Caveolin-enriched membrane signaling complexes in human and murine osteoblasts.

Osteoblasts receive regulatory signals from hormones, growth factors, calcium, extracellular matrix, and other cells through a variety of receptors that utilize an array of signaling pathways and cytoplasmic messengers. This article addresses the nonuniform distribution of important signaling molecules (platelet-derived growth factor receptors [PDGFRs], nonreceptor tyrosine kinases, tyrosine kinase adaptor proteins, G proteins, and nitric oxide synthases [NOSs]) in the surface membranes of human and murine osteoblasts. We show that particular inner leaflet signaling molecules (e.g., heterotrimeric G proteins and Src family tyrosine kinases) are clustered and concentrated in Triton X-100-insoluble membranes that are enriched in caveolin, the major structural component of caveolae (50- to 100-nm flask-shaped invaginations of the plasma membrane that apparently are organized by oligomers of the protein caveolin). In addition, we show that a subset of highly ligand-responsive PDGFRs and mitogen-activated protein (MAP) kinase pathway effectors are present in the caveolin-enriched membrane fraction of osteoblasts.     

Anesthetic effects on muscarinic signal transduction

A wealth of pharmacological and physiological evidence has established that anesthetics disrupt synaptic transmission at muscarinic and other synapses. The sequence of molecular events precipitated by agonist binding to the receptors is under intense scrutiny. It appears that at the majority of synapses G proteins serve to mediate the transfer of information from receptors to intracellular mechanisms. The major exception to this scheme is the situation in which an ion channel is incorporated directly in the receptor structure. Binding of an agonist to these receptors produces a conformational change in the receptors which opens an intrinsic ion channel. This situation occurs in nicotinic acetylcholine gamma-amino butyric acid type A (GABAA, and 5-hydroxytryptamine type 3 (5-HT3) receptors). Assays have been developed to evaluate several steps in the cascade of events involved in synaptic signal transduction, and these assays have been employed to determine the step at which anesthetics act to disrupt synaptic transmission. We have demonstrated that several volatile anesthetics alter the interaction of muscarinic receptors with transducer G proteins. Ligand-binding experiments suggest that receptor-G protein complexes are stabilized, thereby disrupting G protein GTPase activity and muscarinic control of cellular activity. This "stabilization" does not appear to involve an inhibition of guanine nucleotide binding, the proximal event in receptor-G protein dissociation. Two possibilities warrant further consideration: (1) that GDP release from inactive G protein trimers, which is normally catalyzed by the receptor, is inhibited, and (2) that receptor-G protein complexes fail to dissociate even in response to GTP binding. We are currently examining these possibilities using purified G proteins and receptors in reconstituted systems.     

Notch Signaling in Mammary Development and Oncogenesis

With the discovery of an activated Notch oncogene as a causative agent in mouse mammary tumor virus induced breast cancer in mice, the potential role for Notch signaling in normal and pathological mammary development was revealed. Subsequently, Notch receptors have been found to regulate normal development in many organ systems. In addition, inappropriate Notch signaling has been implicated in cancer of several tissues in humans and animal model systems. Here we review important features of the Notch system, and how it may regulate development and cancer in the mammary gland. A large body of literature from studies in Drosophila and C. elegans has not only revealed molecular details of how the Notch proteins signal to control biology, but shown that Notch receptor activation helps to define how other signaling pathways are interpreted. In many ways the Notch system is used to define the context in which other pathways function to control proliferation, differentiation, cell survival, branching morphogenesis, asymmetric cell division, and angiogenesis--all processes which are critical for normal development and function of the mammary gland.     

