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Biomedical subjects

L Birnbaumer

Publications and source records attributed to L Birnbaumer.

At least 73 records · Page 4Linked to original sources
PubMed1995

STE2/SCG1-dependent inhibition of STE4-induced growth arrest by mutant STE4 delta C6 in the yeast pheromone response pathway.

The yeast pheromone response pathway involves the activation of a heterotrimeric G protein composed by SCG1 (alpha) (also GPA1), STE4 (beta), and STE18 (gamma) subunits by the pheromone-activated receptors STE2 and STE3 in a and alpha cells, respectively. Upon exchange of bound GDP for GTP in the SCG1 subunit, the release of STE4/STE18 dimer occurs which, in turn causes activation of downstream effectors leading growth arrest and mating competence. Over-expression of STE4 also leads to growth arrest in a STE18 dependent manner. Removal of 6 amino acids from the C-terminus of STE4 rendered a subunit incapable of downstream signalling but still able to interact with STE18. This delta C6 mutant acts as a dominant negative because it blocks the growth arresting effect obtained by over-expression of STE4. The inhibitory effect of STE4 delta C6 is dependent on the presence of the SCG1 subunit in a STE2 but not ste2 background. Inhibition of the growth arresting effect of STE4 by the delta C6 mutant is not due to competition at the effector site, but rather involves an intrinsic activity of STE2 that is dependent on SCG1.

GTP-Binding Protein alpha Subunits↗
PubMed1995

Homologous desensitization of the murine luteinizing hormone receptor expressed in L cells.

Using a clonal cell line that stably expresses the murine luteinizing hormone receptor (LHR 11/6 cells), we studied the molecular mechanisms of agonist-induced desensitization of the luteinizing hormone/chorionic gonadotropin-responsive adenylyl cyclase. Exposure of transfected cells to human chorionic gonadotropin (hCG) resulted in a dose-dependent loss of maximal hCG-stimulable adenylyl cyclase activity without a significant shift to the right of the dose-response curve to hCG. This rapid uncoupling of the LH receptor from the cellular adenylyl cyclase system was not accompanied by internalization of receptor sites. A 6-h exposure to hCG led only to minor (ca. 25%) loss of membrane binding sites. The dose-response curve to hCG was not altered by pretreating cells with 8-Br-cAMP or prostaglandin E1. These findings, and the observation that hCG-induced desensitization can still be monitored at Mg2+ concentrations in the assay as high as 10 mM, preclude a significant contribution of protein kinase A to LH receptor uncoupling. The murine LH receptor not only stimulates adenylyl cyclase but also phospholipase C and probably protein kinase C (PKC) via diacylglycerol. Activation of PKC by 4 beta-phorbol 12-myristate 13-acetate failed to desensitize. When PKC was down-regulated hCG could still exert a maximal desensitizing effect. It is concluded that in LHR 11/6 cells there is no evidence for a major role of PKC in homologous desensitization. Thus, it is likely that a second messenger-independent kinase, such as beta-adrenergic receptor kinase, or a different, as yet unknown mechanism is involved in the agonist-induced desensitization of the LH receptor.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Gi2 alpha protein deficiency: a model of inflammatory bowel disease.

Mice deficient for the G protein subunit Gi2 alpha were obtained by gene targeting. They displayed a growth retardation that was apparent at 6 weeks of age. They subsequently developed diffuse colitis with clinical and histopathological features closely resembling those of ulcerative colitis in humans. Seven of 20 Gi2 alpha-deficient mice with colitis also developed adenocarcinomas of the colon. Gi2 alpha-deficient thymocytes displayed two- to fourfold increases in mature CD4+8- and CD4-8+ phenotypes, an approximately threefold increase in high-intensity CD3 staining and enhanced proliferative responses to T-cell receptor stimuli. Stimulation of Gi 2 alpha-deficient peripheral T cells induced a hyperresponsive profile of interleukin-2, tumour necrosis factor, and interferon-gamma production, which may reflect a heightened response of primed cells or a defective negative regulation. We suggest that Gi 2 alpha-deficient mice may represent a useful animal model for dissecting the pathomechanisms of inflammatory bowel disease and also for the development of novel therapeutic strategies.

