Tissue-specific expression of the K-ras allele from the A/J parent in (A/J x TSG-p53) F1 mice

Six vertebrate protein kinases (G-protein-coupled receptor kinases; GRKs) that regulate the function of G-protein-coupled receptors (GPCRs) were recently cloned; several distinct properties set them apart from conventional second-messenger regulated protein kinases. It appears that GRKs bind GPCR* through two separate sites: a high-affinity site, which involves intracellular loops of the activated receptor, and the lower-affinity site, encompassing the phosphorylation region. The high-affinity interaction may involve complementary structural elements of GRKs and GPCRs* rather than precise amino acid alignment, thus allowing broad and overlapping specificities of these kinases, in spite of differences in the sequences of GPCRs. In addition, GRK structures are modified by several posttranslational modifications, including phosphorylation, autophosphorylation, prenylation, carboxymethylation, and palmitoylation, probably affecting properties of these enzymes. While GRKs phosphorylate and inactivate receptor molecules which are engaged in G-protein activation, controversy surrounds whether GRKs might be activated and phosphorylate unstimulated GPCRs, leading to a desensitization of a larger population of the receptors. In this review, mechanistic aspects of GPCR* phosphorylation related to the distinct properties, regulation and modes of action of GRKs are described.     

Role of adhesion molecule cytoplasmic domains in mediating interactions with the cytoskeleton

The past ten years have seen significant progress in cell biology research aimed at understanding how cytoskeletal filaments interact with the plasma membrane. Considerable evidence suggests that both actin microfilaments and intermediate filaments attach to the membrane via the cytoplasmic domains of various membrane proteins including adhesion molecules. Interactions between the cytoskeleton and adhesion molecules appear to be essential for a variety of cellular functions, including cell-cell and cell-extracellular matrix (ECM) interactions, cell motility, receptor-ligand interactions, and receptor internalization. Recently, many of the detailed molecular mechanisms which mediate the associations between actin filaments and adhesion molecules have been identified. Among adhesion molecules that support the attachment of cytoskeletal filaments to their cytoplasmic domains are members of the integrin and cadherin families, the intracellular adhesion molecule-1 (ICAM-1, an immunoglobulin family member), and the glycoprotein Ib/IX complex in platelets. A general conclusion emerging from these studies is that physical associations between cytoskeletal filaments and transmembrane glycoproteins do not occur directly between the filaments and the cytoplasmic tails of adhesion molecules. Instead, these interactions appear to be indirect and involve a complex ensemble of intermediary linker proteins. The severe effects of cytoplasmic domain deletion and mutagenesis on adhesion-dependent functions support the view that receptor cytoplasmic domains play a vital role in regulating receptor function and in mediating communication across the membrane. Transfection studies with mutant and chimeric adhesion molecules, along with protein-binding studies, are clarifying the mechanisms which physically link the cytoskeleton to transmembrane proteins, regulate cytoskeletal organization, mediate signaling across the cell membrane, and regulate the ligand specificity and binding affinity of surface receptors.     

Association of PTPmu with catenins in cancer cells: a possible role for E-cadherin

Protein tyrosine phosphorylation and dephosphorylation is regulated by the action of protein tyrosine kinases (PTK) and phosphatases (PTP) respectively. The receptor type phosphatase, PTPmu, is located at the cell surface where it may function to regulate the phosphoryl status of members of the cadherin adhesion complex and thus cadherin function. We have investigated the association of PTPmu with E-cadherin and catenin molecules in human tumour cells and report that PTPmu; is associated with E-cadherin and alpha and beta-catenin in E-cadherin-positive cell lines. However, no association between PTPmu and catenin members could be detected in E-cadherin negative cells. These observations suggest that the association of PTPmu with catenin molecules may occur via E-cadherin rather than a direct interaction.     

