A small region in phosducin inhibits G-protein betagamma-subunit function

G-protein betagamma-subunits (G(betagamma)) are active transmembrane signalling components. Their function recently has been observed to be regulated by the cytosolic protein phosducin. We show here that a small fragment (amino acids 215-232) contained in the C-terminus of phosducin is sufficient for high-affinity interactions with G(betagamma). Corresponding peptides not only disrupt G(betagamma)-G(alpha) interactions, as defined by G(betagamma)-stimulated GTPase activity of alpha(o), but also other G(betagamma)-mediated functions. The NMR structure of a peptide encompassing this region shows a loop exposing the side chains of Glu223 and Tyr224, and peptides with a substitution of either of these amino acids show a complete loss of activity towards G(o). Mutation of this Tyr224 to Ala in full-length phosducin reduced the functional activity of phosducin to that of phosducin's isolated N-terminus, indicating the importance of this residue within the short, structurally defined C-terminal segment. This small peptide derived from phosducin, may represent a model of a G(betagamma) inhibitor, and illustrates the potential of small compounds to affect G(betagamma) functions.     

Arrestin and phosducin are expressed in a small number of brain cells

Retinal photoreceptor rods and pinealocytes contain well-characterized proteins such as arrestin and phosducin whose expression is highly restricted to these cell types. Transgenic mice having a LacZ gene under the control of an arrestin promoter expressed beta-galactosidase (beta-Gal) in the photoreceptor rods and pinealocytes. In addition, it was expressed in very small numbers of discrete cells in the habenular commissura, amygdala, ventral tegmental area and superior colliculus of the brain. Immunocytochemical studies with antibody probes revealed that high level of arrestin and phosducin were also found in the same cell types. Furthermore melatonin was found in those cells of the habenula commissura. The results indicate that novel cell types are present in the brain tissues. Since high levels of arrestin and phosducin expression are generally restricted to photoreceptor rod cells and pinealocytes, these data suggest that certain brain cells may have functions similar to pinealocytes.     

PhLPs and PhLOPs in the phosducin family of G beta gamma binding proteins

In this study, we identify new isoforms of the retinal phosducin and investigate the expression of the phosducin family, showing that an isoform, PhLP1, has sequence homology with Phd and Gbeta gamma binding capability, whereas two isoforms (phosducin-like orphan proteins, PhLOPs) share sequence homology with Phd but fail to bind Gbeta gamma. Original identification of PhLP1 and the PhLOPs was from a human retina cDNA library, using a PCR product for library hybridization screening that contained a predicted functional epitope domain. The screen identified Phd and three related, but distinct, recombinants (PhLP1, PhLOP1, and PhLOP2). By RT-PCR, all isoforms are expressed in either retina or forskolin-stimulated Y79 retinoblastoma cells; however, the new isoforms are below the level of detection on Northern blot analysis. The predicted amino acid translation of each homologue revealed major differences, arising from either splice variants or gene duplication of Phd. To test the functional interaction of all phosducin isoforms with Gbeta gamma in vitro, a glutathione S-transferase (GST) fusion protein was developed for each member. Biochemical interaction with purified retinal transducin Gbeta gamma was verified for GST-Phd and demonstrated for GST-PhLP1; however, neither GST-PhLOP1 nor GST-PhLOP2 bound Gbeta gamma. Comparable results were observed when the GST-phosducin fusion proteins selectively sequestered Gbeta gammas from retinal extracts or when functional Gbeta gamma interactions were assessed using surface plasmon resonance technology. Phosducin and its isoforms are widely distributed in body tissues where they may participate in signal transduction pathways. Phd and PhLP1 possess an 11-amino acid conserved epitope domain (TGPKGVINDWR) that controls the high-affinity binding of Gbeta gamma; these isoforms are implicated in the G-protein signaling pathway. The phosducin-like orphan proteins (PhLOPs) fail to bind Gbeta gamma, suggesting that the PhLOP isoforms may participate in still unidentified signaling pathways.     

