In mammals, 10 TLRs recognize conserved pathogen-associated molecular patterns, resulting in the induction of inflammatory innate immune responses. One of these, TLR9, is activated intracellularly by bacterial DNA and synthetic oligodeoxynucleotides (ODN), containing unmethylated CpG dinucleotides. Following treatment with CpG ODN, TLR9 is found in lysosome-associated membrane protein type 1-positive lysosomes, and we asked which intracellular compartment contains TLR9 before CpG exposure. Surprisingly, we found by microscopy and supporting biochemical evidence that both transfected and endogenously expressed human TLR9 is retained in the endoplasmic reticulum. By contrast, human TLR4 trafficked to the cell surface, indicating that endoplasmic reticulum retention is not a property common to all TLRs. Because TLR9 is observed in endocytic vesicles following exposure to CpG ODN, our data indicate that a special mechanism must exist for translocating TLR9 to the signaling compartments that contain the CpG DNA.

MD-2, a glycoprotein that is essential for the innate response to lipopolysaccharide (LPS), binds to both LPS and the extracellular domain of Toll-like receptor 4 (TLR4). Following synthesis, MD-2 is either secreted directly into the medium as a soluble, active protein, or binds directly to TLR4 in the endoplasmic reticulum before migrating to the cell surface. Here we investigate the function of the secreted form of MD-2. We show that secreted MD-2 irreversibly loses activity over a 24-h period at physiological temperature. LPS, but not lipid A, prevents this loss in activity by forming a stable complex with MD-2, in a CD14-dependent process. Once formed, the stable MD-2⅐LPS complex activates TLR4 in the absence of CD14 or free LPS indicating that the activating ligand of TLR4 is the MD-2⅐LPS complex. Finally we show that the MD-2⅐LPS complex, but not LPS alone, induces epithelial cells, which express TLR4 but not MD-2, to secrete interleukin-6 and interleukin-8. We propose that the soluble MD-2⅐LPS complex plays a crucial role in the LPS response by activating epithelial and other TLR4 ؉ /MD-2 ؊ cells in the inflammatory microenvironment. Lipopolysaccharide (LPS),1 a component of the outer membrane of Gram-negative bacteria, stimulates an exceptionally potent innate immune response in mammals that can result in septic shock and death (1). The LPS response is mediated by four proteins (2): LPS binding protein extracts single molecules from LPS micelles and transfers them to CD14, a glycosylphosphatidylinositol-anchored cell surface receptor that also exists as a serum protein. In turn, the CD14⅐LPS complex activates two proteins that comprise the essential signaling complex of the LPS response, Toll-like receptor 4 (TLR4) and MD-2 (3-6). TLR4 is a type I integral membrane glycoprotein and is one of 10 TLR paralogs that activate NF-B, mitogen-activated protein kinases, and other transducers of inflammatory signals in response to pathogen-specific structural motifs. MD-2, a small cysteine-rich glycoprotein, binds to the ectodomain of TLR4 (7) in the endoplasmic reticulum and then transits to the cell surface in an active TLR4⅐MD-2 complex. However, MD-2 is also secreted into the medium as a soluble, active protein (sMD-2) by primary cells such as immature dendritic cells (iDC), and by MD-2-transfected cell lines (8). The activity of sMD-2 was shown by its ability to bind to TLR4 and confer LPS responsiveness to cells that express TLR4 but lack MD-2 (8, 9). Forward genetic and gene deletion studies have demonstrated that both MD-2 and TLR4 are required for normal responsiveness to LPS in vitro and in vivo (3)(4)(5)10).Analyses of species specificity differences for various forms of LPS provide strong evidence that LPS interacts directly with the TLR4⅐MD-2 complex (11-16). However, the molecular events leading to LPS binding and TLR4 activation are only partially understood. Photoaffinity labeling (17) and binding (9,18,19) studies have shown that LPS binds directly to MD-2 and TLR4, and that binding of LPS to TLR4...