G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C

The murine G-protein alpha-subunit G alpha 15 and its human counterpart G alpha 16 are expressed in a subset of hematopoietic cells, and they have been shown to regulate beta-isoforms of inositide-specific phospholipase C. We studied the ability of a variety of receptors to interact with G alpha 15 and G alpha 16 by cotransfecting receptors and G-protein alpha-subunits in COS-7 cells. Activation of beta 2 adrenergic and muscarinic M2 receptors in cells expressing the receptors alone or together with G alpha q, G alpha 11, or G alpha 14 led to a very small stimulation of endogenous phospholipase C. However, when the receptors were coexpressed with G alpha 15 and G alpha 16, addition of appropriate ligands caused a severalfold increase in inositol phosphate production which was time- and dose-dependent. A similar activation of phospholipase C was observed when several other receptors which were previously shown to couple to members of the Gi and Gs family were coexpressed with G alpha 15/16. In addition, stimulation of inositol phosphate formation via receptors naturally coupled to phospholipase C was enhanced by cotransfection of G alpha 15 and G alpha 16. These data demonstrate that G alpha 15 and G alpha 16 are unique in that they can be activated by a wide variety of G-protein-coupled receptors. The ability of G alpha 15 and G alpha 16 to bypass the selectivity of receptor G-protein interaction can be a useful tool to understand the mechanism of receptor-induced G-protein activation. In addition, the promiscuous behavior of G alpha 15 and G alpha 16 toward receptors may be helpful in finding ligands corresponding to orphan receptors whose signaling properties are unknown.     

Precoupling of Gi/G(o)-linked receptors and its allosteric regulation by monovalent cations

The ability of receptors (R) to activate G-proteins (G) is promoted by the binding of agonists, reflecting their induction of a receptor conformation which facilitates both the formation of a RG complex and guanine nucleotide exchange. Recent evidence from isolated membrane studies has indicated, however, that some receptors have the inherent ability to form RG complexes and promote GDP/GTP exchange in their unoccupied state. These receptors preferentially activate pertussis toxin-sensitive G-proteins (i.e. Gi/G(o)) and the interactions of R and G are modulated by monovalent cations (most notably Na+) both in the unoccupied and agonist-occupied states. Basal G-protein activation by such receptors is reduced both by increasing levels of cation and by antagonists which may act by inducing receptor conformations which are less favorable for RG complexation. This behaviour conforms to the predictions of a ternary complex model of receptor function and can be considered in structural terms for those receptors such as the alpha-2 adrenergic receptor where ligand binding and G-protein activation regions have been proposed.     

Implicit memory in posttraumatic stress disorder with amnesia for the traumatic event

The regulators of G-protein signaling (RGS) family members contain a conserved region, the RGS domain, and are GTPase-activating proteins for many members of G-protein alpha-subunits. We report here that the core domain of RGS16 is sufficient for in vitro biochemical functions as assayed by its G-protein binding affinity and its ability to stimulate GTP hydrolysis by G alpha(o) protein. RGS16 also requires, in addition to the RGS domain, the divergent N-terminus for its biological function in the attenuation of pheromone signaling in yeast, whereas its C-terminus region is dispensable. Together with other evidence, these data support the notion that RGS proteins interact with other cellular factors and may serve to link specific G-proteins to different downstream effectors in G-protein-mediated signaling pathways.     

Gpa2p, a G-protein alpha-subunit, regulates growth and pseudohyphal development in Saccharomyces cerevisiae via a cAMP-dependent mechanism

The small GTP-binding protein Ras and heterotrimeric G-proteins are key regulators of growth and development in eukaryotic cells. In mammalian cells, Ras functions to regulate the mitogen-activated protein kinase pathway in response to growth factors, whereas many heterotrimeric GTP-binding protein alpha-subunits modulate cAMP levels through adenylyl cyclase as a consequence of hormonal action. In contrast, in the yeast Saccharomyces cerevisiae, it is the Ras1 and Ras2 proteins that regulate adenylyl cyclase. Of the two yeast G-protein alpha-subunits (GPA1 and GPA2), only GPA1 has been well studied and shown to negatively regulate the mitogen-activated protein kinase pathway upon pheromone stimulation. In this report, we show that deletion of the GPA2 gene encoding the other yeast G-protein alpha-subunit leads to a defect in pseudohyphal development. Also, the GPA2 gene is indispensable for normal growth in the absence of Ras2p. Both of these phenotypes can be rescued by deletion of the PDE2 gene product, which inactivates cAMP by cleavage, suggesting that these phenotypes can be attributed to low levels of intracellular cAMP. In support of this notion, addition of exogenous cAMP to the growth media was also sufficient to rescue the phenotype of a GPA2 deletion strain. Taken together, our results directly demonstrate that a G-protein alpha-subunit can regulate the growth and pseudohyphal development of S. cerevisiae via a cAMP-dependent mechanism. Heterologous expression of mammalian G-protein alpha-subunits in these yeast GPA2 deletion strains could provide a valuable tool for the mutational analysis of mammalian G-protein function in an in vivo null setting.     

