The role of Rab5a GTPase in endocytosis and post-endocytic trafficking of the hCG-human luteinizing hormone receptor complex

This study examined the role of Rab5a GTPase in regulating hCG-induced internalization and trafficking of the hCG-LH receptor complex in transfected 293T cells. Coexpression of wild-type Rab5a (WT) or constitutively active Rab5a (Q79L) with LHR significantly increased hCG-induced LHR internalization. Conversely, coexpression of dominant negative Rab5a (S34N) with LHR reduced internalization. Confocal microscopy showed LHR colocalizing with Rab5a (WT) and Rab5a (Q79L) in punctuate structures. Coexpression of Rab5a (WT) and Rab5a (Q79L) with LHR significantly increased colocalization of LHR in early endosomes. Conversely, dominant negative Rab5a (S34N) decreased this colocalization. While Rab5a stimulated internalization of LHR, it significantly decreased LHR recycling to the cell surface and increased degradation. Dominant negative Rab5a (S34N) increased LHR recycling and decreased degradation. These results suggest that Rab5a plays a role in LHR trafficking by facilitating internalization and fusion to early endosomes, increasing the degradation of internalized receptor resulting in a reduction in LHR recycling.     

Moving towards a paradigm: common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes

Chemotaxis, or directed migration of cells along a chemical gradient, is a highly coordinated process that involves gradient sensing, motility, and polarity. Most of our understanding of chemotaxis comes from studies of cells undergoing amoeboid-type migration, in particular the social amoeba Dictyostelium discoideum and leukocytes. In these amoeboid cells the molecular events leading to directed migration can be conceptually divided into four interacting networks: receptor/G protein, signal transduction, cytoskeleton, and polarity. The signal transduction network occupies a central position in this scheme as it receives direct input from the receptor/G protein network, as well as feedback from the cytoskeletal and polarity networks. Multiple overlapping modules within the signal transduction network transmit the signals to the actin cytoskeleton network leading to biased pseudopod protrusion in the direction of the gradient. The overall architecture of the networks, as well as the individual signaling modules, is remarkably conserved between Dictyostelium and mammalian leukocytes, and the similarities and differences between the two systems are the subject of this review.     

Molecular basis for chemoprevention by sulforaphane: a comprehensive review

The consumption of cruciferous vegetables has long been associated with a reduced risk in the occurrence of cancer at various sites, including the prostate, lung, breast and colon. This protective effect is attributed to isothiocyanates present in these vegetables, and sulforaphane (SF), present in broccoli, is by far the most extensively studied to uncover the mechanisms behind this chemoprotection. The major mechanism by which SF protects cells was traditionally thought to be through Nrf2-mediated induction of phase 2 detoxification enzymes that elevate cell defense against oxidative damage and promote the removal of carcinogens. However, it is becoming clear that there are multiple mechanisms activated in response to SF, including suppression of cytochrome P450 enzymes, induction of apoptotic pathways, suppression of cell cycle progression, inhibition of angiogenesis and anti-inflammatory activity. Moreover, these mechanisms seem to have some degree of interaction to synergistically afford chemoprevention. Received: 10 November 2006; received after revision 15 January 2007; accepted 5 February 2007     

Molecular basis of osteoarthritis: biomechanical aspects

The unique biomechanical properties of healthy cartilage ensure that articular cartilage is able to transmit force between the joints while maintaining almost friction-free limb movement. In osteoarthritis, the biomechanical properties are compromised, but we still do not understood whether this precedes the onset of the disease or is a result of it. This review focuses on the physical changes to cartilage with age, disease, and mechanical loading, with specific reference to the increased collagen cross-linking that occurs with age (nonenzymatic glycation), and the response of chondrocytes to physiological and pathological loads. In addition, the biomechanical properties and matrix biosynthesis of cartilage from various joint surfaces of the knee and ankle are compared to elucidate reasons why the ankle is less affected by progressive osteoarthritis than the knee.     

Endothelial signaling and the molecular basis of arteriovenous malformation

Arteriovenous malformations occur when abnormalities of vascular patterning result in the flow of blood from arteries to veins without an intervening capillary bed. Recent work has revealed the importance of the Notch and TGF-β signaling pathways in vascular patterning. Specifically, Notch signaling has an increasingly apparent role in arterial specification and suppression of branching, whereas TGF-β is implicated in vascular smooth muscle development and remodeling under angiogenic stimuli. These physiologic roles, consequently, have implicated both pathways in the pathogenesis of arteriovenous malformation. In this review, we summarize the studies of endothelial signaling that contribute to arteriovenous malformation and the roles of genes implicated in their pathogenesis. We further discuss how endothelial signaling may contribute to vascular smooth muscle development and how knowledge of signaling pathways may provide us targets for medical therapy in these vascular lesions.     

