Cyclic Nucleotide Binding Domain

The cyclic nucleotide binding sites ( 110 amino acids each) in PKAs and PKGs are homologous to the cyclic nucleotide binding domain in the cyclic nucleotide gated cation channels and to that in an evolutionarily related bacterial cAMP-binding protein, catabolite gene activating protein (CAP) (Fig. 4). As such, more is known about these cyclic nucleotide-binding sites than about other unrelated families of cyclic nucleotide-binding sites such as those occurring on cyclic nucleotide...

Effects of Ca2

AC-V and -VI are subject to direct inhibition by micromolar concentrations of Ca2+. In cardiac membranes this inhibition reaches a maximum of 50 of the starting activity. In the intact cardiac myocyte, inhibition of AC-V and -VI by Ca2+ entering through voltage-dependent Ca2+ channels contributes to the initiation of the sequels that reverse sympathetic stimulation. Types I, III, and VIII are enzymes stimulated by increases in cytosolic Ca2+ acting via CaM. Ca-CaM stimulation of type I and III...

Calmodulin Binding to Target Proteins

Calmodulin binds to and regulates a remarkable array of cellular proteins. Some of these targets require Ca2+ binding to calmodulin whereas others bind calmodulin independently of Ca2+. More than 30 molecular targets for Ca2+-calmodulin have been identified, many of which are proteins involved in signal transduction pathways. These molecules include protein kinases and phosphatases, Ca2+-pump-ing ATPases, adenylyl cyclases, cyclic nucleotide phosphodiesterases, proteases, nitric oxide...

Pathophysiology Of Protein Phosphatases

Given the partnership between protein kinases and phosphatases to regulate cell physiology, there are a growing number of situations when deregulation of protein phosphatases leads to disease. For instance, a chromosomal translocation in many acute undiffer-entiated leukemias results in the fusion of I2PP2A, also known as SET, with a nucleoporin. This may deregulate the nuclear pool of PP2A to induce leukemia. Yet other data show that I2PP2A is overexpressed in Wilm's tumor. The chronic...

Pdes As Targets For Pharmacological Intervention

Inhibition of PDE is one of the most ancient pharmacological treatments used by mankind. It is believed that stone-age man discovered the stimulating properties of extracts of leaves and seeds from Thea sinensis, Coffea arabica, and Theobroma cacao, and mention of the stimulating properties of these extracts is found in records of the most ancient civilizations. The active principles in these extracts are alkaloids called xanthines, the most potent being the-ophylline and caffeine. The effect...

PDE Inhibitors and Cardiovascular Disorders

By virtue of their inotropic and vasorelaxant effects, PDE3 inhibitors have been widely tested for cardiovascular disorders (Beavo, 1995). They are currently used in acute settings to treat patients during cardiac surgery or for patients awaiting heart transplantation. They have been also tested as a possible replacement of, or in conjunction with, digoxin in congestive heart failure. Although a large multicentric trial with milrinone was stopped prematurely because of increased death rate in...

IRKassociated PTPs A Key Therapeutic Target

The recognition that the in vivo administration of pVs to rats induced hypoglycemia led to studies in diabetic animals. It was shown that in insulinoprivic BB Wistar diabetic rats the parenteral administration of pV but not vanadate returned blood glucose levels to normal and fully abrogated the associated ketoacidosis (Fig. 8) (Yale et al., 1995). This established the concept that inhibition of the IRK-associated PTP Fig. 8. Effect of bpV (phen) and vanadate on fasting glucose levels in...

Physiological Role of PLD

The physiological role of PLD is not as well defined as that of PLA2 or PLC. The physiologically significant product of PLD action is PA, as its cellular concentration is increased by agonist activation of the enzyme. In contrast, the cellular concentrations of choline and phosphorycholine are already high in resting cells. There are many reports of PA effects on enzymes and other proteins in vitro, but few demonstrations that these are affected in vivo. Reported targets of PA include NADPH...