Recent progress in clinical aspects of receptor research

Clinical receptology encompasses broad areas, including receptor or postreceptor defects due to mutations of receptor or other genes, abnormalities due to receptor antibodies and secondary changes of receptors under various pathological conditions. Recent progress in molecular biology has succeeded in cloning genes of receptors, G-proteins and other cellular proteins that are involved in the signal transduction and clarified their germ-line and somatic mutations. It is of importance that mutations of receptors and G-proteins do not necessarily cause loss of function but sometimes cause gain of function of receptors or G-proteins, thus leading to hyperfunction. Molecular basis that causes either loss or gain of function has been studied but is not completely understood. Some examples of gain of function mutatious of G-protein coupled receptors, tyrosin kinase-type receptors and G alpha protein are shown. Another important aspect in receptor research is that mutation of a single receptor gene sometimes result in different phenotypes and even different modes of inheritance. For example, mutations of rhodopsin (a G-protein coupled receptor) gene cause retinitis pigmentosa of autosomal dominant type and autosomal recessive type and also cause congenital stationary night blindness. Exact mechanisms responsible for such differences are not completely understood. There are polymorphisms in some genes that may be involved in some diseases. An example is a polymorphism in beta 3-adrenergic receptor that is claimed but not clearly demonstrated to be a cause of obesity or type II diabetes. Such polymorphism is possibly a gene in polygenic diseases. Receptology is important for elucidating pathogenesis of complex diseases.     

Concomitant chemoradiotherapy of cancer of the esophagus

Many cell membrane bound receptors communicate with the inside of the cell through guanine nucleotide binding proteins (G-proteins). This holds also for olfactory receptor neurons, which respond to odorants with G-protein mediated increases in the concentration of cyclic adenosine 3', 5'-monophosphate (cAMP) and/or inositol 1,4,5-triphosphate (InsP3). These substances regulate the ionic conductivity of the wall of the cilia. We have studied a similar system, namely G-protein coupled alpha 2-adrenoceptors, present for example in the cells of certain fish scales. These receptors react on, catecholamines and the G-protein mediates a decrease in cAMP, which causes an aggregation of pigment containing granulas to the middle of the cells. The light transmission of the cell increases due to this aggregation. This simple physiological response has been used in a sensitive biosensor for noradrenaline and for pertussis toxin that is based on isolated fish scales from cuckoo wrasse (Labrus ossifagus). The results were obtained with a simple photometer. Measurements can be performed also on single isolated melanophores. The main purpose of this contribution is, however, to point out that G-protein coupled receptors together with a simple physiological response form a principle for biosensing, which could also be an interesting alternative for odour sensing.     

Increased oxidative stress in renal proximal tubules of the spontaneously hypertensive rat: a mechanism for defective dopamine D1A receptor/G-protein coupling

AIM: Defective dopamine D1A dopamine receptor/G-protein coupling has been demonstrated in renal proximal tubules of the spontaneously hypertensive rat (SHR). In the present study, we aimed to analyze the underlying mechanisms through which such defects are introduced into the D1A receptor protein of SHR. MATERIALS AND METHODS: The oxidative state of SHR proximal tubules was analyzed by measuring lipid peroxidation. D1A receptor/G-protein coupling was measured following the induction of oxidative stress in normotensive Wistar-Kyoto (WKY) rats. RESULTS: For the first time, an increased state of oxidative stress was demonstrated in SHR proximal tubules compared with those of normotensive controls, WKY and Sprague-Dawley rats. Lipid peroxidation levels in SHR were significantly higher by 66 and 79%, relative to WKY or Sprague-Dawley rats, respectively. Hydrogen peroxide treatment of proximal tubules from SHR, WKY and Sprague-Dawley rats induced an additional increase in lipid peroxidation in a dose-dependent manner, although the percentage induction was lower in SHR than in WKY and Sprague-Dawley rats. This induction of lipid peroxidation in WKY rats resulted in a loss of D1A/G-protein coupling, with no decrease in receptor protein. Treatment of WKY rat proximal tubules with an antioxidant, ascorbic acid, or a reducing agent, dithiothreitol, induced D1A receptor/G-protein coupling. CONCLUSIONS: These data indicate that D1A receptor/G-protein coupling is modulated by changes in redox states. Therefore, the D1A receptor/G-protein coupling in SHR may have been damaged by reactive oxygen species released as a result of the elevated oxidative stress seen in the proximal tubules.     