Regulation of phosducin phosphorylation in retinal rods by Ca2+/calmodulin-dependent adenylyl cyclase

The phosphoprotein phosducin (Pd) regulates many guanine nucleotide binding protein (G protein)-linked signaling pathways. In visual signal transduction, unphosphorylated Pd blocks the interaction of light-activated rhodopsin with its G protein (Gt) by binding to the beta gamma subunits of Gt and preventing their association with the Gt alpha subunit. When Pd is phosphorylated by cAMP-dependent protein kinase, it no longer inhibits Gt subunit interactions. Thus, factors that determine the phosphorylation state of Pd in rod outer segments are important in controlling the number of Gts available for activation by rhodopsin. The cyclic nucleotide dependencies of the rate of Pd phosphorylation by endogenous cAMP-dependent protein kinase suggest that cAMP, and not cGMP, controls Pd phosphorylation. The synthesis of cAMP by adenylyl cyclase in rod outer segment preparations was found to be dependent on Ca2+ and calmodulin. The Ca2+ dependence was within the physiological range of Ca2+ concentrations in rods (K1/2 = 230 +/- 9 nM) and was highly cooperative (n app = 3.6 +/- 0.5). Through its effect on adenylyl cyclase and cAMP-dependent protein kinase, physiologically high Ca2+ (1100 nM) was found to increase the rate of Pd phosphorylation 3-fold compared to the rate of phosphorylation at physiologically low Ca2+ (8 nM). No evidence for Pd phosphorylation by other (Ca2+)-dependent kinases was found. These results suggest that Ca2+ can regulate the light response at the level of Gt activation through its effect on the phosphorylation state of Pd.     

Identification of two classes of lipid molecule binding sites on the microsomal triglyceride transfer protein

The gene for the microsomal triglyceride transfer protein (MTP) is defective in subjects with the genetic disease abetalipoproteinemia, indicating that MTP is essential for the assembly of apolipoprotein B containing lipoproteins. In vitro, MTP is a lipid molecule binding protein that catalyzes lipid transport between membranes by a shuttle mechanism. In this study, the lipid binding properties of MTP were examined. MTP was incubated with donor phosphatidylcholine vesicles of varying neutral lipid composition. MTP was subsequently reisolated by ultracentrifugation, and MTP-bound lipid was quantitated. When the triolein content of the vesicles was increased up to 4 mol %, neutral lipid binding to MTP increased proportionately, while phosphatidylcholine binding appeared to remain constant around two molecules per MTP. Using phosphatidylcholine emulsions containing 60 mol % triolein as the donor particles resulted in only a slight increase in triolein binding to MTP. The highest triolein:MTP ratio observed was (0.20-0.25):1. Differences in the neutral and phospholipid binding properties of MTP were observed by measuring the transport of lipid from MTP to acceptor vesicles. Transport of triolein was rapid and complete, while phosphatidylcholine transport was biphasic, containing rapid and slow phases. These results indicated that MTP contains more than one class of lipid molecule binding site. Measurements of fluorescent lipid transport from donor vesicles to MTP supported this hypothesis. The transport of pyrene-labeled triglyceride from donor particles to MTP was rapid, while phosphatidylcholine transfer had fast and slow phases. From these data, we propose that MTP contains at least two distinct classes of lipid molecule binding sites that differ in function. The fast site or sites are responsible for lipid transport.     

Outline structure of the human L1 cell adhesion molecule and the sites where mutations cause neurological disorders

The L1 cell adhesion molecule has six domains homologous to members of the immunoglobulin superfamily and five homologous to fibronectin type III domains. We determined the outline structure of the L1 domains by showing that they have, at the key sites that determine conformation, residues similar to those in proteins of known structure. The outline structure describes the relative positions of residues, the major secondary structures and residue solvent accessibility. We use the outline structure to investigate the likely effects of 22 mutations that cause neurological diseases. The mutations are not randomly distributed but cluster in a few regions of the structure. They can be divided into those that act mainly by changing conformation or denaturing their domain and those that alter its surface properties.     

Crystal structure at 2.4 angstroms resolution of the complex of transducin betagamma and its regulator, phosducin

The crystal structure of transducin's betagamma subunits complexed with phosducin, which regulates Gtbetagamma activity, has been solved to 2.4 angstroms resolution. Phosducin has two domains that wrap around Gtbetagamma to form an extensive interface. The N-terminal domain binds loops on the "top" Gtbeta surface, overlapping the Gtalpha binding surface, explaining how phosducin blocks Gtbetagamma's interaction with Gtalpha. The C-terminal domain shows structural homology to thioredoxin and binds the outer strands of Gtbeta's seventh and first blades in a manner likely to disrupt Gtbetagamma's normal orientation relative to the membrane and receptor. Phosducin's Ser-73, which when phosphorylated inhibits phosducin's function, points away from Gtbetagamma, toward a large flexible loop. Thus phosphorylation is not likely to affect the interface directly, but rather indirectly through an induced conformational change.     