Volume Regulation

When cells are exposed to a hypotonic environment they passively swell and then spontaneously shrink over a period of minutes to return to their normal volume. The return to normal volume, the regulatory volume decrease, is mediated through the activation of both K+ and Cl- channels. Movement of salt through the two conductance pathways obligates the osmotic flow of water out of the cell, causing it to shrink and eventually return to normal volume. Anion channels involved in this response may...

Ca2 Is Required For Termination Of Phototransduction

Deactivation of the phototransduction cascade, which is defined as the process of terminating the visual cascade following cessation of the light stimulus, is very rapid and occurs within 100 ms. A critical messenger of deactivation is Ca2+ and it appears to function at multiple steps in the phototransduction cascade. One of the mechanisms through which Ca2+ mediates rapid deactivation is through phosphorylation by the Ca2+-dependent protein kinase, PKC. There are at least two distinct PKC...

Death Receptors

Ceramide Signaling

In contrast to the effect of growth factors, a number of cytokines that bind to cell surface receptors that are members of the TNF receptor superfamily can induce apoptosis. These death receptors possess similar cysteine-rich extracellular domains but lack substantial homology in their cytoplasmic regions except in the death domain, which spans approximately 80 amino acids and which is required for transduction of the death signal. Members of this family include TRAMP (also known as DR3, Apo3,...

Classification Structure and Distribution of VGCCs

Central to the operation of the Vm-dependent Ca2+ signaling pathway is the incorporation of VGCCs into the electrical membrane activity. These channels are large multisubunit proteins that span the plasma membrane to provide a Ca2+-selective pathway into the cytosol. At rest, the channel remains closed, blocking Ca2+ entry into the cell. In response to membrane depolarization, the molecular gates of the channel open and Ca2+ is free to flow through the aqueous pore of the channel. The...

Internalization Of Activated Irks

Following insulin binding to the IRK there rapidly occurred internalization of the insulin-IRK complex into the cell (Bergeron et al., 1979 Posner et al., 1980, 1986 Khan et al., 1985). Internalized insulin-IRK complexes were shown to concentrate in a novel cellular organelle (Khan et al., 1981) that became known as the endosomal apparatus or endosomes (ENs). It was appreciated early that endosomal IRK remained activated for a significant period of time subsequent to internalization and even...

Type Dependent Regulation of ACs

Successful molecular cloning of the nine subtypes of AC and the study of their differential properties as seen when they are over expression in mammalian or Sf9 and insect cells has revealed a bewildering array of regulatory features that, for the most part, had not been appreciated from studying activities in tissues or cells as cells and tissues tend to coexpress more than one AC. An illuminating case in this regard are GH4Cl cells. Analysis of their AC activity by standard methods in crude...

GH Induces Calcium Fluxes and the Activation of Protein Kinase C

GH stimulates increases in intracellular calcium in several GH-responsive cell types. In each instance, the increase can be accounted for, at least in part, by an influx of extracellular calcium. Inhibitor studies implicate voltage-dependent L-type calcium channels found in the plasma membrane in this process, and suggest a possible role for GH in their maintenance. The GH-induced increase in intracellular calcium is believed to underly the refractory nature of GH-stimu-lated cells to the...

Regulation of the Catalytic Activity of Phosphorylation Targets

In their pioneering studies on glycogen metabolism in 1955 Fischer and Krebs showed that activation of phosphorylase by phosphorylase kinase was due to phosphorylation at Ser14, and they were awarded the 1992 Nobel Prize in Medicine for this discovery. In 1968 it was found that phosphorylase kinase itself was phosphorylated and activated by the cAMP-dependent protein kinase. These discoveries formed the foundation for our understanding of many aspects of protein phosphorylation and signal...

Regulation of Cytoskeletal Organization in Chemotaxis

A dramatic rise of the proportion of polymerized actin (F-actin) is one of the most striking changes after addition of extracellular cAMP to aggregation-competent Dictyostelium cells. A schematic diagram for the establishment of the leading edge by redistribution of actin filaments and components involved in the regulation of actin filaments is presented in Fig. 3. Localized actin assembly is required for the psuedo-pod formation during chemotaxis. Sites of actin filament assembly in the cell...

