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S Marullo

Publications and source records attributed to S Marullo.

At least 37 records · Page 2Linked to original sources
PubMed1996

New molecular and structural determinants involved in beta 2-adrenergic receptor desensitization and sequestration. Delineation using chimeric beta 3/beta 2-adrenergic receptors.

As the beta 3-adrenergic receptor (beta3AR) is resistant to short term agonist-promoted desensitization and sequestration, chimeric beta3/beta2 receptors were generated to identify the molecular determinants responsible for these regulatory processes in the beta2AR. By exchanging single or multiple intracellular domains of the beta3AR for the corresponding regions of the beta2AR, we show that specific domains can be identified as additive determinants for desensitization, while sequestration is more dependent on global structural conformation. The carboxyl-terminal tail, the third and the second intracellular loops of the beta2AR provided additive contributions to the desensitization observed upon short term agonist stimulation. The second intracellular loop plays a role which is as important as that of third cytoplasmic loop and carboxyl-terminal tail which had previously been identified as the major determinants of agonist-promoted desensitization. Additive contributions of the cytoplasmic domains of the beta2AR were also observed for agonist-promoted sequestration. The substitution of the first and second intracellular loops and the carboxyl tail were associated with a beta2-like sequestration phenotype. However, in contrast to what is observed for desensitization the co-substitution of the third cytoplasmic loop with any of the other domains completely suppressed sequestration. These results suggest that sequestration depends not only on appropriate interactions of multiple molecular determinants within the cytoplasmic region of the beta2AR but also on conformational determinants that may influence their orientation.

Adenylyl Cyclases↗
PubMed1995

Functional effects of long-term activation on human beta 2- and beta 3-adrenoceptor signalling.

The functional effects of long-term activation of beta-adrenoceptors were investigated by measuring adenylyl cyclase activity, cyclic AMP accumulation and cyclic AMP-dependent protein kinase activity in CHW and L cells expressing either human beta 2- or beta 3-adrenoceptors. Pre-incubation of CHW and L cells expressing beta 2-adrenoceptors with 10 microM isoprenaline for 24 h produced a marked reduction in the total receptor number and dramatically reduced the capacity of the receptor to stimulate adenylyl cyclase maximally. In contrast, the ability of beta 3-adrenoceptors number was observed in L but not in CHW cells. Maximal levels of intracellular cyclic AMP concentrations were reached during the first hour of receptor activation with isoprenaline in all four cell lines. In the absence of phosphodiesterase inhibitors, cyclic AMP decreased to basal levels within 24 h of continuous stimulation. This phenomenon occurred more rapidly in cells expressing the beta 2- than the beta 3-adrenoceptors. These results confirm that, at the level of adenylyl cyclase stimulation and cyclic AMP accumulation, the beta 3-adrenoceptor is more resistant than the beta 2-adrenoceptor to long-term desensitization. However, when cyclic AMP-dependent protein kinase activity was considered, a 24 h stimulation of beta 2- and when cyclic AMP-dependent protein kinase activity was considered, a 24 h stimulation of beta 2- and beta 3-adrenoceptor expressing cells led to the desensitization of the kinase in L but not in CHW cells. In conclusion, long-term desensitization may have distinct functional effects on cell signalling depending on the receptor subtype and the cell type considered. These findings might have practical implications for future strategies involving long-term therapies with receptor agonists.

Adenylyl Cyclases↗
PubMed1995

Quantitative analysis of transforming growth factor beta 1 messenger RNA in the liver of patients with chronic hepatitis C: absence of correlation between high levels and severity of disease.

Transforming growth factor beta 1 (TGF beta 1) is a cytokine involved in liver fibrogenesis. Previous semiquantitative studies of patients with chronic viral hepatitis showed that liver TGF beta 1 messenger RNA (mRNA) was increased, compared with normal controls and with patients with chronic hepatitis C virus (HCV) infection who responded favorably to interferon alfa (IFN alpha) treatment. To evaluate its potential prognostic significance, we measured liver TGF beta 1 mRNA, using a new competitive reverse gene amplification assay, in a total of 35 patients with chronic HCV. This technique was reproducible and sensitive; we could measure as few as 5,000 molecules of TGF beta 1 mRNA per microgram of total liver RNA. In patients with chronic HCV, the mean level of TGF beta 1 mRNA was 200-fold higher than in controls. However, no correlation could be found between TGF beta 1 mRNA and either the biological (serum amino-terminal peptide of type III procollagen) and histological (Knodell scores) indices of liver fibrosis or a favorable response to IFN alpha therapy. In 9 patients, second liver specimens were obtained after treatment; in most cases, TGF beta 1 mRNA levels and hepatic histological findings varied in parallel. These data are consistent with the hypothesis that TGF beta 1 plays a role in stimulating liver fibrogenesis during chronic HCV, despite the lack of prognostic value of TGF beta 1 mRNA levels measured before treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Species difference in the G protein selectivity of the human and bovine A1-adenosine receptor.

