Search

G-protein β subunits perform essential neuronal functions as part of G-protein βγ and Gβ5-regulators of G-protein signaling (RGS) complexes. Both Gβγ and Gβ5-RGS are obligate dimers that are thought to require the assistance of the cytosolic chaperonin CCT and a cochaperone, phosducin-like protein 1 (PhLP1) for dimer formation. To test this hypothesisin vivo, we deleted thePhlp1gene in mouse (Mus musculus) retinal rod photoreceptor cells and measured the effects on G-protein biogenesis and visual signal transduction. In the PhLP1-depleted rods, Gβγ dimer formation was decreased 50-fold, resulting in a >10-fold decrease in light sensitivity. Moreover, a 20-fold reduction in Gβ5and RGS9–1 expression was also observed, causing a 15-fold delay in the shutoff of light responses. These findings conclusively demonstratein vivothat PhLP1 is required for the folding and assembly of both Gβγ and Gβ5-RGS9.

eLiposomes are liposomes encapsulating emulsions and therapeutics for targeted delivery. By applying ultrasound to eLiposomes, emulsion droplets can transform from liquid to gas and rupture the lipid bilayer of the eLiposome to release a drug or plasmid. In this study, perfluoropentane (PFC5) emulsions were encapsulated inside folated eLiposomes carrying a model drug (calcein) or a model GFP plasmid to examine the effects of a folate ligand, PFC5 emulsion and various ultrasonic acoustic parameters in drug delivery and gene transfection into HeLa cells. Confocal microscopy was used to quantify drug delivery and the level of plasmid transfection into HeLa cells. The results showed that drug delivery or transfection was minimal without incorporation of internal PFC5 emulsions and folate ligand on the eLiposome surface. It was also shown that application of ultrasound greatly enhanced the drug delivery and plasmid transfection. Delivery of these therapeutics appears to be to the cytosol, indicating that the expansion of the emulsion droplets disrupted both the eLiposomes and the endosomes.

Background Arthropods have received much attention as a model for studying opsin evolution in invertebrates. Yet, relatively few studies have investigated the diversity of opsin proteins that underlie spectral sensitivity of the visual pigments within the diverse beetles (Insecta: Coleoptera). Previous work has demonstrated that beetles appear to lack the short-wavelength-sensitive (SWS) opsin class that typically confers sensitivity to the “blue” region of the light spectrum. However, this is contrary to established physiological data in a number of Coleoptera. To explore potential adaptations at the molecular level that may compensate for the loss of the SWS opsin, we carried out an exploration of the opsin proteins within a group of beetles (Buprestidae) where short-wave sensitivity has been demonstrated. RNA-seq data were generated to identify opsin proteins from nine taxa comprising six buprestid species (including three male/female pairs) across four subfamilies. Structural analyses of recovered opsins were conducted and compared to opsin sequences in other insects across the main opsin classes—ultraviolet, short-wavelength, and long-wavelength. Results All nine buprestids were found to express two opsin copies in each of the ultraviolet and long-wavelength classes, contrary to the single copies recovered in all other molecular studies of adult beetle opsin expression. No SWS opsin class was recovered. Furthermore, the male Agrilus planipennis (emerald ash borer—EAB) expressed a third LWS opsin at low levels that is presumed to be a larval copy. Subsequent homology and structural analyses identified multiple amino acid substitutions in the UVS and LWS copies that could confer short-wavelength sensitivity. Conclusions This work is the first to compare expressed opsin genes against known electrophysiological data that demonstrate multiple peak sensitivities in Coleoptera. We report the first instance of opsin duplication in adult beetles, which occurs in both the UVS and LWS opsin classes. Through structural comparisons of known insect opsins, we suggest that opsin duplication and amino acid variation within the chromophore binding pocket explains sensitivity in the short-wavelength portion of the visible light spectrum in these species. These findings are the first to reveal molecular complexity of the color vision system within beetles. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0674-4) contains supplementary material, which is available to authorized users.

Follicular dendritic cells (FDCs) increase HIV replication and virus production in lymphocytes by increasing the activation of NF-κB in infected cells. Because α-1-antitrypsin (AAT) decreases HIV replication in PBMCs and monocytic cells and decreases NF-κB activity, we postulated that AAT might also block FDC-mediated HIV replication. Primary CD4+ T cells were infected with HIV and cultured with FDCs or their supernatant with or without AAT, and ensuing viral RNA and p24 production were monitored. NF-κB activation in the infected cells was also assessed. Virus production was increased in the presence of FDC supernatant, but the addition of AAT at concentrations >0.5 mg/ml inhibited virus replication. AAT blocked the nuclear translocation of NF-κB p50/p65 despite an unexpected elevation in associated phosphorylated and ubiquitinated IκBα (Ub-IκBα). In the presence of AAT, degradation of cytoplasmic IκBα was dramatically inhibited compared with control cultures. AAT did not inhibit the proteasome; however, it altered the pattern of ubiquitination of IκBα. AAT decreased IκBα polyubiquitination linked through ubiquitin lysine residue 48 and increased ubiquitination linked through lysine residue 63. Moreover, lysine reside 63-linked Ub-IκBα degradation was substantially slower than lysine residue 48-linked Ub-IκBα in the presence of AAT, correlating altered ubiquitination with a prolonged IκBα t1/2. Because AAT is naturally occurring and available clinically, examination of its use as an inhibitory agent in HIV-infected subjects may be informative and lead to the development of similar agents that inhibit HIV replication using a novel mechanism.