Molecular biology of adrenergic and dopamine receptors and the study of developmental nephrology

Neurotransmitters convey specific messages by binding to receptors on the cell membrane surface. Receptors are linked to membrane-bound, signal-transducing proteins which act as intermediaries in the generation of second messengers that elicit biological responses. Cell surface receptors could be grouped into families that utilize common systems for their signal transmission. These classes include the growth factor receptors, the transporter receptors which internalize their ligands, ion channels, and G-protein-coupled receptors. In the past few years, the cDNAs and/or genes of a number of G-protein-coupled receptors have been cloned. Structural analysis of the G-protein-coupled receptors, as well as the other classes of receptor, shows that those receptors which use a common signaling pathway have similar topographies and share significant sequence homology. Adrenergic and dopamine receptors are examples of receptors coupled to G proteins. This review outlines some strategies in the study of adrenergic and dopamine receptors using molecular biology techniques and how they relate to investigations in developmental nephrology.     

Aberrant G protein signaling in nervous system tumors

OBJECT: Guanosine triphosphate (GTP)-binding proteins, also known as G proteins, play important roles in the regulation of cell growth and differentiation by transmitting intracellular signals from cell surface receptors. In this paper, the authors review G protein signaling in general and its aberrations in four human nervous system tumors. METHODS: In the nervous system, four tumor types have been associated with aberrant G protein signaling. The first tumor type includes astrocytomas, which have increased levels of the activated form of the small G protein, p21-ras, without primary oncogenic p21-ras mutations. The likely source for increased p21-ras activity in sporadically occurring astrocytomas is overexpressed or constitutively activated growth factor receptors, whereas in neurofibromatosis Type 1 (NF1)-associated astrocytomas, the source is a loss of expression of neurofibromin, a major inactivator of p21-ras (ras-GTPase activating protein [GAP]). The second type of tumor associated with aberrant G protein signaling includes sporadic and NF1-associated neurofibromas and malignant peripheral nerve sheath tumors, which also have increased p21-ras activity due to a loss of neurofibromin expression. The third tumor type includes subependymal giant cell astrocytomas as part of the tuberous sclerosis complex (TSC). These tumors display a loss of tuberin expression due to germline mutations in the TSC2 gene. Tuberin functions as an inactivator of the small G protein rap1B (rap1-GAP) and, hence, loss of its expression could lead to increased rap1B activity. In addition to TSC-associated tumors, the authors demonstrate that the majority of sporadically occurring astrocytomas display either loss of tuberin or overexpression of rap1B. This suggests that increased rap1B activity, which can augment p21-ras-mediated signals, also contributes to G protein-mediated aberrant signaling in sporadically occurring astrocytomas. The fourth tumor type includes a significant subset of pituitary adenomas that show constitutive activation of the G alpha subunit of the large heterotrimeric G s protein, which is involved in hormone receptor signaling. The net result of this aberrant activation is increased cyclic adenosine monophosphate and mitogenic tumor-promoting signals. CONCLUSIONS: The authors' review of G protein signaling and aberrations in this process is made with the long-term view that increased understanding of relevant signaling pathways will eventually lead to novel biological targeted therapies against these tumors.     

Preconditioning, anesthetics, and perioperative medication

Insights gained from experimental analysis of cardioprotective signaling pathways has led to advances in understanding how perioperative outcomes may be modulated in high risk patients. Volatile anesthetics are clearly beneficial in ischemic and reperfused myocardium and their use improved outcome in cardiac surgical patients. Opioids that activate delta-1 opioid receptors may also be beneficial, although, the clinical evidence is less clear. K_ATP channel and COX-2 activity are critical components in cardioprotection and drugs that block the activity of these proteins, such as sulfonylureas and selective COX-2 inhibitors, should probably be avoided in patients at risk for myocardial ischemia. Aging and disease states, such as diabetes, also impair cardioprotection. Pharmacological therapies to restore IPC or APC during disease or in aged myocardium remain to be elucidated.     

