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Biomedical subjects

E M Ross

Publications and source records attributed to E M Ross.

At least 37 records · Page 2Linked to original sources
PubMed1997

Inhibition of brain Gz GAP and other RGS proteins by palmitoylation of G protein alpha subunits.

Palmitoylation of the alpha subunit of the guanine nucleotide-binding protein Gz inhibited by more than 90 percent its response to the guanosine triphosphatase (GTPase)-accelerating activity of Gz GAP, a Gz-selective member of the regulators of G-protein signaling (RGS) protein family of GTPase-activating proteins (GAPs). Palmitoylation both decreased the affinity of Gz GAP for the GTP-bound form of Galphaz by at least 90 percent and decreased the maximum rate of GTP hydrolysis. Inhibition was reversed by removal of the palmitoyl group by dithiothreitol. Palmitoylation of Galphaz also inhibited its response to the GAP activity of Galpha-interacting protein (GAIP), another RGS protein, and palmitoylation of Galphai1 inhibited its response to RGS4. The extent of inhibition of Gz GAP, GAIP, RGS4, and RGS10 correlated roughly with their intrinsic GAP activities for the Galpha target used in the assay. Reversible palmitoylation is thus a major determinant of Gz deactivation after its stimulation by receptors, and may be a general mechanism for prolonging or potentiating G-protein signaling.

Animals↗
PubMed1997

Patient-specific decisions about hormone replacement therapy in postmenopausal women.

OBJECTIVE: To examine the effect of hormone replacement therapy on life expectancy in postmenopausal women with different risk profiles for heart disease, breast cancer, and hip fracture. DESIGN: Decision analysis using a Markov model. Published regression models were used to link risk factors to disease incidence and to estimate the lifetime risks of developing coronary heart disease (CHD), breast cancer, hip fracture, and endometrial cancer. The impact of hormone therapy on disease incidence was estimated from published epidemiologic studies. SETTING: Mathematical model applicable to primary care. INTERVENTIONS: Treatment with hormone replacement therapy or no hormone replacement therapy. MAIN OUTCOME MEASURE: Life expectancy. RESULTS: Hormone replacement therapy should increase life expectancy for nearly all postmenopausal women, with some gains exceeding 3 years, depending mainly on an individual's risk factors for CHD and breast cancer. For women with at least 1 risk factor for CHD, hormone therapy should extend life expectancy, even for women having first-degree relatives with breast cancer. Women without any risk factors for CHD or hip fracture, but who have 2 first-degree relatives with breast cancer, however, should not receive hormone therapy. CONCLUSIONS: The benefit of hormone replacement therapy in reducing the likelihood of developing CHD appears to outweigh the risk of breast cancer for nearly all women in whom this treatment might be considered. Our analysis supports the broader use of hormone replacement therapy.

Aged↗
PubMed1997

A Gs-selective analog of the receptor-mimetic peptide mastoparan binds to Gs alpha in a kinked helical conformation.

Mastoparan, a 14-residue peptide, stimulates GDP/GTP exchange on G proteins in a manner strikingly analogous to that of agonist-bound receptors. Presumably, the peptide structurally mimics a receptor's G protein-binding domain. We previously reported that mastoparan-X binds to alpha-subunits of Gi and Go in a predominantly alpha-helical conformation [Sukumar, M., & Higashijima, T. (1992) J. Biol. Chem. 267, 21421-21424]. We have now developed an analogous peptide, INWKGIASM-alpha-aminoisobutyryl (Aib)-RQVL-NH2 (MP-S), which is a selective activator of Gs. We report the conformation of MP-S when it is bound to Gs alpha, determined from distance geometry calculations based on transferred nuclear Overhauser effects (TRNOEs). The Gs-bound conformation of MP-S is an alpha-helix that is kinked at residue 9. The conformations of MP-S when bound to Gi alpha or Go alpha are similar to the Gs alpha-bound conformation. In contrast, the lipid-bound conformation of MP-S is a straight helix. On the basis of the Gs-bound conformation of MP-S, directions for the design of Gs-selective peptidergic mimics of receptors are suggested.