The purified bovine brain A1-adenosine receptor has previously been shown to discriminate among closely related G protein alpha-subunits. To obtain analogous information for the human receptor, the cDNA coding for the human A1-adenosine receptor was inserted into a plasmid placing the synthesis of the receptor protein under the control of the MalE promoter. Following induction by maltose, active receptor accumulated in Escherichia coli membranes. Binding of the antagonist 8-[3H]cyclopentyl-1,3-dipropylxanthine to E. coli membranes (KD approximately 2 nM, Bmax approximately 0.2-0.4 pmol/mg) showed the appropriate pharmacological profile. Incubation of E. coli membranes with purified Go,i-reconstituted guanine nucleotide-sensitive high affinity binding of the agonist (-)[125I] N6-3-(iodo-4-hydroxyphenylisopropyl)adenosine to the receptor (KD approximately 1 nM). In the presence of purified beta gamma-subunit, the recombinant receptor interacted equally well with the recombinant G protein alpha-subunits Gi alpha-1, Gi alpha-2, Gi alpha-3; G(o) alpha displayed a lower affinity for the receptor while Gs alpha was inactive. Parallel experiments were carried out in bovine and human brain membranes pretreated with N-ethylmaleimide to inactivate the endogenous G(o)/Gi proteins; Gi alpha-3 was most potent in reconstituting 125I-HPIA binding to bovine membranes, while Gi alpha-1, Gi alpha-2, and G(o) alpha displayed similar affinities. However, in human membranes, Gi alpha-1, Gi alpha-2, and Gi alpha-3, were equipotent and high concentrations of G(o) alpha were required to promote 125I-HPIA binding. These observations show (i) that functional human A1-adenosine receptors were synthesized in E. coli; (ii) that the pattern of G protein coupling is identical for the recombinant human A1-receptor and its counterpart in the native membrane; (iii) and that species differences between bovine and human receptor exist not only in their pharmacological profile but also in their G protein specificity suggesting that species homologues of receptors may use different signaling mechanisms.

Animals↗
PubMed1994

Cellular signaling by an agonist-activated receptor/Gs alpha fusion protein.

The consequences of agonist-dependent activation of guanine nucleotide-binding protein (G protein)-coupled receptors vary from cell to cell, depending on a complex network of regulations between components of the signaling cascade. Specific interactions between receptors, G proteins, and effectors are difficult to analyze in intact cells. Engineering of receptor-transducer fusion proteins might be an effective strategy to target cellular effectors more efficiently and specifically. As a model, we evaluated the ability of a fusion protein of beta 2-adrenergic receptor bound to the alpha subunit of adenylyl cyclase-stimulatory G protein (Gs alpha) to restore the defective activation of adenylyl cyclase in S49 cyc- cells that lack endogenous Gs alpha. The coupling between the two partners of the fusion protein was functional, and the agonist-dependent activation of the effector was more potent and more productive in transfected than in wild-type S49 cells. The covalent link between receptor and Gs alpha could thus convey an advantage over freely interacting components. Such receptor-G alpha fusion proteins may help to elucidate the complex interactions between members of signaling pathways and may also constitute a useful tool for studying the effects of single effector activation.

Adenylyl Cyclases↗
PubMed1994

Cell-specific down-regulation of the beta 3-adrenergic receptor.