The G protein betagamma subunit dimer (Gbetagamma) and the Gbeta5/regulator of G protein signaling (RGS) dimer play fundamental roles in propagating and regulating G protein pathways, respectively. How these complexes form dimers when the individual subunits are unstable is a question that has remained unaddressed for many years. In the case of Gbetagamma, recent studies have shown that phosducin-like protein 1 (PhLP1) works as a co-chaperone with the cytosolic chaperonin complex (CCT) to fold Gbeta and mediate its interaction with Ggamma. However, it is not known what fraction of the many Gbetagamma combinations is assembled this way or whether chaperones influence the specificity of Gbetagamma dimer formation. Moreover, the mechanism of Gbeta5-RGS assembly has yet to be assessed experimentally. The current study was undertaken to directly address these issues. The data show that PhLP1 plays a vital role in the assembly of Ggamma2 with all four Gbeta1-4 subunits and in the assembly of Gbeta2 with all twelve Ggamma subunits, without affecting the specificity of the Gbetagamma interactions. The results also show that Gbeta5-RGS7 assembly is dependent on CCT and PhLP1, but the apparent mechanism is different from that of Gbetagamma. PhLP1 seems to stabilize the interaction of Gbeta5 with CCT until Gbeta5 is folded, after which it is released to allow Gbeta5 to interact with RGS7. These findings point to a general role for PhLP1 in the assembly of all Gbetagamma combinations and suggest a CCT-dependent mechanism for Gbeta5-RGS7 assembly that utilizes the co-chaperone activity of PhLP1 in a unique way.

Members of the phosducin gene family were initially proposed to act as down-regulators of G protein signaling by binding G protein betagamma dimers (Gbetagamma) and inhibiting their ability to interact with G protein alpha subunits (Galpha) and effectors. However, recent findings have over-turned this hypothesis by showing that most members of the phosducin family act as co-chaperones with the cytosolic chaperonin complex (CCT) to assist in the folding of a variety of proteins from their nascent polypeptides. In fact rather than inhibiting G protein pathways, phosducin-like protein 1 (PhLP1) has been shown to be essential for G protein signaling by catalyzing the folding and assembly of the Gbetagamma dimer. PhLP2 and PhLP3 have no role in G protein signaling, but they appear to assist in the folding of proteins essential in regulating cell cycle progression as well as actin and tubulin. Phosducin itself is the only family member that does not participate with CCT in protein folding, but it is believed to have a specific role in visual signal transduction to chaperone Gbetagamma subunits as they translocate to and from the outer and inner segments of photoreceptor cells during light-adaptation.

In the striatum, signaling via G protein-coupled neurotransmitter receptors is essential for motor control. Critical to this process is the effector enzyme adenylyl cyclase type 5 (AC5) that produces second messenger cAMP upon receptor-mediated activation by G protein Golf. However, the molecular organization of the Golf-AC5 signaling axis is not well understood. In this study, we report that in the striatum AC5 exists in a stable pre-coupled complex with subunits of Golf heterotrimer. We use genetic mouse models with disruption in individual components of the complex to reveal hierarchical order of interactions required for AC5-Golf stability. We further identify that the assembly of AC5-Golf complex is mediated by PhLP1 chaperone that plays central role in neurotransmitter receptor coupling to cAMP production motor learning. These findings provide evidence for the existence of stable G protein-effector signaling complexes and identify a new component essential for their assembly. DOI: http://dx.doi.org/10.7554/eLife.10451.001, eLife digest In the brain, cells called neurons communicate with one another using chemicals called neurotransmitters, which bind to receptors on the surface of cells. In most cases, the binding of neurotransmitter causes a protein known as a G protein to interact with the receptor. G proteins are made up of three subunits: α, β and γ. After binding to the receptor, the α subunit separates from the β/γ subunits – which remain together – and both components then act as signals to activate specific targets in the cell. There are many different α, β and γ subunits, which participate in various signaling pathways in different parts of the brain. In the striatum – a region involved in controlling movement – a particular combination of α, β and γ subunits called Golf is responsible for activating an enzyme called adenylyl cyclase type 5 (AC5). Traditionally, G protein signalling has been thought to occur in stages so that the binding of neurotransmitter to receptors on striatal neurons would lead to the dissociation of Golf into the α and βγ subunits. Then the α and βγ subunits are thought to bind to and activate AC5. However, Xie et al. now show that all three subunits of Golf are already found in a stable group (or complex) with AC5 in striatal neurons. Neurotransmitter binding to the receptors causes the entire Golf-AC5complex to rearrange and this process activates AC5. A particular chaperone protein regulates the assembly of the G protein-AC5complex. Mice that lack the gene that encodes this chaperone in striatal neurons struggle to learn how to balance on a rotating rod. In humans, mutations in the genes that encode Golf and AC5 cause dystonia, which is a disorder characterised by involuntary movements. Given the evidence linking this chaperone protein to the regulation of Golf-AC5 signaling, future experiments should investigate whether it might also contribute to dystonia. DOI: http://dx.doi.org/10.7554/eLife.10451.002