Drug Design↗
PubMed1997

A GTPase-activating protein for the G protein Galphaz. Identification, purification, and mechanism of action.

A GTPase-activating protein (GAP) specific for Galphaz was identified in brain, spleen, retina, platelet, C6 glioma cells, and several other tissues and cells. Gz GAP from bovine brain is a membrane protein that is refractory to solubilization with most detergents but was solubilized with warm Triton X-100 and purified up to 50,000-fold. Activity is associated with at least two separate proteins of Mr approximately 22,000 and 28,000, both of which have similar specific activities. In an assay that measures the rate of hydrolysis of GTP pre-bound to detergent-soluble Galphaz, the GAP accelerates hydrolysis over 200-fold, from 0.014 to 3 min -1 at 15 degrees C, or to >/=20 min-1 at 30 degrees C. It does not alter rates of nucleotide association or dissociation. When co-reconstituted into phospholipid vesicles with trimeric Gz and m2 muscarinic receptor, Gz GAP accelerates agonist-stimulated steady-state GTP hydrolysis as predicted by its effect on the hydrolytic reaction. In the single turnover assay, the Km of the GAP for Galphaz-GTP is 2 nM. Its activity is inhibited by Galphaz-guanosine 5'-O-thiotriphosphate (Galphaz-GTPgammaS) or by Galphaz-GDP/AlF4 with Ki approximately 1.5 nM for both species; Galphaz-GDP does not inhibit. G protein betagamma subunits inhibit Gz GAP activity, apparently by forming a GTP-Galphazbetagamma complex that is a poor GAP substrate. Gz GAP displays little GAP activity toward Galphai1 or Galphao, but its activity with Galphaz is competitively inhibited by both Galphai1 and Galphao at nanomolar concentrations when they are bound to GTPgammaS but not to GDP. Neither phospholipase C-beta1 (a Gq GAP) nor several adenylyl cyclase isoforms display Gz GAP activity.

Animals↗
PubMed1997

Expression of human muscarinic cholinergic receptors in tobacco.

We expressed human m1, m2 and chimeric muscarinic cholinergic receptors (MAChR) in tobacco plants and in cultured BY2 tobacco cells using Agrobacterium-mediated transformation. The membranes of most transgenic plants and calli bound muscarinic ligands with appropriate affinities, kinetics and pharmacologic specificity, as determined by direct and competitive binding measurements using the muscarinic ligand [3H]quinuclidinyl benzylate (QNB). Membranes of untransformed plants and calli or those transformed with vector alone did not bind [3H]QNB. Preliminary experiments did not suggest regulation of endogenous plant G protein signalling pathways by the recombinant receptors. Membranes from one callus clone expressed m1 MAChR at the level of 2.0-2.5 pmol [3H]QNB bound per mg membrane protein, more than the number of m1 MAChR in mammalian brain and comparable to that expressed in Sf9 insect cells using baculovirus vectors. This work demonstrates high level expression of active G protein-coupled receptors in plants, such that signaling might be genetically reconstituted by co-expression of appropriate G proteins and effectors.

Cells, Cultured↗
PubMed1996

Carboxyl-terminal fragments of phospholipase C-beta1 with intrinsic Gq GTPase-activating protein (GAP) activity.