Previous studies have shown that beta 3-adrenergic receptors, in contrast to the beta 1 and beta 2 subtypes, do not undergo desensitization following short term activation (minutes) with agonists. Longer activation (hours) has been shown to induce desensitization of the beta 3-adrenergic receptor in some, but not all, cases, suggesting that cell- or species-specific mechanisms may be involved. We investigated the contribution of the cell type to the pattern of beta 3-adrenergic receptor long term desensitization by studying, in parallel, two cell lines (Chinese hamster fibroblasts and murine Ltk- cells) expressing the human beta 3-adrenergic receptor. Sustained agonist-promoted down-regulation of the beta 3-adrenergic receptor could be induced in Ltk- cells, whereas only a transient and weak reduction of receptor number was observed in Chinese hamster fibroblasts. The half-life of the beta 3-adrenergic receptor was not affected by the agonist activation in either cell line, indicating that in contrast to the beta 2-adrenergic receptor, degradation of preexisting receptor protein does not contribute to down-regulation. Sustained reduction of receptor RNA levels, monitored by reverse transcriptase polymerase chain reaction, was exclusively shown in Ltk- cells and probably accounted for most of the observed down-regulation. Differences in the ability of the receptor to stimulate adenylyl cyclase activity in the two cell lines may be responsible for the distinct patterns of beta 3-adrenergic receptor down-regulation.

Adenylyl Cyclases↗
PubMed1993

[Bacillary peliosis in AIDS. Anatomo-clinical study of 2 cases].

The authors report two cases of peliosis hepatis, occurring in patients with AIDS, who presented a persistent fever and an hepatomegaly. The liver biopsies showed areas of peliosis, where bacilli were observed by Warthin-Starry stain. In one case, techniques of molecular biology allowed the identification of Rochalimaea henselae, pathogen involved in bacillary angiomatosis. This rickettsia has been newly recognized in the United-States, where 17 cases of bacillary peliosis have been published in immunocompromised hosts and mainly in patients with AIDS. These observations illustrate the clinical and histological features of this new opportunistic infection, as it is described in the literature. The clinical signs include an unexplained fever, an hepatomegaly, and in 75% of the cases, a splenomegaly. The cutaneous lesions of bacillary angiomatosis are associated in 40% of the cases. An antibiotic treatment by erythromycin ensures a complete recovery.

AIDS-Related Opportunistic Infections↗
PubMed1993

The human beta 3-adrenergic receptor is resistant to short term agonist-promoted desensitization.

The human beta 3-adrenergic receptor (beta 3AR) lacks most of the structural determinants that, in the beta 2AR, contribute to agonist-induced receptor desensitization. To evaluate the effect of these structural differences on the beta 3AR desensitization profile, the human beta 2- and beta 3AR were stably expressed in Chinese hamster fibroblasts (CHW) and murine Ltk- cells (L cells). Incubation of CHW-beta 2 or L-beta 2 cells with 10 microM isoproterenol for 30 min induced a decrease in the maximal agonist-stimulated adenylyl cyclase activity and a cAMP-dependent reduction in the potency of isoproterenol to stimulate the receptor. In addition, this pretreatment impaired the formation of the high affinity heterotrimeric agonist-receptor-guanine nucleotide-binding protein complex and induced the sequestration of approximately 30% of the beta 2AR away from the cell surface. In contrast, similar treatment of CHW-beta 3 and L-beta 3 cells did not affect the maximal receptor-stimulated adenylyl cyclase activity, nor did it induce any significant sequestration of the beta 3AR. In fact, only a modest cAMP-independent decrease in the potency of isoproterenol to stimulate the receptor could be observed after isoproterenol treatment. The rapid desensitization pattern of a chimeric beta 3AR, in which the third cytoplasmic loop and the carboxyl-terminal tail were exchanged with those of the beta 2AR (which include potential phosphorylation sites and other possible molecular determinants of desensitization), was found to be intermediate between those of the two original receptor subtypes. These results demonstrate that (i) the beta 3AR is less prone than the beta 2AR to undergo rapid agonist-promoted desensitization and, (ii) in addition to the phosphorylation sites located in the third cytoplasmic loop and the carboxyl-terminal tail of the beta 2AR, other molecular determinants contribute to short term desensitization.

Adenylyl Cyclases↗
PubMed1993

Lupoid sclerosis with antiphospholipid and antimyelin antibodies.

Lupoid sclerosis is a rare syndrome associating clinical symptoms of multiple sclerosis (MS), positive false tests for syphilis and positive tests for antinuclear and anticardiolipin antibodies. In a patient with lupoid sclerosis, antimyelin antibodies were detected by indirect immunofluorescence on human sciatic nerve sections. These antibodies were not found in the serum of control patients with MS nor in sera of patients with antiphospholipid autoantibodies and focal ischemic neurologic disease. The presence of such antimyelin antibodies may contribute to the underlying physiopathological mechanism of this syndrome.