Regulation of PLD1

PLDl exists in two splice variants, one of which has a 38 amino acid deletion between conserved 5.2.1. Regulation of PLD1 by Protein Kinase C Numerous studies have shown that phorbol esters activate PLD in a variety of cell types, and, as described in the previous sections, many agonists stimulate PIP2 hydrolysis and thereby activate PKC. These findings indicate that PKC is a major regulator of PLD. Certain PKC isozymes are able to directly activate PLD1 isozymes in vitro. Surprisingly the...

Phosphatase Inhibitors Coordinate Hormone Signals

The first endogenous phosphatase inhibitors to be identified were two thermostable proteins, termed inhibitor-1 (I-1) and inhibitor-2 (I-2). These proteins were isolated from skeletal muscle extracts and shown to inhibit the skeletal muscle phosphorylase phospha-tase (PP1). Nanomolar concentrations of these proteins inhibited PP1 activity but they had little effect on PP2A or other type-2 phosphatases, even at 1000fold higher concentration. What makes these proteins particularly interesting is...

Regulation of Protein Protein Interactions Subcellular Localization and Stability by Protein Phosphorylation

More and more cases are emerging where the interaction of proteins is regulated by protein phosphorylation, subsequently resulting in the regulation of cellular function. Two different mechanisms can account for phosphorylation-dependent protein interactions. First, interacting proteins could directly bind to the phosphorylation site via phosphopeptide binding sequences, or second, a hidden binding site could be indirectly exposed due to a conformational change resulting from phosphorylation....

Role of CD45 in Lymphocyte Activation

CD45 is an RPTP with a glycosylated extracellular domain, a single transmembrane segment, and two tandem PTP catalytic domains. The extracellular domain is thought to have a ligand binding function however, the ligand(s) for CD45 have not yet been identified. The expression of CD45 is limited to nucleated cells of hematopoietic origin, where it occupies about 10 of the cell surface. Relative to many other proteins with signaling functions CD45 is surprisingly abundant. The size of the...

Protein Tyrosine Phosphatases and Cell Adhesion

Cell-cell interactions and contacts that cells make with the surrounding extracellular matrix (ECM) affect cell shape and motility and induce growth and differentiation. In development, these cell contacts are particularly crucial because they are needed to organize cells into tissues and organs. Cell-cell communication also provides cues to neurons as they make connections to one another and innervate muscle or other tissues. Alterations or defects in normal cell adhesion are thought to...

Functions of PLC IsozymesDAG Production and Activation of PKC

The other product of PIP2 hydrolysis, DAG, is a major mediator of PKC activation. PKC occurs in several forms that are grouped into conventional or Ca2+-dependent isozymes (a, pI, pn, y), novel or Ca2+-independent isozymes ( , e, 0), and atypical or DAG-independent isozymes (i, y, and ). Another isozyme ( or D) has an unusual structure and does not belong in the other groups. Schematic structures of the PKC isozymes are depicted in Fig. 10. The conventional forms have an autoinhibitory...

Functions of PLC IsozymesIP3 and Ca2 Mobilization

The physiological substrate of all mammalian PLC isozymes is PIP2 and its products are two signaling molecules inositol 3,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). The function of IP3 is to interact with specific receptors located on the cytosolic surface of certain components of the endoplasmic reticulum (ER). Activation of these receptors causes the release of Ca2+ from the ER and hence a rise in cytosolic Ca2+ (Fig. 9). The IP3 receptors have a transmembrane domain near the...

The Various PKG Isoforms Have Distinct Structures and Functions

Two main classes of PKG isoforms (PKGI and PKGII) occur in mammalian tissues (Fig. 2). These forms differ significantly in their tissue distributions, substrate specificities, affinities for cGMP, and subcellular localizations. In contrast to the PKA system, PKGI and PKGII have not yet been found to coexpress substantially in the same cell type. The PKGs are typically abundant in more selected tissues including smooth muscle, smooth muscle related tissues, platelets, cerebellum, intestinal...