Amino Acid Sequence↗
PubMed1995

Alpha-1 subunits of voltage gated Ca2+ channels in the mesencephalon x neuroblastoma hybrid cell line MES23.5.

The identity of alpha 1 subunits from voltage operated Ca2+ channels was determined in the rat/mouse mesencephalon x N18TG2 hybridoma cell line MES23.5, by sequence analysis of reverse transcription-polymerase chain reaction products and antagonist binding. Sequences were derived from the L-(alpha 1D), Q-(alpha 1A) and omega-conotoxin GVIA sensitive N-type (alpha 1B) Ca2+ channel alpha 1 subunits. The amplified fragments, which are homologous to the region between domain III and IV of known alpha 1 subunits, reveal splice variation in the L- and Q-type alpha 1 subunit of MES23.5 cells. The transcripts of alpha 1 subunits in these cells were quantified by RNAase protection assay. The data show the existence of different Ca2+ channel types in a single cell line and may reflect multiple functions of voltage operated Ca2+ channels during growth, differentiation and transmitter release.

Amino Acid Sequence↗
PubMed1995

Ulcerative colitis and adenocarcinoma of the colon in G alpha i2-deficient mice.

G proteins are involved in cellular signalling and regulate a variety of biological processes including differentiation and development. We have generated mice deficient for the G protein subunit alpha i2 (G alpha i2) by homologous recombination in embryonic stem cells. G alpha i2-deficient mice display growth retardation and develop a lethal diffuse colitis with clinical and histopathological features closely resembling ulcerative colitis in humans, including the development of adenocarcinoma of the colon. Prior to clinical symptoms, the mice show profound alterations in thymocyte maturation and function. The study of these animals should provide important insights into the pathogenesis of ulcerative colitis as well as carcinogenesis.

Adenocarcinoma↗
PubMed1995

Gi proteins and the response to 5-hydroxytryptamine in porcine cultured endothelial cells with impaired release of EDRF.

1. The receptor-mediated release of endothelium-derived relaxing factor(s) (EDRF) requires the presence of different functional G proteins in endothelial cells. Release of EDRF in response to 5-hydroxytryptamine (5-HT), which involves activation of pertussis toxin-sensitive Gi proteins, is impaired in both regenerated endothelium of the coronary artery following balloon catheterization and in porcine cultured endothelial cells. This study used porcine cultured endothelial cells as a model of regenerated endothelium to determine if the abnormal release of EDRF in response to 5-HT may be associated with the loss of functional pertussis toxin-sensitive Gi proteins. 2. Binding studies on porcine cultured endothelial cells demonstrated specific binding sites for [3H]-5-HT. Scatchard analyses revealed a single binding site for [3H]-5-HT with Kd of 7.2 +/- 3.5 nM and maximal binding (Bmax) of 121.4 +/- 51.3 fmol mg-1 protein. Binding of [3H]-5-HT was displaced by methiothepin (5-HT1 and 5-HT2 antagonist; Ki = 6.2 +/- 1.2 nM), but not by ketanserin (preferential 5-HT2 antagonist). 3. Gi alpha 1 protein was expressed in cultured but not in native endothelial cells. Gi alpha 2 and Gi alpha 3 proteins were expressed to significant levels in porcine native and cultured endothelial cells, as detected by Northern and Western blot analysis. 4. In membranes from cultured endothelial cells, two bands of 40 and 41 kDa, which corresponded to the Gi alpha 2 and the combination of Gi alpha 3-Gi alpha 1 proteins, respectively, were ADP-ribosylated by pertussis toxin. The labelling intensity was Gi alpha 2>Gi alpha 3-Gi alpha l and the amount of ADP-ribosylation was not different between porcine native and cultured endothelial cells. Stimulation of the cultured cells with 5-HT (3 x 10-6 M; 4 min) decreased significantly further ADP-ribosylation of Gi alpha 2 by pertussis toxin, but not that of Gi alpha 3 and/or Gi alpha l.5. The present results suggest that porcine endothelial cell culture may lead to the abnormal expression of Gi alpha l protein and that the dysfunctional release of EDRF from cultured porcine endothelial cells in response to 5-HT is not associated with the loss of Gi alpha proteins or the absence of 5-HT binding sites.