G protein β subunits (Gβ) play essential roles in phototransduction as part of G protein βγ (Gβγ) and regulator of G protein signaling 9 (RGS9)-Gβ5 heterodimers. Both are obligate dimers that rely on the cytosolic chaperone CCT and its co-chaperone PhLP1 to form complexes from their nascent polypeptides. The importance of PhLP1 in the assembly process was recently demonstrated in vivo in a retinal rod-specific deletion of the Phlp1 gene. To test whether this is a general mechanism that also applies to other cell types, we disrupted the Phlp1 gene specifically in mouse cones and measured the effects on G protein expression and cone visual signal transduction. In PhLP1-deficient cones, expression of cone transducin (Gt2) and RGS9-Gβ5 subunits was dramatically reduced, resulting in a 27-fold decrease in sensitivity and a 38-fold delay in cone photoresponse recovery. These results demonstrate the essential role of PhLP1 in cone G protein complex formation. Our findings reveal a common mechanism of Gβγ and RGS9-Gβ5 assembly in rods and cones, highlighting the importance of PhLP1 and CCT-mediated Gβ complex formation in G protein signaling.

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein betagamma subunit complex (Gbetagamma) that has been recently shown to catalyze the formation of the Gbetagamma dimer from its nascent polypeptides. Phosphorylation of PhLP at one or more of three consecutive serines (Ser-18, Ser-19, and Ser-20) is necessary for Gbetagamma dimer formation and is believed to be mediated by the protein kinase CK2. Moreover, several lines of evidence suggest that the cytosolic chaperonin complex (CCT) may work in concert with PhLP in the Gbetagamma-assembly process. The results reported here delineate a mechanism for Gbetagamma assembly in which a stable ternary complex is formed between PhLP, the nascent Gbeta subunit, and CCT that does not include Ggamma. PhLP phosphorylation permits the release of a PhLP x Gbeta intermediate from CCT, allowing Ggamma to associate with Gbeta in this intermediate complex. Subsequent interaction of Gbetagamma with membranes releases PhLP for another round of assembly.

Phosducin (Pdc) is a G protein βγ dimer (Gβγ) binding protein, highly expressed in retinal photoreceptor and pineal cells, yet whose physiological role remains elusive. Light controls the phosphorylation of Pdc in a cAMP and Ca2+-dependent manner, and phosphorylation in turn regulates the binding of Pdc to Gtβγ or 14-3-3 proteins in vitro. To directly examine the phosphorylation of Pdc in intact retina, we prepared antibodies specific to the three principal phosphorylation sites (Ser-54, Ser-73, and Ser-106) and measured the kinetics of phosphorylation/dephosphorylation during light/dark adaptation and the subsequent effects on Gtβγ binding. Ser-54 phosphorylation increased slowly (t½ ∼ 90 min) during dark adaptation to ∼70% phosphorylated and decreased rapidly (t½ ∼ 2 min) during light adaptation to less than 20% phosphorylated. Ser-73 phosphorylation increased much faster during dark adaptation (t½ ∼ 3 min) to ∼50% phosphorylated and decreased more slowly during light adaptation (t½ ∼ 9 min) to less than 20% phosphorylated. The Ca2+ chelator BAPTA-AM blocked Ser-54 phosphorylation during dark adaptation but had no effect on Ser-73 phosphorylation. In contrast, Ser-106 was not phosphorylated in either the light or dark. Importantly, Gβγ binding to Pdc was enhanced by Ca2+ chelation and the binding kinetics closely paralleled those of Ser-54 dephosphorylation, indicating that Ser-54 phosphorylation controls Gtβγ binding in vivo. These results suggest a pivotal role of Ser-54 and Ser-73 phosphorylation in determining the interactions of Pdc with its binding partners, Gtβγ and 14-3-3 protein, which may regulate the light-dependent translocation of the photoreceptor G protein.

G proteins (Gαβγ) are essential signaling molecules, which dissociate into Gα and Gβγ upon activation by heptahelical membrane receptors. We have identified the βγ subunit complex of the photoreceptor-specific G protein, transducin (T), as a target of the ubiquitin-proteasome pathway. Ubiquitylated species of the transducin γ-subunit (Tγ) but not the α- or β-subunits were assembled de novo in bovine photoreceptor preparations. In addition, Tγ was exclusively ubiquitylated when Tβγ was dissociated from Tα. Ubiquitylation of Tβγ on Tγ was selectively catalyzed by human ubiquitin-conjugating enzymes UbcH5 and UbcH7 and was coincident with degradation of the entire Tβγ subunit complex in vitroby a mechanism requiring ATP and the proteasome. We also show that Tβγ association with phosducin, a photoreceptor-specific protein of unknown physiological function, blocks Tβγ ubiquitylation and subsequent degradation. Phosphorylation of phosducin by Ca2+/calmodulin-dependent protein kinase II, which inhibits phosducin-Tβγ complex formation, completely restored Tβγ ubiquitylation and degradation. We conclude that Tβγ is a substrate of the ubiquitin-proteasome pathway and suggest that phosducin serves to protect Tβγ following the light-dependent dissociation of Tαβγ.