Something old,something new and something borrowed: emerging paradigm of insulin-like growth factor type 1 receptor (IGF-1R) signaling regulation

The insulin-like growth factor type 1 receptor (IGF-1R) plays a key role in the development and progression of cancer; however, therapeutics targeting it have had disappointing results in the clinic. As a receptor tyrosine kinase (RTK), IGF-1R is traditionally described as an ON/OFF system, with ligand stabilizing the ON state and exclusive kinase-dependent signaling activation. Newly added to the traditional model, ubiquitin-mediated receptor downregulation and degradation was originally described as a response to ligand/receptor interaction and thus inseparable from kinase signaling activation. Yet, the classical model has proven over-simplified and insufficient to explain experimental evidence accumulated over the last decade, including kinase-independent signaling, unbalanced signaling, or dissociation between signaling and receptor downregulation. Based on the recent findings that IGF-1R “borrows” components of G-protein coupled receptor (GPCR) signaling, including β-arrestins and G-protein-related kinases, we discuss the emerging paradigm for the IGF-1R as a functional RTK/GPCR hybrid, which integrates the kinase signaling with the IGF-1R canonical GPCR characteristics. The contradictions to the classical IGF-1R signaling concept as well as the design of anti-IGF-1R therapeutics treatment are considered in the light of this paradigm shift and we advocate recognition of IGF-1R as a valid target for cancer treatment.     

The deiodinase family: selenoenzymes regulating thyroid hormone availability and action

Thyroid hormones control growth, development, differentiation and metabolism in vertebrates. Most of the actions of the active thyroid hormone T3 (3,5,3'-triiodo-L-thyronine) are exerted via ligand-activated nuclear T3 receptors. Activation of the secretory product of the thyroid gland, L-thyroxine (3,3',5,5'-tetraiodo-L-thyronine), or T4, is catalyzed by two enzymes, iodothyronine-5'-deiodinases type I and type II. Inactivation of T4 and T3 occurs via type III iodothyronine-5-deiodinase and to some extent by type I 5'deiodinase. Complementary DNAs (cDNAs) encoding the substrate-binding selenocysteine-containing subunits of the deiodinases were cloned, though some controversy still exists on the type II 5'-deiodinase subunits. Characterization of tissue-specific expression patterns indicates that these selenium-dependent enzymes exert tight control on local and systemic availability of active T3. Thus, deiodinases are envisaged as guardians to the gate of thyroid hormone action mediated by T3 receptors.     

Possible mechanisms and function of nuclear trafficking of the colony-stimulating factor-1 receptor

Receptor tyrosine kinases (RTK) have long being studied with respect to the “canonical” signaling. This includes ligand-induced activation of a receptor tyrosine kinase at the cell surface that leads to receptor dimerization, followed by its phosphorylation in the intracellular domain and activation. The activated receptor then recruits cytoplasmic signaling molecules including other kinases. Activation of the downstream signaling cascade frequently leads to changes in gene expression following nuclear translocation of downstream targets. However, RTK themselves may localize within the nucleus, as either full-length molecules or cleaved fragments, with or without their ligands. Significant differences in this mechanism have been reported depending on the individual RTK, cellular context or disease. Accumulating evidences indicate that the colony-stimulating factor-1 receptor (CSF-1R) may localize within the nucleus. To date, however, little is known about the mechanism of CSF-1R nuclear shuttling, as well as the functional role of nuclear CSF-1R.     

A structural view of nuclear hormone receptor: endocrine disruptor interactions

Endocrine-disrupting chemicals (EDCs) represent a broad class of exogenous substances that cause adverse effects in the endocrine system by interfering with hormone biosynthesis, metabolism, or action. The molecular mechanisms of EDCs involve different pathways including interactions with nuclear hormone receptors (NHRs) which are primary targets of a large variety of environmental contaminants. Here, based on the crystal structures currently available in the Protein Data Bank, we review recent studies showing the many ways in which EDCs interact with NHRs and impact their signaling pathways. Like the estrogenic chemical diethylstilbestrol, some EDCs mimic the natural hormones through conserved protein–ligand contacts, while others, such as organotins, employ radically different binding mechanisms. Such structure-based knowledge, in addition to providing a better understanding of EDC activities, can be used to predict the endocrine-disrupting potential of environmental pollutants and may have applications in drug discovery.     

The role of parathyroid hormone and calcitonin in magnesium absorption in the rat small intestine

Summary We studied duodenal and ileal magnesium (Mg) absorption in intact, parathyroidectomized (PTX), thyroid-(TX) and thyroparathyroidectomized (TPTX) rats with iodine hormones replaced, and, additionally, in PTX rats receiving bovine parathyroid hormone 1–34 and 1,25-dihydroxyvitamin D₃, respectively. Ma absorption was reduced after PTX and TPTX in the duodenum, but not in the ileum, whereas TX had no influence on duodenal or ileal Mg absorption. Both bovine parathyroid hormone 1–34 and 1,25-dihydroxyvitamin D₃ increased Mg absorption in the duodenum and the ileum in PTX rats.Acknowledgments. We are grateful to B. Schreiber, K. Schwille and I. Goldberg for technical and secretarial help. Supported by Deutsche Forschungsgemeinschaft (Schw 210/3). Reprint requests to P.O.S., University Hospital, Maximiliansplatz, D-8520 Erlangen.     