Molecular mechanisms of opioid receptor-dependent signaling and behavior

Opioid receptors have been targeted for the treatment of pain and related disorders for thousands of years and remain the most widely used analgesics in the clinic. Mu (μ), kappa (κ), and delta (δ) opioid receptors represent the originally classified receptor subtypes, with opioid receptor like-1 (ORL1) being the least characterized. All four receptors are G-protein coupled and activate inhibitory G proteins. These receptors form homo- and heterodimeric complexes and signal to kinase cascades and scaffold a variety of proteins.The authors discuss classic mechanisms and developments in understanding opioid tolerance and opioid receptor signaling and highlight advances in opioid molecular pharmacology, behavioral pharmacology, and human genetics. The authors put into context how opioid receptor signaling leads to the modulation of behavior with the potential for therapeutic intervention. Finally, the authors conclude there is a continued need for more translational work on opioid receptors in vivo.     

Fibroblast growth factors and their receptors in urological cancers: basic research and clinical implications

Because therapeutical options for advanced urological cancers are limited, the understanding of key elements responsible for invasion and metastasis is very important. It has been hypothesized that progression to malignant growth is associated with a dysregulation of growth factors and/or their receptors. In the last few years, signaling pathways of the fibroblast growth factor (FGF) family have been subject to intense investigation. Fibroblast growth factors constitute one of the largest families of growth and differentiation factors for cells of mesodermal and neuroectodermal origin. The family comprises two prototypic members, acidic FGF (aFGF) and the basic FGF (bFGF), as well as 21 additionally related polypeptide growth factors that have been identified to date. FGFs are involved in many biological processes during embryonic development, wound healing, hematopoesis, and angiogenesis. In prostate, bladder, and renal cancers, FGFs regulate the induction of metalloproteinases (MMP) that degrade extracellular matrix proteins, thus facilitating tumor metastasis. Probably due to their potent angiogenic properties, aFGF and bFGF have received the most attention. However, there is increasing evidence that other FGFs also play crucial roles in tumors of the prostate, bladder, kidney, and testis. This review will discuss the different elements involved in FGF signaling and summarize the present knowledge of their biological and clinical relevance in urological cancers.     

Opioids in anesthesia

Opioids are the most effective and widely used drugs in the treatment of severe acute and chronic pain. They act through opioid receptors that belong to the family of G protein-coupled receptors. Three classes of opioid receptors (mu, delta, kappa), expressed in the central and peripheral nervous system, have been identified. The analgesic effect of opioids is mediated through multiple pathways of opioid receptor signaling (e.g., G(i/o) coupling, cAMP inhibition, Ca(++) channel inhibition). The standard exogenous opioid analgesics used in the operating room are fentanyl, sufentanil, morphine, alfentanil, and remifentanil. Preclinical pharmacology, clinical applications, and side effects will be reviewed in this chapter.     

Molecular mechanism of morphine tolerance and biological approaches to resolve tolerance

One of the major problems associated with the chronic use of morphine is tolerance. Repeated uses of morphine to relieve pain often cause patients to develop increasing resistance to the effects of the drugs, so that progressively higher doses are required to achieve the same analgesic effects. Acquired tolerance is thought to be different from dependence or addiction, but molecular mechanism underlying the development of tolerance is still unclear. Tolerance has been explained by desensitization of opioid receptor signaling and loss of functional receptors in the cell surface. The classical hypothesis was that phosphorylation and arrestin binding resulted in uncoupling of the receptor from G proteins, and reduced agonist efficacy. The receptor internalization would then result in fewer functional receptors at the cell surface. These events would cause so-called signaling desensitization. However, recent molecular biological studies have led researchers to revise the classical view of tolerance from observations that morphine does not always promote efficient receptor internalization. Among several key processes, the sequestration and subsequent internalization of the opioid receptor may play an important role for morphine tolerance. In fact, recent studies have suggested that receptor internalization can reduce tolerance. In addition, activation of the NMDA subtype of the glutamate receptor has been suggested as an anti-opioid system in the development of morphine tolerance. In this review, we focus on recent research progress on the morphine tolerance, and molecular biological and clinical approaches to resolve morphine tolerance.     