Luteinizing hormone receptor is a G protein-coupled receptor and consists of two halves: the N-terminal extracellular half (exodomain) and C-terminal membraneassociated half (endodomain). Hormone binds to the exodomain, and the resulting hormone-exodomain complex modulates the endodomain to generate signals. There are mutations that impair either hormone binding or signal generation. We report that the coexpression of a binding defective mutant and a signal-defective mutant rescues signal generation to produce cAMP. This rescue requires both types of mutant receptors and is dependent on the human chorionic gonadotropin dose, the surface concentration of mutant receptors, and the amino acid position of mutations. Furthermore, random collisions among mutant receptors are not involved in the rescue. Our observations provide new insights into the mechanisms of the functional and structural relationship of the exo-and endodomain, signal transduction, and receptor genetics, in particular for defective heterozygotes.

Previously, we reported that a liganded LH receptor (LHR) is capable of activating itself (cis-activation) and other nonliganded LHRs to induce cAMP (trans-activation). Trans-activation of the LHR raises two crucial questions. Is trans-activation unique to LHR or common to other G protein-coupled receptors? Does trans-activation stimulate phospholipase Cbeta as it does adenylyl cyclase? To address these questions, two types of novel FSH receptors (FSHRs) were constructed, one defective in hormone binding and the other defective in signal generation. The FSHR, a G protein-coupled receptor, comprises two major domains, the N-terminal extracellular exodomain that binds the hormone and the membrane-associated endodomain that generates the hormone signals. For signal defective receptors, the exodomain was attached to glycosyl phosphatidylinositol (ExoGPI) or the transmembrane domain of CD8 immune receptor (ExoCD). ExoGPI and ExoCD can trans-activate another nonliganded FSH. Surprisingly, the trans-activation generates a signal to activate either adenylyl cyclase or phospholipase Cbeta, but not both. These results indicate that trans-activation in these mutant receptors is selective and limited in signal generation, thus providing new approaches to investigating the generation of different hormone signals and a novel means to selectively generate a particular hormone signal. Our data also suggest that the FSHR's exodomain could not trans-activate LHR.

Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

G protein-coupled receptors (GPCRs) accommodate a wide spectrum of activators from ions to glycoprotein hormones. The mechanism of activation for this large and clinically important family of receptors is poorly understood. Although initially thought to function as monomers, there is a growing body of evidence that GPCR dimers form, and in some cases that these dimers are essential for signal transduction. Here we describe a novel mechanism of intermolecular GPCR activation, which we refer to as trans-activation, in the LH receptor, a GPCR that does not form stable dimers. The LH receptor consists of a 350-amino acid amino-terminal domain, which is responsible for high-affinity binding to human CG, followed by seven-transmembrane domains and connecting loops. This seven-transmembrane domain bundle transmits the signal from the extracellular amino terminus to intracellular G proteins and adenylyl cyclase. Here, we show that binding of hormone to one receptor can activate adenylyl cyclase through its transmembrane bundle, intramolecular activation (cis-activation), as well as trans-activation through the transmembrane bundle of an adjacent receptor, without forming a stable receptor dimer. Coexpression of a mutant receptor defective in hormone binding and another mutant defective in signal generation rescues hormone-activated cAMP production. Our observations provide new insights into the mechanism of receptor activation mechanisms and have implications for the treatment of inherited disorders of glycoprotein hormone receptors.

No abstract

An aromatic amino acid, Tyr or Trp, located in the esterase active site wall, is highly conserved, with hyperthermophilic esterases showing preference for Tyr and lower temperature esterases showing preference for Trp. In this study, we investigated the role of Tyr(182) in the active site wall of hyperthermophilic esterase EstE1. Mutation of Tyr to Phe or Ala had a moderate effect on EstE1 thermal stability. However, a small-to-large mutation such as Tyr to His or Trp had a devastating effect on thermal stability. All mutant EstE1 enzymes showed reduced catalytic rates and enhanced substrate affinities as compared with wild-type EstE1. Hydrogen bond formation involving Tyr(182) was unimportant for maintaining EstE1 thermal stability, as the EstE1 structure is already adapted to high temperatures via increased intramolecular interactions. However, removal of hydrogen bond from Tyr(182) significantly decreased EstE1 catalytic activity, suggesting its role in stabilization of the active site. These results suggest that Tyr is preferred over a similarly sized Phe residue or bulky His or Trp residue in the active site walls of hyperthermophilic esterases for stabilizing the active site and regulating catalytic activity at high temperatures.