Ligandgated Cation Channels

Extracellular ligand-gated receptor channels differ from other Ca2+-conducting plasma membrane channels (see below) in three respects. First, their activation depends on the delivery and binding of a ligand to the extracellular domain of these receptor channels. Second, termination of their activities requires removal of the ligand, which is usually mediated by a specific pathway for ligand degradation and or uptake. Third, they are not highly selective for Ca2+, but conduct other ions as well....

Info

Hormonal regulation of the glycogen-bound phosphatase. PP1 localization to the glycogen granule is mediated by the glycogen-targeting or G-subunit. Adrenaline promotes the phosphorylation of the skeletal muscle G-subunit, GM, to release PP1, which then associates with the PKA-activated cytosolic inhibitor, I-1. Thus adrenaline facilitates the phosphorylation of glcogen synthase, GS, and inhibits glycogen synthesis (top). Insulin promotes the phosphorylation of GM at a different serine...

PDEs Are Homeostatic Regulators of Cyclic Nucleotide Signaling and Cell Adaptation

The in-depth investigation of the cAMP pathway has established that signals that activate cyclase and increase cAMP synthesis are also associated with changes in PDE activity. Depending on the cell, the increase in cAMP triggers either negative feedback mechanisms activating PDEs or feed-forward mechanisms inhibiting PDE activity. In many instances, hormones and neurotransmitters that activate cyclic AMP synthesis also cause an activation of cAMP hydrolysis. Signaling through cAMP may require...

Historical Perspective of Drug Design

Animal Disease And Use Drugs

Discovery of novel drugs requires the execution of several critical steps summarized in Fig. 1. The first requirement is to identify a disease to be targeted. Traditionally, the first step was to develop a suitable animal model for this disease. For example, an in vivo model frequently used for hypertension was the spontaneously hypertensive rat. Compounds, generally hundreds, were tested in this model to search for a hit, which would reduce blood pressure. When such a compound was found,...

Activation And Regulation Of Rhodopsin

Rhodopsin consists of two components an apoprotein moiety (opsin) consisting of seven transmembrane domains and a vitamin A derivative (3-hydroxy-11-cis-retinal in Drosophila and 11-cis-retinal in vertebrates) that binds to the seventh transmembrane segment (Fig. 4A). On exposure to light, there is a cis to trans isomerization around the 11-12 double bond in the retinal. This isomerization, which represents the only light-dependent event in phototransduc-tion, results in an ensuing alteration...

Specificity of STAT Activation Is Determined by Both GH Receptor and JAK2 Kinase

Detailed mapping studies have identified the contributions made by specific regions of GHR and JAK2 kinase to GH-induced STAT activation. GH activation of STATs 1 and 3 minimally requires a membrane-proximal receptor cytoplasmic domain region, which is limited to human GHR residues 271-317 in the case of STAT3, and thus maps to the same minimal region of GHR required for JAK2 activation. While GH activation of STATs 1 and 3 may involve interactions with JAK2, the nature of these interactions...

Classification Of Protein Tyrosine Phosphatases

Enzymes customarily referred to as protein tyro-sine phosphatases employ a similar catalytic mechanism involving formation of a thiophosphate intermediate at an essential Cys residue located within a hallmark active site motif (Cys-X5-Arg). These tyro-sine phosphatases bear no resemblance to the protein Ser Thr phosphatases and have distinct sequences, structures, catalytic mechanisms, and evolutionary ancestry. The protein tyrosine phosphatases are commonly divided into four groups...

Regulation of the Metaphase Anaphase Transition in Meiosis

An understanding of the individual roles of the functional domains of calmodulin-dependent enzymes has allowed the generation of a number of specific biological reagents to assess the roles of these enzymes in cellular processes. One of the best examples of the use of such reagents is in the identification of CaMKII as the primary target of the Ca2+ signal that results from the fertilization of Xenopus laevis eggs. Fully developed Xenopus oocytes are arrested in prophase of meiosis I....