Fragments of the approximately 50 kDa COOH-terminal region of phospholipase C-beta1 (PLC-beta1(1)), ranging in size from 14 to 38 kDa, were expressed in Escherichia coli, purified, and tested for their regulatory activities. As expected, none of the fragments had phospholipase activity. Several fragments, referred to as PLC tails, displayed GTPase-activating protein (GAP) activity for Gq, the G protein class that stimulates the PLC-betas in response to receptors. Gq GAP activity is characteristic of intact PLC-betas. In reconstituted phospholipid vesicles that contained purified Gq and m1 muscarinic cholinergic receptors, the most active tails increased agonist-stimulated, steady-state GTPase activity over 4-fold. Stimulation of steady-state GTPase by the tails depended on receptors for facilitation of GDP-GTP exchange, suggesting that the tails act by accelerating hydrolysis of bound GTP. In addition to intrinsic GAP activity, one tail with high GAP activity and others with low or minimal activity potentiated the GAP activity of intact PLC-beta1. Other tails inhibited PLC-beta1s GAP effect. Both intrinsic GAP activity and potentiation of the PLC-beta1 GAP effect were often biphasic, with maxima as low as 100 nM tail and declining activities at higher concentrations. Several tails inhibited either the phospholipase activity of PLC-beta1, its stimulation by Gq, or both. The tails thus define the region of PLC-beta1 that has Gq GAP activity and suggest a mechanism of action in which the COOH terminus of PLC-betas can interact with Gq and with other PLC-beta1 molecules.

GTPase-Activating Proteins↗
PubMed1996

Purification and characterization of phospholipase C-beta 1 mutants expressed in E. coli.

In the M1-muscarinic receptor-Gq-phospholipase C-beta 1 (PLC-beta 1) pathway, PLC-beta 1 is both the effector regulated by Gq and acts as GTPase activating protein (GAP) for Gq. To rapidly evaluate in vitro PLC-beta 1 mutants constructed by oligonucleotide-directed mutagenesis, we established a quick expression and purification procedure. A pQE60/His6PLC-beta 1 construct was expressed in E. coli SG13009[pREP4]. Purification (approximately 160-fold) was obtained after high-salt extraction and chromatography over Ni(+)-agarose, Mono Q and Mono S columns. Several His6PLC-beta 1 mutants were equally responsive to alpha q. GTP gamma S, although the mutant His6PLC-beta 1-P57 (G758D) had only 2.5% the intrinsic PLC activity of the wild type. Also, His6PLC-beta 1 wild type and mutants acted as GAPs for Gq in a reconstitution assay. Thus, the present procedure provides a method to quickly assess phospholipase activity, alpha q-responsiveness, and GAP activity of PLC-beta 1.

Amino Acid Sequence↗
PubMed1996

Regulation of phospholipase C-beta1 by Gq and m1 muscarinic cholinergic receptor. Steady-state balance of receptor-mediated activation and GTPase-activating protein-promoted deactivation.

The phospholipase C-beta1 (PLC-beta1) signaling pathway was reconstituted by addition of purified PLC to phospholipid vesicles that contained purified recombinant m1 muscarinic cholinergic receptor, Gq, and 2-4 mol % [3H]phosphatidylinositol 4,5-bisphosphate. In this system, the muscarinic agonist carbachol stimulated steady-state PLC activity up to 90-fold in the presence of GTP. Both GTP and agonist were required for PLC activation, which was observed at physiological levels of Ca2+ (10-100 nM). PLC-beta1 is also a GTPase-activating protein for Gq. It accelerated steady-state GTPase activity up to 60-fold in the presence of carbachol, which alone stimulated activity 6-10-fold, and increased the rate of hydrolysis of Gq-bound GTP by at least 100-fold. Despite this rapid hydrolysis of Gq-bound GTP, the receptor maintained >10% of the total Gq in the active GTP-bound form by catalyzing GTP binding at a rate of at least 20-25 min-1, approximately 10-fold faster than previously described. These and other kinetic data indicate that the receptor and PLC-beta1 coordinately regulate the amplitude of the PLC signal and the rates of signal initiation and termination. They also suggest a mechanism in which the receptor, Gq, and PLC form a three-protein complex in the presence of agonist and GTP (stable over multiple GTPase cycles) that is responsible for PLC signaling.

Amino Acid Sequence↗
PubMed1996

Functional importance of the amino terminus of Gq alpha.

