Biomedical subjects

G Weng

Publications and source records attributed to G Weng.

At least 19 recordsLinked to original sources

PubMed2001

The signal transfer regions of G alpha(s).

The crystal structure of soluble functional fragments of adenylyl cyclase complexed with G alpha(s) and forskolin, shows three regions of G alpha(s) in direct contact with adenylyl cyclase. The functions of these three regions are not known. We tested synthetic peptides encoding these regions of G alpha(s) on the activities of full-length adenylyl cyclases 2 and 6. A peptide encoding the Switch II region (amino acids 222-247) stimulated both adenylyl cyclases 2- to 3-fold. Forskolin synergized the stimulation. Addition of peptides in the presence of activated G alpha(s) partially inhibited G alpha(s) stimulation. Corresponding Switch II region peptides from G alpha(q) and G alpha(i) did not stimulate adenylyl cyclase. A peptide encoding the Switch I region (amino acids 199-216) also stimulated AC2 and AC6. The stimulatory effects of the two peptides at saturating concentrations were non-additive. A peptide encoding the third contact region (amino acids 268-286) located in the alpha 3-beta 5 region, inhibits basal, forskolin, and G alpha(s)-stimulated enzymatic activities. Since this region in G alpha(s) interacts with both the central cytoplasmic loop and C-terminal tail of adenylyl cyclases this peptide may be involved in blocking interactions between these two domains. These functional data in conjunction with the available structural information suggest that G alpha(s) activation of adenylyl cyclase is a complex event where the alpha 3-beta 5 loop of G alpha(s) may bring together the central cytoplasmic loop and C-terminal tail of adenylyl cyclase thus allowing the Switch I and Switch II regions to function as signal transfer regions to activate adenylyl cyclase.

Adenylyl Cyclases↗

PubMed1999

Complexity in biological signaling systems.

Biological signaling pathways interact with one another to form complex networks. Complexity arises from the large number of components, many with isoforms that have partially overlapping functions; from the connections among components; and from the spatial relationship between components. The origins of the complex behavior of signaling networks and analytical approaches to deal with the emergent complexity are discussed here.

Animals↗

PubMed1999

Resolution of a signal transfer region from a general binding domain in gbeta for stimulation of phospholipase C-beta2.

Signaling by guanine nucleotide-binding proteins (G proteins) involves sequential protein-protein interactions. G protein-betagamma subunit (Gbetagamma) interactions with phospholipase C-beta2 (PLC-beta2) were studied to determine if all Gbeta contacts are required for signaling. A peptide encoding Gbeta amino acid residues 86 to 105 stimulated PLC-beta2. Six residues (96 to 101) within this sequence could transfer signals and thus constitute a core signal transfer region. Another peptide, encoding Gbeta amino acid residues 115 to 135, did not substantially stimulate PLC-beta2 by itself but inhibited Gbetagamma stimulation, indicating that residues 115 to 135 constitute a general binding domain. Resolution of signal transfer regions from general binding domains indicates that all protein-protein contacts are not required for signal transfer and that it may be feasible to synthesize agonists and antagonists that regulate intracellular signal flow.

Amino Acid Substitution↗

PubMed1998

A novel mutation in the switch 3 region of Gsalpha in a patient with Albright hereditary osteodystrophy impairs GDP binding and receptor activation.

Albright hereditary osteodystrophy (AHO), a disorder characterized by skeletal abnormalities and obesity, is associated with heterozygous inactivating mutations in the gene for Gsalpha. A novel Gsalpha mutation encoding the substitution of tryptophan for a nonconserved arginine within the switch 3 region (Gsalpha R258W) was identified in an AHO patient. Although reverse transcription-polymerase chain reaction studies demonstrated that mRNA expression from wild type and mutant alleles was similar, Gsalpha expression in erythrocyte membranes from the affected patient was reduced by 50%. A Gsalpha R258W cDNA, as well as one with arginine replaced by alanine (Gsalpha R258A), was generated, and the biochemical properties of in vitro transcription/translation products were examined. When reconstituted with cyc- membranes, both mutant proteins were able to stimulate adenylyl cyclase normally in the presence of guanosine- 5'-O-(3-thiotriphosphate) (GTPgammaS) but had decreased ability in the presence of isoproterenol or AlF4- (a mixture of 10 microM AlCl3 and 10 mM NaF). The ability of each mutant to bind and be activated by GTPgammaS or AlF4- was assessed by trypsin protection assays. Both mutants were protected normally by GTPgammaS but showed reduced protection in the presence of AlF4-. The addition of excess GDP (2 mM) was able to rescue the ability of AlF4- to protect the mutants, suggesting that they might have reduced affinity for GDP. A Gsalpha R258A mutant purified from Escherichia coli had decreased affinity for GDP and an apparent rate of GDP release that was 10-fold greater than that of wild type Gsalpha. Sucrose density gradient analysis demonstrated that both Gsalpha R258W and Gsalpha R258A were thermolabile at higher temperatures and that denaturation of both mutants was prevented by the presence of 0.1 mM GTPgammaS or 2 mM GDP. The crystal structure of Gsalpha demonstrates that Arg258 interacts with a conserved residue in the helical domain (Gln170). Arg258 substitutions would be predicted to open the cleft between the GTPase and helical domains, allowing for increased GDP release in the inactive state, resulting in enhanced thermolability and reduced AlF4--induced adenylyl cyclase stimulation and trypsin protection, since activation by AlF4- requires bound GDP.