Adult↗
PubMed1992

Functional expression of the human serotonin 5-HT1A receptor in Escherichia coli. Ligand binding properties and interaction with recombinant G protein alpha-subunits.

Signaling through serotonin 5-HT1A receptors involves multiple pathways. We have investigated the functional coupling of the human 5-HT1A receptor to different G proteins using an in vitro reconstitution system based on the expression of recombinant receptor (r5-HT1A) and G alpha-subunits (rG alpha) in Escherichia coli. The r5-HT1A receptor was expressed by insertion in a vector allowing its active expression in E. coli inner membranes. Binding of the selective agonist [3H] +/- 8-hydroxy-(2-N-dipropylamine)tetralin ([3H]8-OH-DPAT) to intact bacteria or E. coli membranes was saturable with a KD of approximately 8 nM and an average of 120 sites/bacterium. Binding properties of several serotoninergic ligands to r5-HT1A receptors were comparable with those measured in mammalian cells. Incubation of rG alpha.beta gamma with E. coli membranes resulted in high affinity agonist [3H]8-OH-DPAT binding (KD = 0.7 nM) and titration with a panel of rG alpha subtypes showed the order of potency: rGi alpha-3 greater than rGi alpha-2 greater than rGi alpha-1 much greater than rGo alpha, while rGs alpha appeared incapable of interacting with 5-HT1A receptors. Moreover, agonist-mediated enhancement of [35S]guanosine 5'-O-(3-thiotriphosphate) binding to rGi alpha confirmed the achievement of the functional interaction between receptor and G proteins. Our findings are in agreement with the in vivo ability of 5-HT1A receptors to activate Gi alpha related to adenylyl cyclase inhibition or K+ channel activation, but do not support previously reported adenylyl cyclase stimulation through interaction with Gs alpha.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

The human beta 3-adrenergic receptor: relationship with atypical receptors.

Atypical beta-adrenergic receptors (beta AR), different from beta 1 and beta 2ARs, have been suggested to modulate energy expenditure. We have characterized a gene coding for a third human beta AR, beta 3AR, whose sequence is 402 amino acids long and is 50.7% and 45.5% homologous to that of the human beta 1 and beta 2AR, respectively. The KD of [125I]-iodocyanopindolol for beta 3AR is 10-fold higher than for beta 1 or beta 2AR. The receptor has an apparent molecular weight of 65,000. Agonists for the beta 3AR induce cyclic AMP accumulation. Among 11 beta antagonists tested, only ICI118551 and CGP20712A, previously classified as, respectively, beta 1 and beta 2 selective, inhibit this effect. The beta 1 and beta 2 antagonists pindolol, oxprenolol, and CGP12177 are agonists of the beta 3AR. The potency order of beta agonists at beta 3 sites correlates with that for stimulation of lipolysis in rat fat tissues. Moreover, because beta 3AR mRNA was detected in rodent adipose tissues, liver, and muscle, we propose that the beta 3AR participates to the control by catecholamines of energy expenditure.

Amino Acid Sequence↗
PubMed1991

Expression of two human beta-adrenergic receptors in Escherichia coli: functional interaction with two forms of the stimulatory G protein.

When expressed in Escherichia coli, the human beta 1- and beta 2-adrenergic receptors retain their ligand binding specificity. Their functional integrity was investigated by analyzing receptor-guanine nucleotide-binding regulatory (G) protein coupling by using two splice variants of the alpha subunit of the stimulatory G protein Gs synthesized in E. coli (rGs alpha-S and rGs alpha-L) and the beta gamma subunits of G protein purified from bovine brain. In competition binding experiments with (-)-[125I]iodocyanopindolol and (-)-isoproterenol, rGs alpha-S.beta gamma and rGs alpha-L.beta gamma reconstituted guanine nucleotide-sensitive high-affinity agonist binding with comparable affinities, whereas rGs alpha PT, a mutant of rGs alpha-L with an altered carboxyl terminus, and a recombinant subtype of the alpha subunit of the inhibitory G protein, rGi alpha-1, were approximately 20- and approximately 200-fold less potent, respectively. A comparison of the beta 1- and beta 2-adrenergic receptor expressed in E. coli with the beta 2-receptor in S49 murine lymphoma cyc- cell membranes revealed a similar affinity of rGs alpha-S and rGs alpha-L for the recombinant and native receptors. After stable incorporation of rGs alpha-S.beta gamma into E. coli membranes, receptor-G protein coupling was also verified by determining the isoproterenol-mediated acceleration of the rate for guanine 5'-[gamma-[35S]thio]triphosphate binding. These results show that (i) receptor-G protein coupling can be reconstituted in E. coli using recombinant components and that (ii) such an approach may be more generally used to evaluate coupling preferences between defined molecular species of receptors and G-protein subunits.

