-regulated during adipogenesis and confer unique characteristics to these cells, including high capacity to store triglyceride and release fatty acids.Fat-specific protein (FSP)27, also denoted as CIDEC for the human homolog (15), was discovered over 15 years ago to be strikingly up-regulated during adipogenesis and is highly expressed in both white and brown adipose tissues (15-18). However, the question of FSP27/Cidec function has remained unsolved. The results we present here demonstrate FSP27/Cidec to be a novel lipid droplet protein that shares many features characteristic of the lipid droplet protein perilipin (19,20), including the ability to enhance neutral lipid accumulation when expressed in 3T3-L1 preadipocytes or even COS cells. Our data also reveal that a mechanism whereby FSP27/Cidec functions to promote triglyceride deposition in adiocytes is by inhibiting lipolysis. FSP27 is thus a major new modulator of lipid droplet function that is required for optimal storage of triglycerides by adipocytes. EXPERIMENTAL PROCEDURESMaterials-C57BL/6J (male, 10 weeks old) mice were obtained from The Jackson Laboratory. Human insulin was obtained from Lilly. Fetal bovine serum was purchased from Atlanta Biologicals, Inc. (Lawrenceville, GA). Other reagents ...

Lipid second messengers generated by phosphoinositide (PI) 3-kinases regulate diverse cellular functions through interaction with pleckstrin homology (PH) domains in modular signaling proteins. The PH domain of Grp1, a PI 3-kinase-activated exchange factor for Arf GTPases, selectively binds phosphatidylinositol 3,4,5-trisphosphate with high affinity. We have determined the structure of the Grp1 PH domain in the unliganded form and bound to inositol 1,3,4,5-tetraphosphate. A novel mode of phosphoinositide recognition involving a 20-residue insertion within the beta6/beta7 loop explains the unusually high specificity of the Grp1 PH domain and the promiscuous 3-phosphoinositide binding typical of several PH domains including that of protein kinase B. When compared to other PH domains, general determinants of 3-phosphoinositide recognition and specificity can be deduced.

Transforming growth factor (TGF)β is an important physiological regulator of cellular growth and differentiation. It activates a receptor threonine/serine kinase that phosphorylates the transcription factor Smad2, which then translocates into the nucleus to trigger specific transcriptional events. Here we show that activated type I and II TGFβ receptors internalize into endosomes containing the early endosomal protein EEA1. The extent of TGFβ-stimulated Smad2 phosphorylation, Smad2 nuclear translocation, and TGFβ-stimulated transcription correlated closely with the extent of internalization of the receptor. TGFβ signaling also requires SARA (Smad anchor for receptor activation), a 135-kD polypeptide that contains a FYVE Zn++ finger motif. Here we show that SARA localizes to endosomes containing EEA1, and that disruption of this localization inhibits TGFβ-induced Smad2 nuclear translocation. These results indicate that traffic of the TGFβ receptor into the endosome enables TGFβ signaling, revealing a novel function for the endosome as a compartment specialized for the amplification of certain extracellular signals.