Adenosine Diphosphate Ribose↗
PubMed1995

Dual activation of the cardiac Ca2+ channel alpha 1C-subunit and its modulation by the beta-subunit.

Ca2+ channels are heteromultimeric proteins in which the alpha 1-subunit forms the voltage-dependent Ca(2+)-selective ionic channel. We reported recently that coexpression of the beta-subunit with the cardiac alpha 1-subunit (alpha 1C) facilitates channel opening without affecting either the amplitude or the time course of the gating currents (13). Here we present evidence for the existence of two modes of channel opening. Xenopus oocytes expressing the alpha 1C-subunit alone display two modes of activation as indicated by the double-exponential time course of macroscopic ionic currents and the two open-time distributions of single channels. Coexpression of the beta-subunit potentiates Ca2+ currents by a relative increase of the fast-activating component, an acceleration of the slow component, and a larger proportion of long openings. We propose that multiple modes of gating are encoded in the alpha 1-subunit and that the beta-subunit increases Ca2+ channel opening by favoring a willing mode of gating in which the final transitions leading to channel opening are facilitated. In addition, we show that the carboxy terminus of alpha 1C also modulates the channel-gating behavior.

Animals↗
PubMed1994

Requirement for intramolecular domain interaction in activation of G protein alpha subunit by aluminum fluoride and GDP but not by GTP gamma S.

An ion-counterion interaction between the lysine of the NKXD motif in the GTPase domain and an aspartate in the inserted helical domain of alpha subunits of heterotrimeric G proteins, Lys-278 and Asp-158, respectively, of Gs alpha is shown to be essential for activation by AlF4- and partially so for interaction with beta gamma dimers and activation by GTP and receptor. However, this domain interaction is not required for activation by the non-hydrolyzable analog guanosine 5'-3-O-(thio)triphosphate. Proximity of the helical domain to the GTPase domain is thus involved in the fundamental inactive-->active transition of the protein in a way that further distinguishes alpha subunits of heterotrimeric G proteins from ras and ras-like GTPases that lack helical domains and are neither activated by AlF4- nor combine with beta gamma dimers.

Aluminum Compounds↗
PubMed1994

Appearance of a novel Ca2+ influx pathway in Sf9 insect cells following expression of the transient receptor potential-like (trpl) protein of Drosophila.

Activation of phospholipase C, elevation of free cytosolic Ca2+ concentration ([Ca2+]i) and stimulation of Ca2+ influx have been implicated in Drosophila phototransduction. Electrophysiological studies suggest that trp and trpl proteins may be important for the light-activated Ca2+ current found in Drosophila photoreceptor cells. Although these proteins exhibit homologies to voltage-gated Ca2+ and Na+ channels, their actual function in insect cells and their relation to proteins involved in mammalian cell Ca2+ signaling remains unknown. In the present study, [Ca2+]i was examined in fura-2-loaded Sf9 insect cells infected with recombinant baculovirus containing cDNA for the trpl protein. Ca2+ influx was examined by use of Ba2+, a Ca2+ surrogate that is not a substrate for Ca(2+)-pumps or carriers and by measurement of whole-cell membrane currents. The results suggest that expression of trpl is associated with appearance of a Ca2+ permeable, non-selective cation channel formed by the trpl protein.