Cell Membrane↗
PubMed1991

Expression of three human beta-adrenergic-receptor subtypes in transfected Chinese hamster ovary cells.

The genes coding for three pharmacologically distinct subtypes of human beta-adrenergic receptors (beta 1 AR, beta 2 AR and beta 3 AR) were transfected for expression in Chinese hamster ovary (CHO) cells. Stable cell lines expressing each receptor were analyzed by ligand binding, adenylate cyclase activation and photoaffinity labeling, and compared to beta AR subtypes observed in previously described tissues, primary cultures and transfected cell lines. Each of the three receptor subtypes displayed saturable [125I]iodocyanopindolol-binding activity. They showed the characteristic rank order of potencies for five agonists, determined by measuring the accumulation of intracellular cAMP. These recombinant cell lines express a homogeneous population of receptors and display the known pharmacological properties of beta 1 AR and beta 2 AR, in human tissues. It is therefore likely that the pattern of ligand binding and adenylate cyclase activation, mediated by the new beta 3 AR in CHO cells, also reflects the yet-undetermined pharmacological profile in humans.

Adenylyl Cyclases↗
PubMed1990

Localization and characterization of three different beta-adrenergic receptors expressed in Escherichia coli.

After fusion with the N-proximal portion of the outer membrane protein LamB, three beta-adrenergic receptors, the human beta 1- and beta 2- and turkey beta 1-adrenergic receptor, were expressed in Escherichia coli with retention of their own specific pharmacological properties. Molecular characterization and localization of the three receptors in bacteria and comparison of the behaviour of each hybrid protein are reported. The bacteria were lysed and fractionated on a sucrose gradient. Saturable [125I]iodocyanopindolol binding activity was found associated mainly with the inner membrane fraction, suggesting that the receptor is correctly folded in this membrane. Binding activity was also found in the outer membrane fraction but varied according to the receptor type. Photoaffinity labeling experiments revealed that the receptors exhibit binding activity only after proteolytic removal of the LamB moiety from the fusion protein. The three hybrid proteins, detected in immunoblots by anti-peptide antibodies, were found mainly in the outer membrane fraction. Each of them exhibited different susceptibility to intrinsic bacterial proteolytic enzymes; sites of proteolytic cleavage were localized by the use of anti-peptide antibodies. The functional expression in E. coli of three beta-adrenergic receptors with similar structure but different amino acid sequences suggests that this expression system may be a general feature among similar receptors of the family of G-protein-coupled receptors. The level of expressed binding activity of a given receptor will be within the control of proteolytic degradation processes, depending on the primary sequence of the receptor. Constructions of new hybrid proteins, in combination with expression in protease mutants of E. coli, should help in controlling such processes.

Animals↗
PubMed1990

Selective binding of ligands to beta 1, beta 2 or chimeric beta 1/beta 2-adrenergic receptors involves multiple subsites.

The molecular basis of ligand binding selectivity to beta-adrenergic receptor subtypes was investigated by designing chimeric beta 1/beta 2-adrenergic receptors. These molecules consisted of a set of reciprocal constructions, obtained by the exchange between the wild-type receptor genes of one to three unmodified transmembrane regions, together with their extracellular flanking regions. Eight different chimeras were expressed in Escherichia coli and studied with selective beta-adrenergic ligands. The evaluation of the relative effect of each chimeric exchange on ligand binding affinity was based on the analysis of delta G values, calculated from the equilibrium binding constants, as a function of the number of substituted beta 2-adrenergic receptor transmembrane domains. The data showed that the contribution of each exchanged region to subtype selectivity varies with each ligand; moreover, while several regions are critical for the pharmacological selectivity of all ligands, others are involved in the selectivity of only some compounds. The selectivity displayed by beta-adrenergic compounds towards beta 1 or beta 2 receptor subtypes thus results from a particular combination of interactions between each ligand and each of the subsites, variably distributed over the seven transmembrane regions of the receptor; these subsites are presumably defined by the individual structural properties of the ligands.

Adrenergic beta-Agonists↗