Adenylyl Cyclases↗

PubMed1998

Molecular basis for interactions of G protein betagamma subunits with effectors.

Both the alpha and betagamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) communicate signals from receptors to effectors. Gbetagamma subunits can regulate a diverse array of effectors, including ion channels and enzymes. Galpha subunits bound to guanine diphosphate (Galpha-GDP) inhibit signal transduction through Gbetagamma subunits, suggesting a common interface on Gbetagamma subunits for Galpha binding and effector interaction. The molecular basis for interaction of Gbetagamma with effectors was characterized by mutational analysis of Gbeta residues that make contact with Galpha-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-beta2, and beta-adrenergic receptor kinase revealed the Gbeta residues required for activation of each effector and provides evidence for partially overlapping domains on Gbeta for regulation of these effectors. This organization of interaction regions on Gbeta for different effectors and Galpha explains why subunit dissociation is crucial for signal transmission through Gbetagamma subunits.

Adenosine Diphosphate Ribose↗

PubMed1997

Adenylyl cyclase 6 is selectively regulated by protein kinase A phosphorylation in a region involved in Galphas stimulation.

Receptors activate adenylyl cyclases through the Galphas subunit. Previous studies from our laboratory have shown in certain cell types that express adenylyl cyclase 6 (AC6), heterologous desensitization included reduction of the capability of adenylyl cyclases to be stimulated by Galphas. Here we further analyze protein kinase A (PKA) effects on adenylyl cyclases. PKA treatment of recombinant AC6 in insect cell membranes results in a selective loss of stimulation by high (>10 nM) concentrations of Galphas. Similar treatment of AC1 or AC2 did not affect Galphas stimulation. Conversion of Ser-674 in AC6 to an Ala blocks PKA phosphorylation and PKA-mediated loss of Galphas stimulation. A peptide encoding the region 660-682 of AC6 blocks stimulation of AC6 and AC2 by high concentrations of Galphas. Substitution of Ser-674 to Asp in the peptide renders the peptide ineffective, indicating that the region 660-682 of AC6 is involved in regulation of signal transfer from Galphas. This region contains a conserved motif present in most adenylyl cyclases; however, the PKA phosphorylation site is unique to members of the AC6 family. These observations suggest a mechanism of how isoform selective regulatory diversity can be obtained within conserved regions involved in signal communication.

Adenylyl Cyclases↗

PubMed1997

Differential regulation of adenylyl cyclases by Galphas.

Regulation of adenylyl cyclases 1, 2, and 6 by Galphas was studied. All three mammalian adenylyl cyclases were expressed in insect (Sf9 or Hi-5) cells by baculovirus infection. Membranes containing the different adenylyl cyclases were stimulated by varying concentrations of mutant (Q227L) activated Galphas expressed in reticulocyte lysates. Galphas stimulation of AC1 involved a single site and had an apparent Kact of 0.9 nM. Galphas stimulation of AC2 was best explained by a non-interactive two site model with a "high affinity" site at 0.9 nM and a "low affinity" site at 15 nM. Occupancy of the high affinity site appears to be sufficient for Gbetagamma stimulation of AC2. Galphas stimulation of AC6 was also best explained by a two-site model with a high affinity site at 0. 6-0.8 nM and a low affinity site at 8-22 nM; however, in contrast to AC2, only a model that assumed interactions between the two sites best fit the AC6 data. With 100 microM forskolin, Galphas stimulation of all three adenylyl cyclases showed very similar profiles. Galphas stimulation in the presence of forskolin involved a single site with apparent Kact of 0.1-0.4 nM. These observations indicate a conserved mechanism by which forskolin regulates Galphas coupling to the different adenylyl cyclases. However, there are fundamental differences in the mechanism of Galphas stimulation of the different adenylyl cyclases with AC2 and AC6 having multiple interconvertible sites. These mechanistic differences may provide an explanation for the varied responses by different cells and tissues to hormones that elevate cAMP levels.