Gq alpha is palmitoylated at residues Cys9 and Cys10. Removal of palmitate from purified Gq alpha with palmitoylthioesterase in vitro failed to alter interactions of Gq alpha with phospholipase C-beta 1, the G protein beta gamma subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated Gq alpha (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of Gq alpha. However, truncated Gq alpha and the single Cys mutants activated phospholipase C-beta 1 normally, while the double Cys mutants were poor activators. Loss of both Cys residues impaired but did not abolish interaction of Gq alpha with m1 receptors. These Cys residues are thus important regardless of their state of palmitoylation. When expressed in HEK-293 or Sf9 cells, all of the proteins studied associated entirely or predominantly with membranes, although a minor fraction of nonpalmitoylated Gq alpha proteins accumulated in the cytosol of HEK-293 cells. When subjected to TX-114 phase partitioning, a significant fraction of all of the proteins, including those with no palmitate, was found in the detergent-rich phase. Removal of residues 1-34 of Gq alpha caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of Gq alpha are thus important for its interactions with effector and receptor, and the amino terminus conveys a hydrophobic character to the protein distinct from that contributed by palmitate.

Animals↗
PubMed1995

Carboxyl-terminal domains in the avian beta 1-adrenergic receptor that regulate agonist-promoted endocytosis.

Most G protein-coupled receptors, including the mammalian beta 2-adrenergic receptor, are endocytosed to an intracellular, vesicular compartment upon continued exposure to agonist. The long form of the avian beta 1-adrenergic receptor, which contains a carboxyl-terminal 59-amino acid extension, does not undergo agonist-promoted endocytosis. We constructed and expressed turkey beta 1-adrenergic receptor cDNAs with regularly spaced carboxyl-terminal truncations and studied their agonist-promoted endocytosis. Removal of 34-86 amino acids from the carboxyl terminus of the turkey receptor allowed its efficient endocytosis, with optimal endocytosis observed upon removal of 59 residues. Removal of only 18 residues allowed some endocytosis. A receptor that lacks the entire carboxyl-terminal region (124 residues) was not endocytosed. We also constructed a chimeric hamster beta 2-adrenergic receptor with the added 59-residue carboxyl-terminal domain of the turkey receptor. The chimera was not significantly endocytosed. These data indicate that residues 450-465 in the carboxyl-terminal region of the beta 1-adrenergic receptor can act independently to block agonist-promoted endocytosis and that other carboxyl-terminal structures nearer to the seventh membrane span are required for endocytosis.

Animals↗
PubMed1995

The carboxyl-terminal anchorage domain of the turkey beta 1-adrenergic receptor is encoded by an alternatively spliced exon.

The originally described cDNA of the turkey beta 1-adrenergic receptor encodes a receptor with a carboxyl-terminal, 59-amino acid extension that was not found in several mammalian beta 1-adrenergic receptors. This extension blocks agonist-promoted endocytosis and down-regulation of the receptor. This carboxyl-terminal domain is encoded by an exon distinct from that which encodes the body of the receptor, and the originally described cDNA results from removal of an 849-nucleotide intron. Unspliced mRNA encodes a shorter open reading frame whose translated carboxyl terminus is identical with that of the mammalian beta 1-adrenergic receptors. There is no evidence for other introns in the coding region. Splicing of the intron to produce the non-endocytosing receptor is highest in fetal blood cells, is appreciable in adult brain and heart, and is detectable in other tissues. Thus, different tissues use alternative splicing to express beta-adrenergic receptors that either do or do not endocytose and down-regulate in response to agonist.

Alternative Splicing↗
PubMed1995

Fetal oxygenation at cordocentesis, maternal smoking and childhood neuro-development.