Animals↗
PubMed1994

Human-Xenopus chimeras of Gs alpha reveal a new region important for its activation of adenylyl cyclase.

G proteins are heterotrimeric GTPases that play a key role in signal transduction. The alpha subunit of Gs bound to GTP is capable of activating adenylyl cyclase. The amino acid sequences derived from two X. laevis cDNA clones that apparently code for Gs alpha subunits are 92% identical to those found in the short form of human Gs alpha. Despite this high homology, the X. laevis Gs alpha clones expressed in vitro, yielded a protein that are not able to activate the adenylyl cyclase present in S49 cyc- membranes in contrast with human Gs alpha similarly expressed. This finding suggested that the few amino acid substitutions found in the amphibian subunit are important in defining the functionality of the human Gs alpha. The construction of chimeras composed of different fractions of the cDNAs of the two species was adopted as an approach in determining the regions of the molecule important in its functionality in this assay. Four pairs of chimeras were constructed using reciprocal combinations of the cDNAs coding for human and Xenopus Gs alpha. These eight constructs were expressed in vitro and equivalent amounts of the resulting proteins were assayed in the activation of adenylyl cyclase with GTP gamma and isoproterenol. The results obtained here clearly indicate that the G alpha sequence that extends from amino acid 70 to 140, is important for the functionality of human Gs alpha in activating adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1994

Modification of Ca2+ channel activity by deletions at the carboxyl terminus of the cardiac alpha 1 subunit.

Voltage-sensitive Ca2+ channels are multisubunit complexes that include, among others, a large alpha 1 subunit, which by itself is sufficient to form a channel. Several alpha 1 genes encoding L-, N-, and P-type Ca2+ channels have been cloned. These alpha 1 genes share a high degree of sequence homology in the putative transmembrane regions, but vary substantially in the putative intracellular loops and the flanking amino and carboxyl termini. In the present study, we investigated the functional roles of the 665-amino acid long carboxyl terminus of a cardiac alpha 1 by constructing deletion mutants. Expression in Xenopus oocytes of delta C1856, delta C1733, and delta C1700, which lack from 307 to 472 amino acids at the carboxyl terminus, led to inward Ba2+ currents that were 4- to 6-fold greater than observed with the 2171-amino acid long wild type alpha 1. Ionic currents increased without a change in the amount of charge moved during voltage-dependent gating, suggesting that the increase in ionic currents was not due to an increase in the number of channels that were expressed. Single channel analysis revealed an unaltered unitary conductance. Thus, removal of up to 70% of the carboxyl terminus increased current density by facilitating the coupling between the voltage-dependent gating and channel opening, leading to an increased opening probability of the channel.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

The amino terminus of a calcium channel beta subunit sets rates of channel inactivation independently of the subunit's effect on activation.

There is molecular diversity in both alpha 1 and beta subunits of voltage-gated Ca2+ channels. Coupling between voltage sensing and pore opening of the C-type alpha 1 (alpha 1c) is improved by the type 2 beta subunit (beta 2), and E-type alpha 1 beta complexes inactivate at different rates depending on the nature of beta. We compared the effects of type 1 and 2 beta subunits on activation of the human E-type alpha 1 (alpha 1E) with the effects they have on inactivation, as seen in Xenopus oocytes. The beta subtypes stimulated activation in similar fashion but affected inactivation differently, and even in opposing directions. beta subunits have a common central core but differ in their N- and C-termini and in a central region. N-terminal chimeras between beta 1 and beta 2 subunits that have opposing effects on inactivation resulted in the reciprocal transfer of their effects. We conclude that regulation of activation and inactivation of alpha 1 by beta are separable events and that the N-terminus of beta is one of the structural determinants important in setting the rate and voltage at which an alpha 1 inactivates.

Amino Acid Sequence↗
PubMed1994

Ca(2+)-dependent inactivation of a cloned cardiac Ca2+ channel alpha 1 subunit (alpha 1C) expressed in Xenopus oocytes.