Adenylyl Cyclases↗

PubMed1997

A surface on the G protein beta-subunit involved in interactions with adenylyl cyclases.

Receptor activation of heterotrimeric G proteins dissociates G alpha from the G betagamma complex, allowing both to regulate effectors. Little is known about the effector-interaction regions of G betagamma. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto Gbeta to identify residues on Gbeta that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (Gbeta 86-105) and 115-135 (Gbeta 115-135) from Gbeta. The Gbeta 86-105 peptide inhibited G betagamma stimulation of AC2 and blocked G betagamma inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the Gbeta 115-135 peptide were similar to those of Gbeta 86-105 but Gbeta 115-135 was less efficacious in blocking G betagamma inhibition of AC1. Substitution of Tyr-124 with Val in the Gbeta 115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of Gbeta as a surface that is involved in transmitting signals to effectors.

Adenylyl Cyclases↗

PubMed1996

Gbeta subunit interacts with a peptide encoding region 956-982 of adenylyl cyclase 2. Cross-linking of the peptide to free Gbetagamma but not the heterotrimer.

The region encoded by amino acids 956-982 of adenylyl cyclase 2 is important for Gbetagamma stimulation. Interactions of a peptide encoding the 956-982 region of adenylyl cyclase 2 (QEHAQEPERQYMHIGTMVEFAYALVGK (QEHA peptide)) with Gbetagamma subunits were studied. QEHA peptide was covalently attached to beta subunit of free Gbetagamma by the cross-linker N-succinimidyl(4-iodoacetyl)aminobenzoate. Cross-linking was proportional to the amount of QEHA peptide added; other control peptides cross-linked minimally. When Go was used, very little cross-linking was observed with GDP and EDTA, but upon activation by guanosine 5'-3-O-(thio)triphosphate and Mg2+, specific cross-linking of the QEHA peptide to Gbeta was observed. We conclude that beta subunits of G proteins contain effector interaction domains that are occluded by Galpha subunits in the heterotrimer. Molecular modeling studies used to dock the QEHA peptide on to Gbeta indicate that amino acids 75-165 of Gbeta may be involved in effector interactions.

Adenylyl Cyclases↗

PubMed1996

A resonance Raman study of the C=N configurations of octopus rhodopsin, bathorhodopsin, and isorhodopsin.

The resonance Raman spectra of octopus rhodopsin, bathorhodopsin, and isorhodopsin at 120 K have been obtained as well as those of pigments regenerated with isotopically labeled retinals near the C14-C15 bond. Deuteration of the Schiff base nitrogen induces relatively large changes in the C-C stretch region between 1100 and 1300 cm-1, including a large frequency shift of the C14-C15 stretch mode located at 1206-1227 cm-1 in the three octopus species, as revealed by the Raman spectra of their 14,15-(13)C2 derivatives. Such results are different compared to those of the bovine pigments, in which no significant frequency shift of the C14-C15 stretch mode was observed upon Schiff base N deuteration. In an earlier Raman study of a Schiff base model compound which contained only one single bond adjacent to two double bonds, we have found that the stretch mode of this C-C single bond at 1232 cm-1 shifts up by 15 cm-1 and its intensity is also greatly reduced upon Schiff base N deuteration when the C=N configuration is anti [Deng et al., (1994) J. Phys. Chem. 98, 4776-4779]. The same study has also shown that when the C=N configuration is syn, the C-C stretch mode should be at about 1150 cm-1. Since the C14-C15 stretch mode frequency is relatively high in the spectra of octopus rhodopsin and bathorhodopsin (> 1200 cm-1) and since the normal mode pattern near the Schiff base is similar to the model, we suggest that the C=N configuration in these two species is anti. The different responses of the C14-C15 stretch mode to the Schiff base nitrogen deuteration in bovine and octopus pigments are due to the fact that the coupled C14-C15 stretch and the C12-C13 stretch motions in the model compound or in bovine rhodopsin are altered in octopus rhodopsin so that the stretch motion of the C14-15 bond is more localized, similar to the C-C stretch motion in the small Schiff base model compound. In clear contrast with the bovine rhodopsin Raman spectrum, which is very similar to that for the 11-cis-retinal Schiff base, the drastically different octopus rhodopsin spectrum indicates large protein perturbations on the C11=C12-C13 moiety, either by steric or by electrostatic interactions. Further studies are required to determine if such spectral differences indicate a difference of the energy conversion mechanism in the primary photochemical event of these two pigments.