Phosducin and phosducin-like protein (PhLP) bind G protein βγ subunits and regulate their activity. This report describes a previously uncharacterized binding partner unique to PhLP that was discovered by coimmunoprecipitation coupled with mass spectrometric identification. Chaperonin containing tailless complex polypeptide 1 (CCT), a cytosolic chaperone responsible for the folding of many cellular proteins, binds PhLP with a stoichiometry of one PhLP per CCT complex. Unlike protein-folding substrates of CCT, which interact only in their nonnative conformations, PhLP binds in its native state. Native PhLP competes directly for binding of protein substrates of CCT and thereby inhibits CCT activity. Overexpression of PhLP inhibited the ability of CCT to fold newly synthesized β-actin by 80%. These results suggest that the interaction between PhLP and CCT may be a means to regulate CCT-dependent protein folding or alternatively, to control the availability of PhLP to modulate G protein signaling.

Human tissues that express the angiotensin II (Ang II) type 1 receptor (hAT(1)R) can synthesize four distinct alternatively spliced hAT(1)R mRNA transcripts. In this study, we show that the relative abundance of these mRNA transcripts varies widely in human tissues, suggesting that each splice variant is functionally distinct. Here we demonstrate, for the first time, that the hAT(1)R-B mRNA splice variant encodes a novel long hAT(1)R isoform in vivo that has significantly diminished affinity for Ang II (i.e. >3-fold) when compared with the short hAT(1)R isoform (encoded by hAT(1)R-A mRNA splice variant). This reduced agonist affinity caused a significant shift to the right in the dose-response curve for Ang II-induced inositol trisphosphate production and Ca(2+) mobilization of the long hAT(1)R when compared with that of the short hAT(1)R. The functional differences between these isoforms allows Ang II responsiveness to be fine-tuned by regulating the relative abundance of the long and short hAT(1)R isoform expressed in a given human tissue.

Programmed cell death protein 5 (PDCD5) has been proposed to act as a pro-apoptotic factor and tumor suppressor. However, the mechanisms underlying its apoptotic function are largely unknown. A proteomics search for binding partners of phosducin-like protein, a co-chaperone for the cytosolic chaperonin containing tailless complex polypeptide 1 (CCT), revealed a robust interaction between PDCD5 and CCT. PDCD5 formed a complex with CCT and β-tubulin, a key CCT-folding substrate, and specifically inhibited β-tubulin folding. Cryo-electron microscopy studies of the PDCD5·CCT complex suggested a possible mechanism of inhibition of β-tubulin folding. PDCD5 bound the apical domain of the CCTβ subunit, projecting above the folding cavity without entering it. Like PDCD5, β-tubulin also interacts with the CCTβ apical domain, but a second site is found at the sensor loop deep within the folding cavity. These orientations of PDCD5 and β-tubulin suggest that PDCD5 sterically interferes with β-tubulin binding to the CCTβ apical domain and inhibits β-tubulin folding. Given the importance of tubulins in cell division and proliferation, PDCD5 might exert its apoptotic function at least in part through inhibition of β-tubulin folding.

Phosducin (Pd) and phosducin-like protein (PhLP) have been shown to regulate G-protein signaling by binding Gβγ subunits. To better define the function and regulation of PhLP, and to begin to investigate its potential role in human pathophysiological states, we have cloned the human PhLP (hPhLP) cDNA. The hPhLP shows 92% identity with the rat PhLP (rPhLP). However, unlike the rPhLP, no evidence of hPhLP isoforms were detected in the human tissues investigated. Additionally, unlike the rPhLP, alternative polyadenylation sites were detected in hPhLP cDNA clones which corresponded with two distinct mRNA transcripts, 1.2 kb and 3.1 kb, respectively. Interestingly, the predominantly expressed long transcript contains multiple AU-rich elements (AREs) in its 3′-untranslated region (3′-UTR) which have been shown to correlate with rapid mRNA turnover and translational control. This study shows that the hPhLP AREs are functional both in vitro and in vivo, with the long transcript exhibiting a much shorter mRNA half-life. We also demonstrate that subcloning of either the full-length 3′-UTR or the ARE-rich region of the long transcript immediately following the stop codon of luciferase reporter gene confers instability to the luciferase mRNA and results in a ninefold reduction of luciferase activity in the cell types investigated. Taken together, these findings suggest that the AREs present in the long hPhLP mRNA may play a critical role in the regulation of hPhLP gene expression.

Cholic acid derivatives appended with amine groups have been prepared for use as permeabilizers of the outer membranes of Gram-negative bacteria. These compounds interact synergistically with antibiotics such as erythromycin and novobiocin to inhibit growth of Gram-negative bacteria. When a hydrophobic chain is included on the permeabilizers, they can be converted into potent antibiotics. The role of the hydrophobic chain is to facilitate self-promoted transport of the cholic acid derivatives across the outer membrane of Gram-negative bacteria. These compounds share activities found with polymyxin B and derivatives.