Translocation of phosducin in living neuroblastoma x glioma hybrid cells (NG 108-15) monitored by red-shifted green fluorescent protein

Activation of G protein-coupled receptors triggers translocation of certain proteins from cytoplasm to cell membrane located targets. One of these cytosolic proteins is phosducin (Phd) which has been described to compete with G protein-coupled receptor kinases for Gbetagamma dimers attached to the cell membrane, thereby attenuating desensitization of activated receptors. These features of protein redistribution prompted us to examine whether stimulation of membrane associated E-prostaglandin receptors coupled to Gs causes Phd to migrate towards the plasma membrane. We made use of enhanced green fluorescence protein (EGFP), a reporter protein, to follow redistribution of Phd both by means of confocal microscopy and biochemical techniques in living neuronal NG 108-15 hybrid cells challenged with prostaglandin E1 (PGE1). The cells were transiently transfected to express Phd fused to the C-terminus of EGFP, or to express EGFP only. Overexpression of the proteins is implied by FACS analysis as well as by western blot technique, and the functional integrity of EGFP-tagged Phd was confirmed by its ability to elevate cAMP accumulation. Time-lapse imaging of single living cells by means of confocal microscopy revealed that exposure to prostaglandin causes EGFP/Phd, which is evenly spread throughout the cell, to relocate towards the membrane within few minutes. Fluorescence associated with the cell nucleus displayed little rearrangement. The principle finding that prostaglandin triggers translocation of Phd from cytosol to the cell periphery was verified with membranes prepared from EGFP/Phd expressing cells. We found maximal concentrations of membrane associated fluorescent material 5 to 7 min upon prostaglandin exposure. The present study reports for living NG 108-15 hybrid cells that PGE1 stimulation causes cytosolic Phd to translocate towards the membrane, where it is believed to bind to G protein subunits such as Gbetagamma and Galphas.     

Analysis of anatomical sites at which galanin impairs delayed nonmatching to sample in rats.

Galanin, a neuropeptide that coexists with acetylcholine in the septohippocampal pathway of the rat, impairs operant delayed nonmatching to sample (DNMTS) when administered intracerebroventricular/ly (icv). Microinjection experiments were conducted with 40 male rats to determine the anatomical site or sites at which galanin acts to disrupt DNMTS. Galanin (0.1, 0.4, or 1.6 nmol) was microinjected into the ventral hippocampus, amygdala, nucleus basalis magnocellularis, prefrontal cortex, or entorhinal cortex. Galanin disrupted DNMTS in a dose-dependent manner when microinjected into the ventral hippocampus but not at the other sites tested. These findings are consistent with the ability of galanin to inhibit physiological and biochemical actions of acetylcholine in the ventral hippocampus. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The phosphorylation state of phosducin determines its ability to block transducin subunit interactions and inhibit transducin binding to activated rhodopsin

Heterotrimeric GTP-binding proteins (G-proteins) serve many different signal transduction pathways. Phosducin, a 28-kDa phosphoprotein, is expressed in a variety of mammalian cell types and blocks activation of several classes of G-proteins. Phosphorylation of phosducin by cyclic AMP-dependent protein kinase prevents phosducin-mediated inhibition of G-protein GTPase activity (Bauer, P. H., Müller, S., Puzicha, M., Pippig, S., Obermaier, B., Helmreich, E. J. M., and Lohse, M. J. (1992) Nature 358, 73-76). In retinal rods, phosducin inhibits transducin (Gt) activation by binding its beta gamma subunits. While rod phosducin is phosphorylated in the dark and dephosphorylated after illumination (Lee, R.-H., Brown, B. M., and Lolley, R. N. (1984) Biochemistry 23, 1972-1977), the significance of these reactions is still unclear. The data presented here permit a more precise characterization of phosducin function and the consequences of its phosphorylation. Dephosphophosducin blocked binding of the Gt alpha 1 subunit to activated rhodopsin in the presence of stoichiometric amounts of Gt beta gamma, whereas phosphophosducin did not. Surprisingly, the binding affinity of phosphophosducin for Gt beta gamma was not significantly reduced compared with the binding affinity of dephosphophosducin. However, the association of phosducin with Gt beta gamma in a size exclusion column matrix was dependent on the phosphorylation state of phosducin. Moreover, the ability of phosducin to compete with Gt alpha for binding to Gt beta gamma was also dependent on the phosphorylation state of phosducin. No interaction was found between phosducin and Gt alpha. These data indicate that phosducin decreases rod responsiveness by binding to the beta gamma subunits of Gt and preventing their interaction with Gt alpha, thereby inhibiting Gt alpha activation by the activated receptor. Moreover, phosphorylation of phosducin blocks its ability to compete with Gt alpha for binding to Gt beta gamma.     

