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Biomedical subjects

L Birnbaumer

Publications and source records attributed to L Birnbaumer.

At least 109 records · Page 6Linked to original sources
PubMed1992

Guanosine 5'-O-(3-thiotriphosphate) causes endothelium-dependent, pertussis toxin-sensitive relaxations in porcine coronary arteries.

To determine whether direct stimulation of endothelial G-proteins causes relaxations of the underlying vascular smooth muscle, the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and sodium fluoride were studied in porcine coronary arteries and endothelial cells. Isometric tension was measured in coronary rings contracted with prostaglandin F2 alpha. GTP gamma S (in the presence of saponin) and sodium fluoride (in the presence of AlCl3) relaxed rings with, but not those without endothelium. The responses were inhibited by nitro-L-arginine and pertussis toxin. In membrane fractions of coronary endothelial cells, GTP gamma S and sodium fluoride inhibited the ADP-ribosylation of G-proteins catalyzed with [32P]-NAD and pertussis toxin. These data suggest that direct stimulation of G-proteins in endothelial cells by GTP gamma S and sodium fluoride causes a pertussis toxin-sensitive relaxation which may be attributed to the release of nitric oxide.

Adenosine Diphosphate Ribose↗
PubMed1992

Xenopus laevis oocyte G alpha subunits mRNAs. Detection and quantitation during oogenesis and early embryogenesis by competitive reverse PCR.

The expression of mRNAs coding for different Xenopus laevis oocyte G alpha subunits was analyzed by the PCR technique. Using the nucleotide sequences of five previously cloned cDNAs for oocyte G alpha subunits [FEBS Lett. 244, 188-192, 1989; FEBS Lett. 268, 27-31, 1990] and the highly sensitive reverse PCR reaction we found that G alpha o, G alpha i-1, G alpha i-3 and G alpha s species are present in oocyte stage VI, G alpha o mRNA being the most abundant transcript. G alpha o mRNA was further quantitated through oogenesis, unfertilized eggs and early embryogenesis stages by a competitive PCR reaction using an 'in vitro' deleted G alpha o mRNA as the internal standard. Using this approach we found that Xenopus G alpha o mRNA levels were constant during oogenesis and unfertilized eggs at a concentration of 3.5 pg of mRNA/stage (5 x 10(5) molecules) and diminish gradually during early embryogenesis, reaching a level of 0.3 pg in the gastrula stage. These findings show that oocyte G alpha o, and perhaps the rest of the alpha subunits, are expressed as maternal mRNAs and could play an important role in signal transduction at the beginning of oocyte cell differentiation.

Animals↗
PubMed1992

Molecular cloning of a human serotonin receptor (S12) with a pharmacological profile resembling that of the 5-HT1D subtype.

We report the molecular cloning of a fragment of human genomic DNA called S12, containing an open reading frame of 1170 nucleotides, which encodes a receptor for serotonin of 390 amino acids. The receptor function of the S12 protein was demonstrated by functional expression in mouse LS12 cells obtained by stable transfection of Ltk- cells, and LM5S12 cells, derived from LM5 cells (Ltk- cells previously transfected with the M5 muscarinic acetylcholine receptor). Adenylyl cyclase studies showed that the S12 receptor is able to mediate inhibition of adenylyl cyclase in response to serotonin in both types of cells. As studied in LM5S12 cells, the S12 receptor did not promote Ca2+ mobilization from internal stores, nor did it significantly modulate the sustained increase in [Ca2+]i elicited by stimulation of the phospholipase C stimulating M5 acetylcholine receptor. The pharmacologic profile of S12 as seen in adenylyl cyclase assays is as follows: (EC50 in nM): serotonin, full agonist (37 nM), 5-carboxamidotryptamine, full agonist (10 nM), sumatriptan, full agonist (50 nM), metergoline, partial agonist (10 nM), methysergide, partial agonist (40 nM), yohimbine, partial agonist (150 nM), metitepin, antagonist (KB = 0.7 to 1.2 nM). We propose that the human S12 serotonin receptor is a receptor of the 5-hydroxytryptamine1D subtype.

Adenylyl Cyclase Inhibitors↗
PubMed1992

Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells.