A bacterial biosensor for benzene, toluene, and similar compounds has been constructed, characterized, and field tested on contaminated water and soil. The biosensor is based on a plasmid incorporating the transcriptional activator xylR from the TOL plasmid of Pseudomonas putida mt-2. The XylR protein binds a subset of toluene-like compounds and activates transcription at its promoter, P u . A reporter plasmid was constructed by placing the luc gene for firefly luciferase under the control of XylR and P u . When Escherichia coli cells were transformed with this plasmid vector, luminescence from the cells was induced in the presence of benzene, toluene, xylenes, and similar molecules. Accurate concentration dependencies of luminescence were obtained and exhibited K 1/2 values ranging from 39.0 ± 3.8 μM for 3-xylene to 2,690 ± 160 μM for 3-methylbenzylalcohol (means ± standard deviations). The luminescence response was specific for only toluene-like molecules that bind to and activate XylR. The biosensor cells were field tested on deep aquifer water, for which contaminant levels were known, and were able to accurately detect toluene derivative contamination in this water. The biosensor cells were also shown to detect BETX (benzene, toluene, and xylene) contamination in soil samples. These results demonstrate the capability of such a bacterial biosensor to accurately measure environmental contaminants and suggest a potential for its inexpensive application in field-ready assays.

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.

G protein signaling depends on the ability of the individual subunits of the G protein heterotrimer to assemble into functional complexes. Formation of the G protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings the Gβ and Gγ subunits together. This system includes the cytosolic chaperonin containing TCP-1 (CCT) and its co-chaperone phosducin-like protein 1 (PhLP1). CCT assists Gβ in achieving its β-propeller structure, while PhLP1 releases Gβ from CCT and facilitates its interaction with Gγ. Once Gβγ is formed, PhLP1 remains bound until it is displaced by the Gα subunit and the G protein heterotrimer is brought together. Another obligate dimer is the complex between the G protein β(5) subunit and a regulator of G protein signaling protein (Gβ(5)-RGS). Gβ(5)-RGS also requires CCT for Gβ(5) folding, but PhLP1 plays a different role. It stabilizes the interaction between Gβ(5) and CCT, perhaps to increase folding efficiency. After Gβ(5) folding PhLP1 must subsequently release, allowing the RGS protein to bind and form the Gβ(5)-RGS dimer directly on CCT. Gβ(5)-RGS is then freed from CCT to interact with its membrane anchoring protein and form a stable complex that turns off the G protein signal by catalyzing GTP hydrolysis on Gα.

Direct measurements of the binding between light-activated rhodopsin (Rho*) and transducin, the retinal rod G-protein, revealed a strongly cooperative interaction. Cooperativity was assessed by measuring the association of 125I-labeled transducin (Gt) to Rho* in urea-stripped rod outer segment membranes at equilibrium. Analysis of 125I-Gt binding curves gave a Hill coefficient of 1.8. These data were consistent with a two-site model in which binding of the first 125I-Gt to Rho* increased the binding of the second 125I-Gt approximately 40-fold (Kd values were 80 +/- 30 and 1.9 +/- 0.7 nM, respectively). The effects of GDP on the binding were also investigated. GDP decreased the affinity between Rho* and Gt approximately 100-fold but did not decrease the degree of cooperativity. Binding curves of 125I-Gt in the presence of 1 mM GDP showed a Hill coefficient of 1.9. The data were also consistent with a two-binding site model in which binding of the first 125I-Gt increased the binding of the second 125I-Gt approximately 70-fold (Kd values were 13.7 +/- 5.4 and 0.20 +/- 0.08 microM, respectively). The Gt alpha subunit in the absence of Gt beta gamma also bound Rho* in a cooperative manner. These data implicate a role for the cooperative association of Rho* and Gt in the light activation cascade of retinal rods.

Cholic acid derivatives appended with amine groups have been prepared for use as permeabilizers of the outer membranes of Gram-negative bacteria. These compounds interact synergistically with antibiotics such as erythromycin and novobiocin to inhibit growth of Gram-negative bacteria. When a hydrophobic chain is included on the permeabilizers, they can be converted into potent antibiotics. The role of the hydrophobic chain is to facilitate self-promoted transport of the cholic acid derivatives across the outer membrane of Gram-negative bacteria. These compounds share activities found with polymyxin B and derivatives.