Molecular basis of localized responses during chemotaxis in amoebae and leukocytes

Free-living amoebae as well as mammalian leukocytes sense chemoattractants with seven helix receptors linked to G-proteins. The cells respond by extending pseudopods and moving in the direction of the highest concentration. Recent studies using GFP-tagged proteins in Dictyostelium have shown that the directional response becomes sharply localized downstream of the receptors and G-proteins but upstream of the actin cytoskeleton. These studies together with the isolation novel genes by insertional mutagenesis in Dictyostelium are leading to a new understanding of chemotaxis in eucaryotic cells.     

Extended and bent conformations of the mannose receptor family

In mammals, the mannose receptor family consists of four members, Endo180, DEC-205, phospholipase A₂ receptor and the mannose receptor. The extracellular domains of all these receptors contain a similar arrangement of domains in which an Nterminal cysteine-rich domain is followed by a single fibronectin type II domain and eight or ten C-type lectin-like domains. This review focuses on the threedimensional structure of the receptors in the mannose receptor family and its functional implication. Recent research has revealed that several members of this family can exist in at least two configurations: an extended conformation with the N-terminal cysteinerich domain pointing outwards from the cell membrane and a bent conformation where the N-terminal domains fold back to interact with C-type lectin-like domains at the middle of the structure. Conformational transitions between these two states seem to regulate the interaction of these receptors with ligands and their oligomerization. Received 25 October 2007; received after revision 23 November 2007; accepted 7 December 2007     

The role of glycosylation in ionotropic glutamate receptor ligand binding, function, and trafficking

Members of the ionotropic glutamate receptor (iGluR) family have between 4 and 12 consensus asparagine (N)-linked glycosylation sites. They are localized on the extracellular N-termini, and the loop between the penultimate and last transmembrane domains. These regions also contain the essential elements for formation of the ligand binding site. N-linked glycosylation does not appear to be essential for formation of the ligand binding site per se, but there are demonstrated interactions between glycosylation state and ligand binding affinity, receptor physiology, susceptibility to allosteric modulation and, in some cases, trafficking. There is no indication of a general role for N-linked glycosylation in iGluRs; instead the effects of glycosylation vary among glutamate receptor subtypes and splice variants, with specific effects on structure or function with different subunits.     

LDL receptor relatives at the crossroad of endocytosis and signaling

For many years, the low-density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP) have been considered to be prototypes of cargo receptors which deliver, via endocytosis, macromolecules into cells. However, the recent identification of additional members of this gene family and examination of their biology has revealed that at least some of these proteins are also signaling receptors. Very low density lipoprotein receptor and ApoER2 transmit the extracellular reelin signal into migrating neurons, and thus are key components of the reelin pathway which governs neuronal layering of the forebrain during embryonic brain development. LRP5 and LRP6 are integral components of the Wnt signaling pathway which is central to many processes of metazoan development, cell proliferation, and tumor formation. Adaptor proteins interacting with the cytosolic domains of these receptors might orchestrate their ability to deliver their cargo or a signal.     

Effects of parathyroid hormone and calcitonin on carbonic anhydrase location in osteoclasts of cultured embryonic chick bone

Summary The ultrastructural location of carbonic anhydrase has been examined in osteoclasts of cultured embryonic chick metatarsi. In untreated cultures and in those to which parathyroid hormone (PTH) was added, the enzyme was found in close association with the plasma membrane of the ruffled border. After brief calcitonin treatment (10 min) the ruffled border disappeared and the association of carbonic anhydrase with the plasma membrane was diminished. The results indicate that the hormones employed act directly on embryonic bone to alter osteoclast structure and the intracellular location of carbonic anhydrase.Acknowledgments. This work was supported by NIH grants DE04345 and AM20374. Dr Cao was a visiting scientist from the Department of Biology, East China Normal University, Shanghai, The Peoples Republic of China. We thank Kathryn Majarwitz for assisting with the morphometric analysis. The PTH was provided by the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases of the National Institutes of Health, US Department of Health and Human Services.     

Pharmacological versus binding analysis of receptor systems: how do they interplay? Myometrial cell receptors for oxytocin as a paradigm.

Binding studies in various biological systems frequently indicate the presence of several binding sites for a biologically active ligand. They differ in their affinity for the ligand in question, binding capacity, and Hill coefficient, which suggests differences in the mechanisms of the binding site-ligand interactions. Identification of the 'true' receptors (sites initiating a cellular response) appears to be difficult. Three clusters of binding sites for oxytocin were found on rat myometrial cells. The oxytocin receptor seems to be linked to the medium-affinity site; the cooperation between the high- and medium-affinity sites in eliciting the uterotonic response seems likely, but lacks experimental proof. Dose-response analysis in partially irreversibly inhibited uterus preparations, the method of equipotent doses (Furchgott-Bursztyn method), and structure-activity analysis of oxytocin-like peptides acting as competitive inhibitors of oxytocin, turned out to be suitable for pharmacological analysis of this receptor system.