Cell membrane receptors and regulation of cell function in ticks and blood-sucking insects

Immunoglobulins cross the midgut epithelium and enter the haemolymph of many blood-feeding arthropods without losing their immunological properties. Antigens essential to the survival of the blood-sucking arthropods which may be affected by the small amounts of specific antibody that cross the gut epithelium include membrane receptors or other factors which regulate cell function. Membrane receptors implicated in transmembrane signalling in response to specific neural and endocrine factors fall into three major classes: (1) gated ion channels, (2) agonist-stimulated tyrosine kinases and (3) receptors that interact with GTP-binding (G) proteins. Examples of all three types have been found in insects and ticks. A dopamine receptor interacts with a G-protein essential for controlling fluid secretion by the salivary glands of ixodid ticks. Another receptor in the ixodid tick salivary gland binds a neuropeptide from the tick synganglion and stimulates turnover of plasma membrane phosphoinositides, but its mechanisms of transmembrane signalling and function remain elusive. Another large class of membrane receptors are those concerned with endocytosis. Examples of receptor-mediated endocytosis include incorporation of vitellogenin by developing oocytes in mosquitoes and ticks and uptake of lysed blood-meal components by digest cells of the tick gut. Many cell membrane receptors and possibly hormones could serve as targets for vaccines in blood-feeding insects and ticks. The major challenge is to identify and characterize essential internal receptors and cellular components that are accessible to and affected by specific antibodies that are introduced into the body of blood-feeding arthropods.     

A negative regulatory region in the intracellular domain of the human interferon-alpha receptor

Interferon-alpha (IFN-alpha)-mediated intracellular signaling is initiated by ligand-induced receptor dimerization, tyrosine phosphorylation of the Tyk2 and Jak1 tyrosine kinases, and subsequent phosphorylation of the Stat1 and Stat2 proteins. The IFN-alpha receptor consists of at least two distinct subunits. One subunit, IFNAR1, has low affinity binding for interferon yet is required for signal transduction. We introduced mutations in the cytoplasmic domain of human IFNAR1 in order to identify residues involved in the mediation of biological responses. We took advantage of the species specificity of the interferon receptors by analyzing human IFN-alpha-induced major histocompatibility complex class I antigen expression in mouse L929 cells stably transfected with mutant human receptors. The membrane proximal 60-amino acids were insufficient to signal a biological response even though within these residues Tyk2 and Stat2 binding sites have been identified. IFN-alpha-induced receptor tyrosine phosphorylation was not critical for signaling because mutation of Tyr residues to Phe did not prevent the biological response to IFN-alpha. The deletion of a 16-amino acid region highly homologous between species created a receptor which signals an enhanced response. Tyrosine dephosphorylation is a component of this enhanced response as mutation of the Tyr residues within this region to Phe resulted in a receptor with increased sensitivity to IFN. The known signaling molecules that interact with IFNAR1 are positive regulators of IFN-alpha function. The presence of this domain in the COOH-terminal region suggests that the receptor may interact with signaling molecules that negatively regulate interferon responses.     

Evidence that the nucleotide exchange and hydrolysis cycle of G proteins causes acute desensitization of G-protein gated inward rectifier K+ channels

The G-protein gated inward rectifier K+ channel (GIRK) is activated in vivo by the Gbeta gamma subunits liberated upon Gi-coupled receptor activation. We have recapitulated the acute desensitization of receptor-activated GIRK currents in heterologous systems and shown that it is a membrane-delimited process. Its kinetics depends on the guanine nucleotide species available and could be accounted for by the nucleotide exchange and hydrolysis cycle of G proteins. Indeed, acute desensitization is abolished by nonhydrolyzable GTP analogues. Whereas regulators of G-protein signaling (RGS) proteins by their GTPase-activating protein activities are regarded as negative regulators, a positive regulatory function of RGS4 is uncovered in our study; the opposing effects allow RGS4 to potentiate acute desensitization without compromising GIRK activation.     