Wnt signaling and orthopedics, an overview

Wnt signaling is a ubiquitous system for intercellular communication, with multiple functions during development and in homeostasis of the body. It comprises several ligands, receptors, and inhibitors. Some molecules, such as sclerostin, appear to have bone-specific functions, and can be targeted by potential drugs. Now, ongoing clinical trials are testing these drugs as treatments for osteoporosis. Animal studies have also suggested that these drugs can accelerate fracture healing and implant fixation. This brief overview focuses on currently available information on the effects of manipulations of Wnt signaling on bone healing.     

Rezeptoren und Signalproteine des angeborenen Immunsystems als neue Zielstrukturen der Sepsistherapie

During polymicrobial sepsis,microbial pathogens and their products activate the innate immune system through signaling receptors of the Toll-like receptor (TLR) family, resulting in hyperinflammation and organ injury.The analysis of preclinical mouse models has shown that inactivation of the common TLR signaling adaptor protein MyD88 prevents the hyperinflammatory response and improves survival.Importantly, MyD88 deficiency does not impair antibacterial defense mechanisms.Thus,TLRs and proteins involved in TLR signaling may represent interesting targets for the development of new drugs for reprogramming pathophysiological immune responses during sepsis.     

Inhibition of nitric oxide synthase activity and nitric oxide-dependent calcium influx in renal epithelial cells by cyclic adenosine monophosphate: implications for cell injury.

Cell injury frequently occurs in the setting of tissue destruction and inflammation and is associated with a rise in intracellular calcium (Cai) and increased NO production. The mechanisms that trigger rises in Cai and NO during cell injury are not fully defined, but they may involve activation of G protein-coupled receptors for substances such as bradykinin, Ang II, thromboxane, and thrombin. These receptors act through G proteins from different families that have distinct functions. Receptors for bradykinin and Ang II act through members of the G alpha i and G alpha q families, whereas receptors for thrombin and thromboxane act through members of the G alpha i, G alpha q, and G alpha 12/13 families. These G proteins cooperate to regulate Cai and NO in epithelial cells through distinct mechanisms. In a number of experimental settings, activators of the adenylyl cyclase system reduce the severity of cell injury. To understand the mechanisms by which G protein-dependent signaling systems may contribute to cell injury and to define the role of adenylyl cyclase in ameliorating cell injury, the effects of adenylyl cyclase on bradykinin-stimulated Ca influx and NO in cultured renal epithelial cells that stably overexpress G alpha q and G alpha 13 were studied. This system allowed for the separation of different components of the signals initiated by receptors for thromboxane and thrombin. G alpha 13 increased bradykinin-stimulated Ca influx by a mechanism that depends on NO and cGMP. The increased Ca influx was blocked by inhibitors of NO synthase and guanylyl cyclase and by activation of adenylyl cyclase. NO production was inhibited by activators of cAMP-dependent protein kinase, which indicated that cAMP blocks Ca influx by inhibiting NO production. Expression of G alpha q, the G protein that regulates phospholipase C, also increased bradykinin-stimulated Ca influx, but by an NO, cGMP-independent mechanism that was insensitive to inhibition by adenylyl cyclase. The authors conclude that Ca influx is modulated by NO-dependent and independent mechanisms, and that to the extent that increased NO production contributes to increased Ca influx and cell injury, cell injury may be reduced by agents that activate adenylyl cyclase.     

Subcellular Compartmentalization of Cyclic Adenosine Monophosphate in Heart Failure and Inotropic Pharmacology

Cyclic adenosine monophosphate (cAMP) is a second messenger downstream of many G-protein coupled receptors, including the β1-adrenoceptor, which is the target of many clinically used inotropic agents. When the Gαs subunit of a heterotrimeric G-protein is activated, it causes a localized elevation of cAMP. The significance of the spatial distribution of the elevation in cAMP is increasingly recognized, as is the disturbance of these microdomains in diseased states. Herein, the spatial compartmentalization of inotropic signaling is explored, including from internalized receptors.     