Genetic Approaches To Drosophila Phototransduction

To identify the molecules and mechanisms underlying Drosophila phototransduction, electrophysio-logical, cell biological, and pharmacological approaches have been combined with genetic approaches. Many mutations affecting phototransduc-tion have been identified using a relatively simple electrophysiological assay, electroretinogram recordings (ERGs). ERGs are extracellular recordings that measure the summed responses of all the cells in the retina to light. There are two main components of the...

Four STATs Can Be Activated by GH

Signal transducers and activators of transcription (STATs) are latent transcription factors that reside in the cytoplasm in an inactive form until converted to active, nuclear transcription factors by a tyrosine phosphorylation reaction. This tyrosine phosphoryla-tion is JAK kinase-dependent, and in the case of GH signaling is generally presumed to be catalyzed by JAK2, rather than by an intermediate tyrosine kinase, such as fyn kinase. Of the seven known mammalian STATs (STATs 1-4, 5a, 5b, and...

Ryanodine Receptor Channels

Ryanodine receptor channels (RyRs) were originally identified as Ca2+ release channels expressed in the SR of skeletal muscle fibers and cardiac myocytes, where they play a central role in excitation-contraction coupling. These channels are also expressed in neurons, chromaffin cells, sea urchin eggs, and several nonexcitable cell types. Mammalian tissues express three isoforms RyR1 is expressed predominantly in skeletal muscle, RyR2 is expressed in cardiac muscle, and RyR3 has a wide tissue...

Activation loop

Protein kinase C is regulated by two phosphorylation switches, one at the activation loop and one at the C-terminus. The former is phosphorylated by a heterologous kinase, the phosphoinositide-dependent kinase 1 (PDK-1) or related kinase. This phosphorylation is required to process catalyti-cally competent protein kinase C its role appears to be to correctly align residues for catalysis to promote the phosphor-ylation of the two C-terminal sites. Once the C-terminal sites are...

STAT5b Plays a Key Role in the Sexually Dimorphic Responses to Plasma GH Secretory Patterns Seen in Rodent Liver

As discussed earlier in this chapter, pituitary GH secretion is regulated by neuroendocrine factors, which are in turn established by gonadal steroid imprinting, and this leads to sex differences in the temporal pattern of circulating GH. Gender differ Fig. 11. DNA binding activity of STAT5b in response to gender-specific plasma GH pattern. Pulsatile GH secretion in male rats leads to intermittent activation of liver STAT5b and is hypothesized to stimulate transcription of male-expressed genes...

Gh Signal Transduction Initial Events

Sie Signaling

Multiple biochemical events and intracellular signals can be detected shortly after GH-responsive target cells are stimulated with GH. Dimerization of GHR following GH binding initiates a series of protein phosphorylation cascades that lead to the activation of several enzymes and transcription factors, followed by the induction of gene expression (Fig. 6). In several instances, a detailed understanding of how these initial signaling events translate into the biological effects of GH is...

Purinergic Receptor Channels

ATP-gated receptors, also known as purinorecep-tors, belong to two major groups G protein-coupled receptors (P2YRs), whose activation causes Ca2+ release from intracellular stores, and plasma membrane channels (P2XRs), whose activation promotes cation influx (Table 1). In contrast to nAChRs and GluRs, P2XRs have been identified in both excitable and nonexcitable cells. Seven channels from this family have been cloned and named P2X1 to P2X7. In addition, several spliced forms of P2X2Rs were...

Nicotinic Acetylcholine Receptor Channels

Nicotine Receptor

The native nAChR was initially identified as a pentamer protein of about 300,000 MW from the fish electrical organ. Recombinant DNA studies revealed close homologies between nAChR subunit sequences derived from electrical organ and skeletal muscle tissue, as well as between muscle and neuronal nAChRs. Five peripheral nAChR subunits, labeled as a Pj, 5, Y, and e, and 10 neuronal nAChR subunits, labeled as a2-a9 and P2-P4, were identified (Fig. 6A). In both muscle and neuronal nAChRs, the large...