Screening lab tests of Chlamydia trachomatis. Do they show declining trend of the infections?

Peptides are flexible molecules and can adopt local structural features of protein, such as secondary structure, hydrophobicity, and distribution of electrostatic charges, and so forth, and mimic their functions. Therapeutic peptidomimetics that are immunologically relevant are developed by engineering the surface loop structures in the proteins and receptors. The class of molecules targeted include immunoglobulin fold-containing molecules: antibody, cell-surface CD4 receptors and cystine-knot-containing receptor family members: tumor necrosis factor (TNF), CD40, and p185/Neu receptors. We have used the loops involved in the molecular recognition as a template and developed peptidomimetics that interfere with the functions of the target molecules. In this article, two molecular targets are discussed: (1) immunoglobulin fold-containing CD4 receptor and (2) cystine-knot-containing TNF receptor (TNFR).     

Adhesion molecules in kidney diseases (part I)

Adhesive molecules are (glyco)proteins of the cellular membranes. All of them have their extramembranous, transmembranous and intracytoplasmatic parts. As receptor molecules, their extracellular parts bind the specific ligand. The ligand can be found on the surface of the other cell or in the extracellular matrix (basal membranes). The following families of adhesion molecules are: cadherins, selectins, integrins and members of immunoglobuline supergene family. Different members of the same family could have different times (in ontogenesis, in adult form) and space distribution (in different tissues, different tissue structures). The contact between the cells and basal membranes with these molecules is important for cell division, maintaining the tissue architecture, polarization and function of cells, migration of cells, endo- and exo-cytosis as well as for maintaining the structure and function of basal membranes. As above stated all this is important in the occurrence morphogenesis, haemostasis, inflammation, malignant cell transformation and metastasis. This knowledge is important for the better understanding of renal diseases.     

Association between a transmembrane protein tyrosine phosphatase and the cadherin-catenin complex

Cadherins are calcium-dependent cell adhesion molecules that play fundamental roles in embryonic development, tissue morphogenesis, and cancer. A prerequisite for their function is association with the actin cytoskeleton via the catenins. Tyrosine phosphorylation of beta-catenin, which correlates with a reduction in cadherin-dependent cell adhesion, may provide cells with a mechanism to regulate cadherin activity. Here we report that beta-catenin immune precipitates from PC12 cells contain tyrosine phosphatase activity which dephosphorylates beta-catenin in vitro. In addition, we show that a member of the leukocyte antigen-related protein (LAR)-related transmembrane tyrosine phosphatase family (LAR-PTP) associates with the cadherin-catenin complex. This association required the amino-terminal domain of beta-catenin but does not require the armadillo repeats, which mediate association with cadherins. The interaction also is detected in PC9 cells, which lack alpha-catenin. Thus, the association is not mediated by alpha-catenin or by cadherins. Interestingly, LAR-PTPs are phosphorylated on tyrosine in a TrkA-dependent manner, and their association with the cadherin-catenin complex is reduced in cells treated with NGF. We propose that changes in tyrosine phosphorylation of beta-catenin mediated by TrkA and LAR-PTPs control cadherin adhesive function during processes such as neurite outgrowth.     

Visual pigment: G-protein-coupled receptor for light signals

The visual pigment present in photoreceptor cells is a prototypical G-protein-coupled receptor (GPCR) that receives a light signal from the outer environment using a light-absorbing chromophore, 11-cis-retinal. Through cis-trans isomerization of the chromophore, light energy is transduced into chemical free energy, which is in turn utilized for conformational changes in the protein to activate the retinal G-protein. In combination with site-directed mutagenesis, various spectroscopic and biochemical studies identified functional residues responsible for chromophore binding, color regulation, intramolecular signal transduction and G-protein coupling. Extensive studies reveal that these residues are localized into specific domains of visual pigments, suggesting a highly manipulated molecular architecture in visual pigments. In addition to the recent findings on dysfunctional mutations in patients with retinitis pigmentosa or congenital night blindness, the mechanism of intramolecular signal transduction in visual pigments and their evolutionary relationship are discussed.