Animals↗

PubMed1996

Neuropeptide Y receptor gene regulation in mouse adrenocortical Y-1 cells.

The mouse adrenocortical Y-1 cell line expresses a high level of neuropeptide Y1 receptor (NPY-Y1). Moreover the receptor density can be up-regulated by dexamethasone or down-regulated by cAMP. To determine whether such regulation occurs at the level of gene expression, Y1 receptor mRNA was measured using a reverse transcriptase-competitive PCR method. Dexamethasone treatment increased Y1 mRNA in Y-1 cells, whereas the cAMP and ACTH decreased it. We also observed that the amount of Y1 receptor RNA was unaffected by phorbol 12-myristate 13-acetate, a protein kinase C stimulator, but was abolished in a cell line expressing apolipoprotein E (apoE). The results indicated that NPY-Y1 receptor mRNA in Y-1 cells is highly regulated by several intracellular messengers. The role of apoE in such regulation is of particular interest in view of evidence that the isoform of the molecule is highly correlated to the age of onset of Alzheimer's disease. The effect observed in the Y-1 cell line which expresses apoE may implicate a possible role of this protein in the process of neuronal death that occurred in the Alzheimer's disease.

Adrenal Cortex↗

PubMed1996

p21ras supports the survival of chick embryonic motor neurones.

Various trophic factors present in muscle extract can promote the survival of cultured motor neurones. However, little is known about the signal transduction pathways used in these cells. The proto-oncogene product p21ras has been shown to play an important role in proliferative and differentiative signalling pathways. We report here that cytoplasmic introduction of its oncogenic form, p21ras(G12V), fully supports the in vitro survival of chick embryonic motor neurones. The proto-oncogenic form of p21ras also showed a dose-dependent survival effect, while a C-terminally truncated counterpart of p21ras(G12V) was ineffective. These results suggest an involvement of p21ras in signal transduction pathways leading to motor neurone survival and may be of relevance for the development of therapeutic strategies for motor neurone disease.

Animals↗

PubMed1995

Studies on neuropeptide Y receptors in a mouse adrenocortical cell line.

The mouse adrenocortical Y-1 cell line has been found to express high affinity binding sites for neuropeptide Y (NPY). Pharmacological studies have shown that these NPY binding sites are of the Y1 type. Reverse transcription-polymerase chain reaction using primers specific for the rat Y1 receptor revealed that the NPY Y1 receptor mRNA is present in Y-1 cells. The Kd of the receptor for NPY was found to be 1.75 +/- 0.20 nM and the Bmax was 265 +/- 18 fmol/mg. The NPY Y1 receptors in this adrenocortical cell line were shown to be coupled to pertussis toxin-sensitive G proteins. Stimulation of Y1 receptors resulted in the inhibition of forskolin- and adrenocorticotropic hormone (ACTH)-stimulated cAMP synthesis. NPY had no effect on basal steroid release from the Y-1 cells. At an ACTH concentration of 0.1 microM, NPY did not affect ACTH-stimulated steroid release, although NPY did inhibit cAMP production under the same hormonal conditions. cAMP profoundly affected the density of the NPY receptors in Y-1 cells. Treatment of the cells with N6,2'-O-dibutyryl-cAMP or ACTH reduced the Y1 receptor density by > 50%. On the other hand the steroid dexamethasone increased the density of Y1 receptors by 35%. Although additional detailed studies are necessary, these results may have interesting implications for the functions of ACTH, steroids, and NPY in the pituitary-adrenocortical axis.

Adrenal Cortex↗

PubMed1995

[Preparation of oral berberine bisulfate liposomes].

The co-precipitate has been made from berberine bisulfate and polyvingyl pyrrolidone (PVP) with the ratio of 1 : 5. The liposomes entrapped berberine bisulfate have been prepared with the ration of drug to lipid 1 : 20 and the ratio of cholesterol to soy phosphatide 2 : 7. The percentage of encapsulation is 48.72%. The test in vivo of small intestinal absorption in rats shows that liposomes can increase the absorption by 4-fold odd compared with free ber-berine bisulfate.

Animals↗

PubMed1994

Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides.