Role of Protein Tyrosine Phosphatases in Growth Factor and Cytokine Signaling

The binding of growth factors, cytokines, and certain hormones to their cell surface receptors results in the rapid activation of PTKs. These initial tyrosine phosphorylation events result in diverse changes in cell physiology and metabolism, cell growth and differentiation, and cell motility and shape. The receptors for many growth factors and certain hormones have intrinsic tyrosine kinase activity within their intracel-lular domains. Ligand-induced oligomerization activates these receptor...

Calmodulin Action in Cells

As calmodulin is involved in interacting with more than 30 separate proteins and enzymes, and Ca2+ is one of the two most frequently used second messengers in cells, a number of strategies have been developed to identify calmodulin-dependent signal transduction pathways. The initially used and most frequently employed inhibitory agents are the small-molecule calmodulin antagonists. The first such agent championed as a calmodulin antagonist was chlorpromazine. This substance effectively serves...

PDEs Are Integrators of Different Signaling Pathways

Initially, it was thought that the different signaling pathways were organized as a linear sequence of steps functioning in parallel and that each extracellular signal would activate its own pathway. The last 10 yr of investigation of signaling have completely revolutionized this view. Signaling pathways are composed of nodes and branches, and different pathways are meshed into an intricate network of crosstalks. The concept of combinatorial signaling has been put forward to underscore the...

Regulation of Protein Kinase Activity by Phosphorylation

Many serine threonine protein kinases are regulated by changes in their phosphorylation state. Most of these protein kinases require phosphorylation of a conserved threonine residue within the so-called activation loop of the catalytic domain, which is located in the proximity of the kinase active site cleft, and in the linear sequence found between the DFG and APE motifs (Fig. 2A). This phosphorylated residue then interacts with multiple residues within the catalytic domain to modify the...

The Gjm Dna Damage Checkpoint

When DNA is damaged, one of the physiological responses of the cell is to arrest in G2, prior to mitotic entry. The presumed purpose of this arrest, or pause, in the cell cycle is to allow the cell time to repair the damage before proceeding through mitosis. Were this not the case, damaged DNA and or broken chromosomes would be passed on to daughter cells at the time of division. The molecular pathway inhibiting entry into mitosis until DNA damage is repaired is referred to as the G2 DNA damage...

Trp And Trpl Represent The Archetypal Phospholipase Cdependent Ion Channels

The major ion channel responsible for light-dependent cation influx is TRP, a protein with six putative transmembrane domains (Fig. 4C). trp mutant flies display a dramatic decrease in Ca2+ influx and moderate reduction in Na+ influx. Moreover, the response in trp flies is only transient. The remaining influx in trp appears to be due to a second ion channel, TRPL, which is highly related to TRP. Flies that are missing both TRP and TRPL are completely unresponsive to light. The mechanism whereby...

CAR Androstane Signaling Pathway

CARP is a novel steroid receptor that appears to participate in a unique mode of ligand-dependent deactivation in response to steroids. It exhibits significant constitutive activity when combined with RXR, but in response to the androstane metabolites, androstenol and androstanol, this activity is switched off. These ligands are naturally occurring, and are thought to function as inverse agonists owing to their ability to reverse transcriptional activation by nuclear receptors. This effect is...

Regulation of Cell Proliferation

Based on the information presented in this chapter, it should come as no surprise that calmodulin is an essential protein in all eukaryotic cells. Deletion of the unique calmodulin gene in the genetically tractable organisms Saccharomyces cerevisiae, Schizosaccha-romyces pombe, Aspergillus nidulans, and Drosoph-ila melanogaster is lethal. A common phenotypic consequence of the absence of calmodulin is inhibition of cell proliferation. Calmodulin is required for quiescent cells to enter the cell...

The ROR Family

The retinoic acid-related orphan receptor ROR family, also called retinoid Z receptor RZR , consists of three subtypes, a, P, and y. Their DBDs are about 70 homologous to the RAR however, they do not appear to be activated by retinoids. The RORa gene gives rise to multiple transcripts RORa1-4 that share a common DBD and LBD but contain distinct N-terminal domains that can modulate their DNA binding activity. RORa isoforms bind as monomers to HREs ROREs and function as constitutive activators in...