Of the several proteins that bind along the cytoplasmic domain of erythrocyte membrane band 3, only the sites of interaction of proteins 4.1 and 4.2 remain to be at least partially localized. Using five independent techniques, we have undertaken to map and characterize the binding site of band 4.1 on band 3. First, transfer of a radioactive cross-linker (125I-2-(p-azido-salicylamido)ethyl-1-3-dithiopropionate) from purified band 4.1 to its binding sites on stripped inside-out erythrocyte membrane vesicles (stripped IOVs) revealed major labeling of band 3, glycophorin C, and glycophorin A. Proteolytic mapping of the stripped IOVs then demonstrated that the label on band 3 was confined largely to a fragment comprising residues 1-201. Second, competitive binding experiments with Fab fragments of monoclonal and peptide-specific polyclonal antibodies to numerous epitopes along the cytoplasmic domain of band 3 displayed stoichiometric competition only with Fabs to epitopes between residues 1 and 91 of band 3. Weak competition was also observed with Fabs to a sequence of the cytoplasmic domain directly adjacent to the membrane-spanning domain, but only at 50-100-fold excess of Fab. Third, band 4.1 protected band 3 from chymotryptic hydrolysis at tyrosine 46 and to a much lesser extent at a site within the junctional peptide connecting the membrane-spanning and cytoplasmic domains of band 3. Fourth, ankyrin, which has been previously shown to interact with band 3 both near a putative central hinge and at the N terminus competed with band 4.1 for band 3 in stripped IOVs. Since band 4.1 does not associate with band 3 near the flexible central hinge, the competition with ankyrin can be assumed to derive from a mutual association with the N terminus. Finally, a synthetic peptide corresponding to residues 1-15 of band 3 was found to mildly inhibit band 4.1 binding to stripped IOVs. Taken together, these data suggest that band 4.1 binds band 3 predominantly near the N terminus, with a possible secondary site near the junction of the cytoplasmic domain and the membrane.

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein betagamma subunit dimer (Gbetagamma). However, its physiological role is poorly understood. To investigate PhLP function, its cellular expression was blocked using RNA interference, resulting in inhibition of Gbetagamma expression and G protein signaling. This inhibition was caused by an inability of nascent Gbetagamma to form dimers. Phosphorylation of PhLP at serines 18-20 by protein kinase CK2 was required for Gbetagamma formation, while a high-affinity interaction of PhLP with the cytosolic chaperonin complex appeared unnecessary. PhLP bound nascent Gbeta in the absence of Ggamma, and S18-20 phosphorylation was required for Ggamma to associate with the PhLP-Gbeta complex. Once Ggamma bound, PhLP was released. These results suggest a mechanism for Gbetagamma assembly in which PhLP stabilizes the nascent Gbeta polypeptide until Ggamma can associate, resulting in membrane binding of Gbetagamma and release of PhLP to catalyze another round of assembly.

The three-dimensional structure of the complex formed between the cytosolic chaperonin CCT (chaperonin containing TCP-1) and phosducin (Pdc)-like protein (PhLP), a regulator of CCT activity, has been solved by cryoelectron microscopy. Binding of PhLP to CCT occurs through only one of the chaperonin rings, and the protein does not occupy the central folding cavity but rather sits above it through interactions with two regions on opposite sides of the ring. This causes the apical domains of the CCT subunits to close in, thus excluding access to the folding cavity. The atomic model of PhLP generated from several atomic structures of the homologous Pdc fits very well with the mass of the complex attributable to PhLP and predicts the involvement of several sequences of PhLP in CCT binding. Binding experiments performed with PhLP/Pdc chimeric proteins, taking advantage of the fact that Pdc does not interact with CCT, confirm that both the N- and C-terminal domains of PhLP are involved in CCT binding and that several regions suggested by the docking experiment are indeed critical in the interaction with the cytosolic chaperonin.

Phosducin-like protein (PhLP) is a broadly expressed member of the phosducin (Pd) family of G protein betagamma subunit (Gbetagamma)-binding proteins. Though PhLP has been shown to bind Gbetagamma in vitro, little is known about its physiological function. In the present study, the effect of PhLP on angiotensin II (Ang II) signaling was measured in Chinese hamster ovary cells expressing the type 1 Ang II receptor and various amounts of PhLP. Up to 3.6-fold overexpression of PhLP had no effect on Ang II-stimulated inositol trisphosphate (IP(3)) formation, whereas further increases caused an abrupt decrease in IP(3) production with half-maximal inhibition occurring at 6-fold PhLP overexpression. This threshold level for inhibition corresponds to the cellular concentration of cytosolic chaperonin complex, a recently described binding partner that preferentially binds PhLP over Gbetagamma. Results of pertussis toxin sensitivity, GTPgammaS binding, and immunoprecipitation experiments suggest that PhLP inhibits phospholipase Cbeta activation by dual mechanisms: (i) steric blockage of Gbetagamma activation of PLCbeta and (ii) interference with Gbetagamma-dependent cycling of G(q)alpha by the receptor. These results suggest that G protein signaling may be regulated through controlling the cellular concentration of free PhLP by inducing its expression or by regulating its binding to the chaperonin.

Phosducin-like protein (PhLP) is an inducible Gbetagamma binding protein which is hypothesized to be a ubiquitous G protein regulator. To elucidate the mechanisms regulating the expression of the human PhLP (hPhLP) gene, we have cloned and characterized its 5'-flanking region. Primer extension analysis identified a major transcription initiation site 172 bp upstream of the ATG start codon. Analysis of the 5'-flanking region revealed that, although it lacked a TATA box element, the hPhLP promoter did contain several consensus binding motifs including AP4, CCAAT, CREB, NF-kappaB, SP1 and E2F. Transient transfection analyses using a series of 5'-flanking deletion/luciferase reporter gene constructs identified a 25 bp sequence (-80 to -55 bp) that is necessary for basal level transcription of the hPhLP gene in all the cell lines investigated. Interestingly, dependent upon the cell line, distinct transcription factors bind to this region suggesting that basal level hPhLP gene transcription may be regulated in a tissue-specific manner.