The alpha 1 subunit of cardiac Ca2+ channel, expressed alone or coexpressed with the corresponding beta subunit in Xenopus laevis oocytes, elicits rapidly inactivating Ca2+ currents. The inactivation has the following properties: 1) It is practically absent in external Ba2+; 2) it increases with Ca2+ current amplitudes; 3) it is faster at more negative potentials for comparable Ca2+ current amplitudes; 4) it is independent of channel density; and 5) it does not require the beta subunit. These findings indicate that the Ca2+ binding site responsible for inactivation is encoded in the alpha 1 subunit and suggest that it is located near the inner channel mouth but outside the membrane electric field.

Alternative Splicing↗
PubMed1994

Pertussis toxin-sensitive G proteins in regenerated endothelial cells of porcine coronary artery.

Endothelium-dependent, pertussis toxin-sensitive relaxations are impaired selectively after regeneration of endothelial cells following balloon denudation of the porcine coronary artery. The present study was designed to examine the hypothesis that there is a difference in G proteins modified by pertussis toxin between regenerated and intact endothelial cells. Yorkshire pigs, fed a high-cholesterol diet, underwent balloon denudation of the endothelium of the left anterior descending coronary arteries (LAD). Four weeks after the denudation the animals were killed to detect G proteins by ADP ribosylation catalyzed with pertussis toxin and [32P]NAD, separated on a urea gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In membrane fractions of endothelial cells obtained from previously denuded LAD, G alpha i-1/G alpha i-3 (41 kDa) and G alpha 1-2 (40 kDa) proteins were labeled. The two bands revealed on the gel were the same as those obtained from intact left circumflex coronary arteries (LCX). However, the intensity of the bands was less prominent in the LAD than the LCX. These results suggest that either a decreased amount or a reduced functionality of Gi proteins in the regenerated endothelial cells may account for the impairment in the pertussis toxin-sensitive relaxations after balloon injury of coronary arteries in the pigs.

Adenosine Diphosphate Ribose↗
PubMed1994

Dual signaling potential is common among Gs-coupled receptors and dependent on receptor density.

We studied the relative ability to activate phospholipase C (PLC) of four Gs-coupled receptors expressed in L cells at different densities. Stable cell lines expressing various levels of the luteinizing hormone receptor (LHR), the type 2 vasopressin receptor (V2R), or the type 1 or type 2 beta-adrenergic receptor (beta 1- or beta 2AR) were isolated. The PLC activity was assessed by the measurement of free intracellular Ca2+ concentrations and the accumulation of inositol phosphates. We previously reported that, at 24,000 sites/cell, the LHR in L cells stimulated adenylyl cyclase by 10-fold over basal levels and PLC by 50% over basal levels. The EC50 for stimulation was 20-fold higher for PLC than for adenylyl cyclase. We now report that LHR tends to stimulate PLC more at a higher receptor density and less at a lower density. EC50 values for accumulation of inositol phosphates remained unchanged. The human V2R and the human beta ARs are strong adenylyl cyclase stimulators, and their potential for dual signaling was unknown. Expressing the V2R at 100,000 sites/cell or more and the beta ARs at 300,000 sites/cell resulted in stimulation of PLC by these receptors. As with the LHR, higher concentrations of vasopressin or isoproterenol were needed to reach 50% stimulation of PLC, compared with that of adenylyl cyclase. The beta 1AR was a stronger PLC stimulator than was the beta 2AR. The orders of potency for isoproterenol, epinephrine, and norepinephrine to stimulate adenylyl cyclase and PLC were the same for each of the two beta ARs. These results indicate that the ability of Gs-coupled receptors to stimulate PLC is dependent on the levels of receptor expression, and they suggest that dual signaling potential is a common property of Gs-coupled receptors and possibly also of G(i)-coupled receptors.

Adenylyl Cyclases↗