We have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF-Tu (elongation factor Tu from Escherichia coli) and the c-Harvey ras protein, p21 (the gene product of the human c-H-ras proto-oncogene). Raman spectra of proteins complexed with GDP (guanosine 5' diphosphate), IDP (inosine 5' diphosphate), 6-thio-GDP, and 6-18O-GDP were measured, and the various difference spectra were determined. These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding. Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6-keto and 2-amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins. These shifts are interpreted as being proportional to the local energy of interaction (delta H) between the two groups and protein residues at the nucleotide binding site. Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water. In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF-Tu. Despite the structural similarity of the binding sites of EF-Tu and p21, the strengths of the observed hydrogen bonds at the 6-keto and 2-amino positions vary substantially, by up to a factor of 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1994

New triterpenoid saponins from Mussaenda pubescens.

Three new triterpenoidal saponins, mussaendosides O [1], P [2], and Q [3] were isolated from whole plants of Mussaenda pubescens. These structures were elucidated on the basis of chemical and spectral methods, such as their 1H-1H COSY, HMQC, HMBC, TOCSY, and NOESY nmr spectra.

Carbohydrate Sequence↗

PubMed1993

A proposed bovine neuropeptide Y (NPY) receptor cDNA clone, or its human homologue, confers neither NPY binding sites nor NPY responsiveness on transfected cells.

Receptors with seven transmembrane domains (7TM) constitute a large family of structurally and functionally related proteins which respond to various types of ligands. We describe here the cloning and expression of a human 7TM receptor, denoted hFB22 (human Fetal Brain 22), which is the homologue (92% amino acid identity) of a bovine receptor (LCR1) reported by others to bind neuropeptide Y (NPY) with a pharmacological profile of the Y3 receptor subtype. However, upon expression in COS1 (confirmed by Northern analysis), COS7 or CHO-K1 cells, the hFB22 receptor did not confer specific 125I-Bolton-Hunter-NPY, 3H-propionyl-NPY or 125I-peptide YY (PYY) binding sites, in either intact cells or in membrane preparations. Similarly, cells transfected with the corresponding bovine clone (LCR1) did not show specific NPY/PYY binding exceeding that resulting from endogenous binding sites; mock-transfected COS7 cells, used frequently for heterologous expression of receptors, were found to have endogenous specific 125I-NPY binding sites (Bmax = 112 fmol/mg protein; Kd = 0.25 nM). Moreover, the hFB22 transfected cells, when compared to control transfected cells, did not display de novo NPY- or PYY-induced second messenger responses, i.e., (1) inhibition of forskolin-stimulated cAMP accumulation or (2) 45Ca2+ influx. The presence of hFB22 mRNA was detected in several human neuroblastoma cell lines, none of which was found to express Y3-like NPY binding sites. hFB22 displays 39% amino acid sequence identity (in the transmembrane regions) to the human interleukin-8 receptor, and 32-36% amino acid identity to the human receptors of angiotensin II, bradykinin, and n-formylpeptide, but only 23% amino acid identity to the previously described human NPY/PYY receptor of the Y1 receptor subtype. Our results show that hFB22 and LCR1 do not encode NPY receptors, and their true ligand(s) remains to be identified.

Amino Acid Sequence↗

PubMed1993

In vitro decalcification of aortic valve leaflets with the Er:YSGG laser, Ho:YAG laser, and the Cavitron ultrasound surgical aspirator.

This study was designed to compare the efficacy of the erbium:yttrium-scandium-gallium-garnet (Er:YSGG) laser and the holmium:yttrium-aluminum-garnet (Ho:YAG) lasers in debriding calcium from freshly explanted aortic valve leaflets and to compare the Er:YSGG laser with the Cavitron ultrasonic surgical aspirator (CUSA). Aortic valve leaflets were freshly explanted from patients undergoing aortic valve replacement for aortic stenosis. Initially, 4 leaflets each were debrided with the Er:YSGG and the Ho:YAG lasers to attempt removal of calcium deposits while preserving the underlying integrity of the leaflets and minimizing thermal damage. The Er:YSGG laser was more effective in doing so with less thermal and photoacoustic damage when compared with the Ho:YAG laser. Twelve more leaflets each were then debrided with the Er:YSGG laser and the CUSA. The Er:YSGG laser again proved less injurious to the underlying leaflet. The CUSA-treated leaflets demonstrated shattering and disruption of adjacent tissue as well as collagen fiber exposure. These changes were not seen with the Er:YSGG laser. Because of these properties, the Er:YSGG laser merits further evaluation as a tool for aortic valvuloplasty procedures in selected patients with senescent calcific aortic stenosis.

Aortic Valve↗