Phosducin and phosducin-like protein regulate G protein signaling pathways by binding the betagamma subunit complex (Gbetagamma) and blocking Gbetagamma association with Galpha subunits, effector enzymes, or membranes. Both proteins are composed of two structurally independent domains, each constituting approximately half of the molecule. We investigated the functional roles of the two domains of phosducin and phosducin-like protein in binding retinal G(t)betagamma. Kinetic measurements using surface plasmon resonance showed that: 1) phosducin bound G(t)betagamma with a 2. 5-fold greater affinity than phosducin-like protein; 2) phosphorylation of phosducin decreased its affinity by 3-fold, principally as a result of a decrease in k(1); and 3) most of the free energy of binding comes from the N-terminal domain with a lesser contribution from the C-terminal domain. In assays measuring the association of G(t)betagamma with G(t)alpha and light-activated rhodopsin, both N-terminal domains inhibited binding while neither of the C-terminal domains had any effect. In assays measuring membrane binding of G(t)betagamma, both the N- and C-terminal domains inhibited membrane association, but much less effectively than the full-length proteins. This inhibition could only be described by models that included a change in G(t)betagamma to a conformation that did not bind the membrane. These models yielded a free energy change of +1.5 +/- 0.25 kcal/mol for the transition from the G(t)alpha-binding to the Pd-binding conformation of G(t)betagamma.

Visual signal transduction is a nearly noise-free process that is exquisitely well regulated over a wide dynamic range of light intensity. A key component in dark/light adaptation is phosducin, a phosphorylatable protein that modulates the amount of transducin heterotrimer (Gt alpha beta gamma) available through sequestration of the beta gamma subunits (Gt beta gamma). The structure of the phosphophosducin/Gt beta gamma complex combined with mutational and biophysical analysis provides a stereochemical mechanism for the regulation of the phosducin-Gt beta gamma interaction. Phosphorylation of serine 73 causes an order-to-disorder transition of a 20-residue stretch, including the phosphorylation site, by disrupting a helix-capping motif. This transition disrupts phosducin's interface with Gt beta gamma, leading to the release of unencumbered Gt beta gamma, which reassociates with the membrane and Gt alpha to form a signaling-competent Gt alpha beta gamma heterotrimer.

Phosducin (Pd) is a widely expressed phosphoprotein that regulates G-protein (G) signaling. Unphosphorylated Pd binds to Gbetagamma subunits and blocks their interaction with Galpha. This binding sequesters Gbetagamma and inhibits both receptor-mediated activation of Galpha and direct interactions between Gbetagamma and effector enzymes. When phosphorylated by cAMP-dependent protein kinase, Pd does not affect these functions of Gbetagamma. To further understand the role of Pd in regulating G-protein signaling in retinal rod photoreceptor cells, we have measured the abundance of Pd in rods and examined factors that control the rate of Pd phosphorylation. Pd is expressed at a copy number comparable to that for the rod G-protein, transducin (Gt). The ratio of rhodopsin (Rho) to Pd is 15. 5 +/- 3.5 to 1. The rate of Pd phosphorylation in rod outer segment preparations was dependent on [cAMP]. K1/2 for cAMP was 0.56 +/- 0. 09 microM, and the maximal rate of phosphorylation was approximately 500 pmol PO4 incorporated/min/nmol Rho. In the presence of Gtbetagamma this rate was decreased approximately 50-fold. From these data, one can estimate a t1/2 of approximately 3 min for the rephosphorylation of Pd in rods during the recovery period after a light response. This relatively slow rephosphorylation of the Pd.Gtbetagamma complex may provide a period of molecular memory in which sensitivity to further light stimuli is reduced as a result of sequestration of Gtbetagamma by Pd.

Publisher Summary This chapter describes a method for labeling G t and measuring light-induced association of G t with Rho* in rod disk membranes. As molecular cloning techniques make a variety of G proteins and their receptors available in larger quantities, this approach may become more widely applicable to the study of receptor-G protein interactions. Among the many questions that will be addressed is whether cooperative interactions are a general feature of receptor-G protein binding or whether they are specific to Rho* and G t . The unique ability of rods to amplify and detect very small signals constitutes one of the most remarkable examples of G-protein-dependent signal transduction. There are number of purification schemes for G tα and G tβγ . The procedures described are a modification of those purification schemes, which have found to give consistently high yields of very pure G ta and G tβγ without detectable loss in activity.

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., Muller, 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.

Cyclic nucleotides can regulate the sensitivity of retinal rods to light through phosducin. The phosphorylation state of phosducin determines the amount of G available for activation by Rho*. Phosducin phosphorylation is regulated by cyclic nucleotides through their activation of cAMP-dependent protein kinase. The regulation of phosphodiesterase activity by the noncatalytic cGMP binding sites as well as Ca2+/calmodulin dependent regulation of cGMP binding to the cation channel are also discussed.

