Morphologic polarity is necessary for chemotaxis of mammalian cells. As a probe of intracellular signals responsible for this asymmetry, the pleckstrin homology domain of the AKT protein kinase (or protein kinase B), tagged with the green fluorescent protein (PHAKT-GFP), was expressed in neutrophils. Upon exposure of cells to chemoattractant, PHAKT-GFP is recruited selectively to membrane at the cell's leading edge, indicating an internal signaling gradient that is much steeper than that of the chemoattractant. Translocation of PHAKT-GFP is inhibited by toxin-B from Clostridium difficile, indicating that it requires activity of one or more Rho guanosine triphosphatases.

In gradients of external chemo-attractant, mammalian neutrophilic leukocytes (neutrophils) and Dictyostelium discoideum amoebae adopt a polarized morphology and selectively accumulate lipid products of phosphatidylinositol-3-OH kinases (PI(3)Ks), including PtdIns(3,4,5)P(3), at their up-gradient edges; the internal PtdIns(3,4,5)P(3) gradient substantially exceeds that of the external attractant. An accompanying report presents evidence for a positive feedback loop that amplifies the gradient of internal signal: PtdIns(3,4,5)P(3) at the leading edge stimulates its own accumulation by inducing activation of one or more Rho GTPases (Rac, Cdc42, and/or Rho), which in turn increase PtdIns(3,4,5)P(3) accumulation. Here we show that interruption of this feedback by treatment with PI(3)K inhibitors reduces the size and stability of pseudopods and causes cells to migrate in jerky trajectories that deviate more from the up-gradient direction than do those of controls. Moreover, amplification of the internal PtdIns(3,4,5)P(3) gradient is markedly impaired by latrunculin or jasplakinolide, toxins that inhibit polymerization or depolymerization of actin, respectively. Thus reciprocal interplay between PtdIns(3,4,5)P(3) and polymerized actin initiates and maintains the asymmetry of intracellular signals responsible for cell polarity and directed motility.

Morphologic polarity is necessary for the motility of mammalian cells. In leukocytes responding to a chemoattractant, this polarity is regulated by activities of small Rho guanosine triphosphatases (Rho GTPases) and the phosphoinositide 3-kinases (PI3Ks). Moreover, in neutrophils, lipid products of PI3Ks appear to regulate activation of Rho GTPases, are required for cell motility and accumulate asymmetrically to the plasma membrane at the leading edge of polarized cells. By spatially regulating Rho GTPases and organizing the leading edge of the cell, PI3Ks and their lipid products could play pivotal roles not only in establishing leukocyte polarity but also as compass molecules that tell the cell where to crawl.

Persistent directional movement of neutrophils in shallow chemotactic gradients raises the possibility that cells can increase their sensitivity to the chemotactic signal at the front, relative to the back. Redistribution of chemoattractant receptors to the anterior pole of a polarized neutrophil could impose asymmetric sensitivity by increasing the relative strength of detected signals at the cell's leading edge. Previous experiments have produced contradictory observations with respect to receptor location in moving neutrophils. To visualize a chemoattractant receptor directly during chemotaxis, we expressed a green fluorescent protein (GFP)-tagged receptor for a complement component, C5a, in a leukemia cell line, PLB-985. Differentiated PLB-985 cells, like neutrophils, adhere, spread, and polarize in response to a uniform concentration of chemoattractant, and orient and crawl toward a micropipette containing chemoattractant. Recorded in living cells, fluorescence of the tagged receptor, C5aR-GFP, shows no apparent increase anywhere on the plasma membrane of polarized and moving cells, even at the leading edge. During chemotaxis, however, some cells do exhibit increased amounts of highly folded plasma membrane at the leading edge, as detected by a fluorescent probe for membrane lipids; this is accompanied by an apparent increase of C5aR-GFP fluorescence, which is directly proportional to the accumulation of plasma membrane. Thus neutrophils do not actively concentrate chemoattractant receptors at the leading edge during chemotaxis, although asymmetrical distribution of membrane may enrich receptor number, relative to adjacent cytoplasmic volume, at the anterior pole of some polarized cells. This enrichment could help to maintain persistent migration in a shallow gradient of chemoattractant.

Positive allosteric modulators of the human sweet taste receptor have been developed as a new way of reducing dietary sugar intake. Besides their potential health benefit, the sweet taste enhancers are also valuable tool molecules to study the general mechanism of positive allosteric modulations of T1R taste receptors. Using chimeric receptors, mutagenesis, and molecular modeling, we reveal how these sweet enhancers work at the molecular level. Our data argue that the sweet enhancers follow a similar mechanism as the natural umami taste enhancer molecules. Whereas the sweeteners bind to the hinge region and induce the closure of the Venus flytrap domain of T1R2, the enhancers bind close to the opening and further stabilize the closed and active conformation of the receptor.positive allosteric modulators | sweet taste receptor | T1R H umans can detect at least five basic taste qualities, including sweet, umami, bitter, salty, and sour. The sweet and umami taste are mediated by closely related G protein-coupled receptors (GPCRs). The three members of the T1R family form two heteromeric taste receptors: umami (T1R1/T1R3) (1, 2) and sweet (T1R2/T1R3) (1, 3). T1R receptors belong to the class C GPCRs, along with metabotropic glutamate receptors (mGluRs), γ-aminobutyric acid receptor B (GABA B R), calcium sensing receptors (CaSR), and others. The defining motif in these receptors is an extracellular Venus flytrap (VFT) domain (4), which consists of two globular subdomains connected by a threestranded flexible hinge. The VFT domain contains the orthosteric ligand binding site. The crystal structures of mGluR VFT domains (5, 6) revealed that the bilobed architecture can form an "open" or "closed" conformation. Glutamate binding stabilizes both the "closed" and the "active" dimer conformation. This scheme in the initial receptor activation has been applied generally to other class C GPCRs.Over the years, researchers have been developing noncaloric sweeteners to reduce dietary sugar intake. Unfortunately, all existing noncaloric sweeteners are characterized by their off taste (7,8) and fail to mimic the real sugar taste. Since the identification of the sweet taste receptor, a new approach became available, which is to develop positive allosteric modulators (PAMs) of the receptor. These molecules work as sweet taste "enhancers," which possess no taste of their own but potentiate the sweet taste of sugars. Examples of taste enhancers can be found in umami taste, which is known for its unique characteristic of synergism (9). Purinic ribonucleotides such as inosine-5′-monophosphate (IMP) and guanosine-5′-monophosphate (GMP) can strongly potentiate the umami taste intensity of glutamate and are rare examples of naturally occurring GPCR PAMs. In taste tests, 200 μM IMP, which does not elicit any umami taste by itself, can increase human umami taste sensitivity to glutamate by 15-fold (1). We recently illustrated the molecular mechanism of IMP/GMP (10). Our data indicate that glutamate binds close to the hinge region of the VFT ...