OBJECTIVE: To assess the relationship between subsequent neuro-development and measurements in small for gestational age (SGA) fetuses, including oxygenation at cordocentesis. DESIGN: A longitudinal study of a cohort of SGA fetuses using those with normal oxygenation as controls. SUBJECTS: 65 chromosomally normal children aged 12-66 months who, as fetuses, had been small for gestational age, had undergone cordocentesis and had been delivered after 32 weeks' gestation. RESULTS: Griffiths' developmental quotient (DQ) was significantly associated with fetal blood pH (r = 0.41, P = 0.0008), PO2 (r = 0.25, P = 0.047) and PCO2 (r = -0.28, P = 0.027) at cordocentesis but not with the severity of growth retardation (r = 0.18, P = 0.17). However, the significant correlations between DQ and both PO2 and PCO2 were explained by their co-correlations with pH and neither remained significant after removing the effect of pH by analysis of variance. The association between DQ and pH was still significant after removing the effect of confounding variables. Maternal smoking during pregnancy was significantly associated with DQ (F to remove = 6.89, P = 0.011) even after allowing for the effects of possible confounding variables including fetal blood pH. CONCLUSIONS: Both fetal pH at cordocentesis and maternal smoking correlate significantly with subsequent neuro-development. Further investigation into the causes of these associations is required.

Child, Preschool↗
PubMed1994

Chimeric muscarinic cholinergic:beta-adrenergic receptors that are functionally promiscuous among G proteins.

We evaluated the G protein selectivity of chimeric M1 and M2 muscarinic cholinergic receptors in which either the third intracellular (I3) loop or the N-terminal portion of this loop (the I3N peptide) was replaced by the corresponding sequence from the beta 1-adrenergic receptor. The chimeras retained agonist-dependent G protein regulatory activity, but were completely promiscuous among potential G protein targets. When expressed in transfected cells, the chimeric receptors activated adenylyl cyclase, the major target of the beta-adrenergic receptor, and activated phospholipase C via a pertussis toxin-insensitive G protein, presumably a Gq. Gs is not a target of either muscarinic receptor, and Gq is not a cellular target of either the M2 muscarinic or beta-adrenergic receptor. When co-reconstituted into phospholipid vesicles with purified G proteins, the chimeric receptors were completely nonselective among all G proteins tested. They activated Gi, G(o), Gz, and Gs with similar efficiencies. This promiscuity was largely suppressed, both in transfected cells and in reconstituted vesicles, by the additional replacement of the second intracellular (I2) loop of the beta-adrenergic receptor. Such double substitutions created receptors specific for Gs, the target of the beta-adrenergic receptor. These findings suggest that G protein specificity depends on the proper combination of multiple regions on a receptor's cytoplasmic surface. In addition, the promiscuous receptors described here may be useful for regulating novel G proteins whose natural regulators are not yet known.

Adenylate Cyclase Toxin↗
PubMed1994

Gs regulation of endosome fusion suggests a role for signal transduction pathways in endocytosis.

Work from several laboratories indicates that guanine nucleotide-binding proteins (GTP-binding proteins) are required for intracellular vesicular transport. In a previous report we presented evidence indicating that one or more heterotrimeric G proteins regulate fusion between endosomes (Colombo, M. I., Mayorga, L. S., Casey, P. J., and Stahl, P. D. (1992) Science 255, 1695-1697). We now report on experiments showing that Gs plays a role in endosome fusion. We have used several reagents known to modulate Gs function including (i) peptides corresponding to the cytoplasmic domains of G protein-coupled receptors and peptides that mimic interaction of receptors with G proteins, (ii) anti-G protein antibodies, and (iii) cholera toxin. Synthetic peptides corresponding to the third cytoplasmic loop of the beta 2-adrenergic receptor which putatively interact with G alpha s inhibited endosomal fusion. The inhibitory effect of these peptides was prevented by a short preincubation of endosomes with guanosine-5'-3-O-(thio)triphosphate or by phosphorylating the peptide with cAMP-dependent protein kinase. The involvement of Gs in endosome recognition and/or the fusion process was assessed by testing an antibody against the COOH terminus of G alpha s. Anti-G alpha s IgG completely abolished fusion between endosomes. Lastly, preincubation of endosomal vesicles with cholera toxin abrogated fusion in the presence of NAD, whereas no effect was observed in the absence of the cofactor. Taken together these findings indicate a role for Gs in either the mechanism or the regulation of fusion among endosomes. These results raise the possibility that signal transduction through cytoplasmic domains of receptors may participate in the regulation of endocytic trafficking.

Amino Acid Sequence↗