Previous studies have demonstrated that modification of erythrocyte membrane cysteine residues via disulfide cross-briding or direct derivatization with thiol reagents promotes massive morphological, rheological, and structural changes in the cell. To determine whether disruption of the band 3-ankyrin interaction, the major membrane-cytoskeletal linkage, might contribute to the above lesions, we quantitatively measured the band 3-ankyrin interaction following modification of Cys-201 and/or Cys-317 of the cytoplasmic domain of band 3. It was observed that irreversible alkylating agents (e.g. N-ethylmaleimide or iodoacetamide and its derivatives), reversible derivatizing compounds (.e.g. p-chloromercuribenzenesulfonate or glutathione), and native disulfide bond formation all blocked the ankyrin interaction. Comparison of the extent of sulfhydryl modification with the degree of inhibition of ankyrin binding further confirmed that cysteine modification was directly responsible for the inhibition. However, analysis of the site of sulfhydryl derivatization revealed that inhibition of ankyrin binding could be initiated in some cases with derivatization of Cys-201, while in other cases obstruction of Cys-317 appeared to be essential. This apparent discrepancy was resolved by demonstrating that Cys-201 of one strand of the cytoplasmic domain of band 3 dimer could disulfide bond with Cys-317 of the opposite strand, thus demonstrating that all four cysteines of the band 3 dimer are clustered at the interface between subunits. We argue that derivatization or disulfide cross-linking of these cysteines can block ankyrin binding by both conformational and steric mechanisms.

The predominant attachment site of the spectrin-based cytoskeleton to the erythrocyte membrane occurs via the interaction of ankyrin with the cytoplasmic domain of band 3 (cdb3). In order to further characterize this interaction, we have conducted experiments to localize the ankyrin-binding site on cdb3. Four monoclonal and three antipeptide polyclonal antibodies were raised against cdb3 and used in competition studies to identify regions of close association of cdb3 with ankyrin. Antibodies to regions of cdb3 near the cytoplasmic domain-membrane spanning domain junction had no effect on 125I-ankyrin binding. Likewise, an antibody to a highly conserved region between residues 142 and 154 did not inhibit ankyrin binding. However, antibodies at or near the cysteine 201-317 cluster and the proposed proline-rich hinge in the center of cdb3 were potent inhibitors of ankyrin association, as were antibodies to the acidic NH2 terminus. Additional evidence for interaction of ankyrin with the NH2-terminal region of cdb3 was obtained by demonstrating the ability of ankyrin to inhibit tyrosine phosphorylation of cdb3 at its NH2 terminus by a purified calf thymus tyrosine kinase. These studies reveal two regions of cdb3, distant in primary sequence, which interact with ankyrin. A specific conformation of cdb3 may be required to permit these regions to simultaneously associate with ankyrin and allow binding to occur.

In earlier model studies we demonstrated that artificially denatured hemoglobin binds to and clusters the protein, band 3, in the plane of the erythrocyte membrane. To determine whether denatured hemoglobin also clusters band 3 in vivo, we have compared the locations of denatured hemoglobin aggregates (Heinz bodies) with band 3 in sickle cells using phase contrast and immunofluorescence microscopy. We report that where Heinz bodies are found associated with the cytoplasmic surface of the membrane, clusters of band 3 are usually colocalized within the membrane. In contrast, normal erythrocyte membranes and regions of sickle cell membranes devoid of Heinz bodies display an uninterrupted staining of band 3. Similarly, ankyrin and glycophorin are periodically seen to aggregate at Heinz body sites, but the degree of colocalization is lower than for band 3. These data demonstrate that the binding of denatured hemoglobin to the membrane forces a redistribution of several major membrane components.

In an effort to evaluate the role of the band 3-ankyrin linkage in maintenance of red blood cell membrane integrity, solution conditions were sought that would selectively dissociate the band 3-ankyrin linkage, leaving other membrane skeletal interactions intact. For this purpose erythrocytes were equilibrated overnight in nutrient-containing buffers at a range of elevated pHs and then examined for changes in mechanical stability and membrane skeletal composition. Band 3 was found to be released from interaction with the membrane skeleton over a pH range (8.4 to 9.5) that was observed to dissociate the band 3- ankyrin interaction in vitro. In contrast, all other membrane skeletal associations appeared to remain intact up to pH 9.3, after which they were also seen to dissociate. Whereas hemolysis of mechanically unstressed cells did not begin until approximately pH 9.3, where the membrane skeletons began to disintegrate, enhanced fragmentation of shear stressed membranes was seen to begin near pH 8, where band 3 dissociation was first observed. Furthermore, the shear-induced fragmentation rate was found to reach a maximum at pH 9.4, ie, where band 3 dissociation was essentially complete. Based on these correlations, we hypothesize that the band 3-ankyrin linkage of the membrane skeleton to the lipid bilayer is essential for red blood cell stability in the face of mechanical distortion but not for cellular integrity in the absence of mechanical stress.