Wnt signaling and orthopedics,an overview

Abstract

Wnt signaling is a ubiquitous system for intercellular communication, with multiple functions during development and in homeostasis of the body. It comprises several ligands, receptors, and inhibitors. Some molecules, such as sclerostin, appear to have bone-specific functions, and can be targeted by potential drugs. Now, ongoing clinical trials are testing these drugs as treatments for osteoporosis. Animal studies have also suggested that these drugs can accelerate fracture healing and implant fixation. This brief overview focuses on currently available information on the effects of manipulations of Wnt signaling on bone healing.     

Integrated physiology of proximal tubular organic anion transport

PURPOSE OF REVIEW: Renal organic anion transport proteins play important roles in the reabsorption and the secretion of endogenous and exogenous compounds. This review focuses on the interpretation of the physiological integration of identified transport molecules in the renal proximal tubules. RECENT FINDINGS: To date, molecular identification of organic anion transport proteins is still continuing: rodent organic anion transporter 5, organic anion-transporting polypeptide 4C1, voltage-driven organic anion transporter 1, multidrug resistance-associated protein 4, and sodium-coupled monocarboxylate transporter have yielded additional information in this field. In addition, particularly at the apical membrane of the proximal tubules, the importance of the PDZ (PSD-95, DglA, and ZO-1) binding domain proteins has emerged in the formation of the multimolecular complex as a functional unit of membrane transport. Finally, discovery of dicarboxylate receptors in the renal tubular cells raises the possibility that dicarboxylate anions function as intrarenal signaling molecules. This novel aspect of renal organic anion transport, the potential modulation of signaling via dicarboxylate receptors, may be of significant relevance to renovascular hypertension and other renal diseases. SUMMARY: Comprehensive understanding of the multimolecular complex, which is composed of transporters and their related signaling elements and is supported by the scaffold proteins underneath the plasma membrane, may be useful in clarifying complex transport phenomena such as renal apical organic anion handling. In addition to the recent proteomics approaches and conventional molecular physiology, it is necessary to develop novel methods to analyze the overall function of the multimolecular complex for the post-genomic era.     

G proteins and modulation of insulin secretion

Guanine nucleotide-binding proteins (G proteins) are critically important mediators of many signal-transduction systems. Several important sites regulating stimulus-secretion coupling and release of insulin from pancreatic beta-cells are modulated by G proteins. Gs mediates increases in intracellular cAMP associated with hormone-induced stimulation of insulin secretin. Gi mediates decreases in intracellular cAMP caused by inhibitors of insulin secretion, e.g., epinephrine, somatostatin, prostaglandin E2, and galanin. G proteins also regulate ion channels, phospholipases, and distal sites in exocytosis. Cholera and pertussis toxins irreversibly ADP ribosylate G proteins and are important tools that can be used both to manipulate G-protein-dependent modulators of insulin secretion and detect and quantify G proteins by electrophoretic techniques. The stage is set to pursue these initial observations in greater depth and ascertain whether G-protein research will provide important new insights into normal and abnormal regulation of insulin secretion.     

Mechanistic Aspects of Crosstalk Between GH and PRL and ErbB Receptor Family Signaling

Growth hormone (GH) and prolactin (PRL) are anterior pituitary hormones that have multiple roles in growth and metabolism. Both hormones are important in mammary development and breast cancer. The epidermal growth factor (EGF) family of peptides and the receptors that they activate (the ErbB family) are also major players in mammary biology and pathophysiology. Recent studies in signal transduction have highlighted the interplay between signaling pathways referred to as crosstalk. In this review, cell biological and signaling studies related to crosstalk between GH and PRL and the ErbB family are discussed. In particular, the role of GH- and PRL-induced phosphorylation of ErbB receptors in regulating EGF responsiveness is highlighted with attention to potential pathophysiological relevance.