Adhesion molecule expression in basal cell carcinoma

OBJECTIVE: Extending recall intervals can be an important strategy for making children's dental care more efficient. The purpose of this study was to describe the recall intervals that the clinicians decided were appropriate for children and adolescents when they were instructed to extend and individualise the routines based on clinical judgement. In addition, the effect on recall interval of the profession of the clinician (dentist or dental hygienist), the child's age and the need for fillings were studied. DESIGN: In a four week period in 1995, all dentists and dental hygienists in one county in Norway reported recall intervals for 2,513 children aged 3 to 18 years. RESULTS: The mean current interval since the previous examination was 17.1 months (SD = 4.7 months) and the mean proposed interval until the next examination was 16.4 months (SD = 4.4 months). Approximately 50% of children were evaluated by the clinicians to be suitable for recall intervals of 20 months or more and 10% were assessed as requiring a new examination within 12 months. The length of the current recall interval, the age of the child, whether or not the child received fillings, and whether the decision-maker was a dentist or a dental hygienist were statistically significantly associated with the length of the proposed recall interval. CONCLUSIONS: Basing recall intervals on clinical judgement resulted in intervals longer than 12 months for the majority of the children.     

Participation of fragile sites in cancer

Structural and numerical chromosomal abnormalities are frequent findings in neoplastic cells. The origin of structural rearrangements is probably due to the existence of specific labile areas on human chromosomes. These areas, named fragile sites (FS), are prone to chromosomal breakage and rearrangements, playing an important role in the first steps of carcinogenesis. The classification of FS, the mechanisms involved in FS induction and their biological significance, specially their relationship with cancer development, are discussed.     

DNA-protein interaction sites in differentiating cells. I. Two-dimensional mapping of modulated sites

We have previously shown that DNA-protein attachment sites form during the induction of hematopoietic cell differentiation. Affinity phase-partitioning studies of DNA/protein complexes demonstrated that the DNA involved is not randomly distributed throughout the genome. The object of this study was to use filter binding followed by two-dimensional (2D) polyacrylamide gel electrophoresis (using a neutral 6% gel in the first dimension and a denaturing gradient gel in the second dimension) to gain insight into changes in DNA-protein interactions during induced granulocytic and monocytic differentiation of HL60 cells. Nitrocellulose filter-binding enriched samples for protein-associated DNA sufficiently to change the pattern of DNA spots on 2D gels. The patterns of spots obtained was reasonably reproducible between experiments and highly reproducible within experiments. Gels obtained from cells induced to differentiate by either phorbol ester or all-trans retinoic acid (RA) showed identical patterns for the majority of spots but changes in a small proportion of spots with respect to uninduced controls. Both intensification and reduction/disappearance of spots was observed, demonstrating the existence of both invariant and variant DNA/protein attachment sites during the early stages of hematopoietic cell differentiation. Previous studies have implicated DNA topoisomerase II in chromatin structural changes that are necessary for induction of granulocytic differentiation. We therefore examined the filter-binding DNA preparation by 5'-exonuclease digestion (since topoisomerase II is known to bind covalently to the 5'termini on either side of its cleavage sites). The filter-associated DNA exhibited increased 5' exonuclease protection (with respect to filter flow-through DNA), and the degree of protection increased significantly with exposure to phorbol ester and less markedly with retinoic acid. However, since not all filter DNA was 5' protected, it remains unresolved whether the specific differentiation-associated DNA-protein interactions revealed here involve DNA topoisomerase II or some other protein.