The murine receptor for luteinizing hormone (LHR) was cloned and expressed in L cells. This LHR (mature protein of 674 amino acids) is very similar to that of the rat (same length, 36 amino acid differences) but differs significantly more from that of man (673 amino acids, 109 differences). Expression of the murine LHR in L cells led to the appearance of binding sites for human chorionic gonadotropin (hCG) with a Kd of 150 pM and an LH- and hCG-stimulable adenylyl cyclase activity (EC50 = 50-100 pM hCG). Upon labeling pools of phosphoinositides with [3H]myo-inositol, L cells expressing the murine LHR responded to hCG with an increase in their rate of phosphoinositide hydrolysis (EC50 = 2,400 pM hCG). This was accompanied by an increase in intracellular Ca2+ [( Ca2+]i), as determined by the Fura2 method. This increase in [Ca2+]i in response to hCG was dependent on the LHR, for HCG did not affect [Ca2+]i in L cells not expressing the LHR. The effect was not due to the cAMP-forming activity of the LH receptor, for neither forskolin nor prostaglandin E1, which both increase cAMP levels in L cells, had a similar effect in either control or LHR-expressing cells and isoproterenol had no effect in L cells expressing a functionally active hamster beta-adrenergic receptor. The effect was also not due to overexpression of a Gs-coupled receptor, for L cells expressing 8-fold higher levels of the human V2 vasopressin receptor did not mimic the Ca(2+)-mobilizing response of the LH receptor. We conclude that the LH receptor has the capability of activating two intracellular signaling pathways: one leading to stimulation of adenylyl cyclase and resulting in increases in cAMP and a second leading to stimulation of phospholipase C and resulting in formation of inositol phosphates and elevations in [Ca2+]i. These data correlate positively with and provide a mechanistic explanation for previous reports on the ability of hCG to mobilize phosphoinositides and increasing [Ca2+]i in luteal and granulosa cells (e.g. Davis, J. S., West, L. A., and Farese, R. V. (1984) J. Biol. Chem. 259, 15028-15034).

Adenylyl Cyclases↗
PubMed1992

Cloning and expression of a cardiac/brain beta subunit of the L-type calcium channel.

The skeletal muscle dihydropyridine receptor/Ca2+ channel is composed of five protein components (alpha 1, alpha 2 delta, beta, and gamma). Only two such components, alpha 1 and alpha 2, have been identified in heart. The present study reports the cloning and expression of a novel beta gene that is expressed in heart, lung, and brain. Coexpression of this beta with a cardiac alpha 1 in Xenopus oocytes causes the following changes in Ca2+ channel activity: it increases peak currents, accelerates activation kinetics, and shifts the current-voltage relationship toward more hyperpolarized potentials. It also increases dihydropyridine binding to alpha 1 in COS cells. These results indicate that the cardiac L-type Ca2+ channel has a similar subunit structure as in skeletal muscle, and provides evidence for the modulatory role of the beta subunit.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Molecular cloning of a human gene (S31) encoding a novel serotonin receptor mediating inhibition of adenylyl cyclase.

We report the molecular cloning of human gene (S31) containing an open reading frame of 1095 nucleotides, which encodes a protein of 365 amino acids. The encoded protein contains seven hydrophobic putative transmembrane domains considered the hallmark of G protein-coupled receptors. The amino acid sequence shows highest homology to receptors for serotonin (5-hydroxytryptamine). Expression of this receptor in murine Ltk- cells conferred upon these cells the ability to respond to serotonin by inhibition of adenylyl cyclase. No response was observed to isoproterenol, epinephrine, histamine, dopamine or melatonin in the transfected cells. We propose that the human gene S31 encodes a novel serotonin receptor.

Adenylyl Cyclases↗
PubMed1992

Cloning of a novel alpha 1-subunit of the voltage-dependent calcium channel from the beta-cell.

To study the molecular regulation of voltage-dependent Ca2+ channels (VDCCs) in the beta-cell, we have cloned a cDNA for the alpha 1-subunit from a hamster insulin-secreting cell line (HIT-T15). The cDNA (HCa3a) encodes a 1610-amino acid protein with four repeating membrane domains and an overall structure characteristic of other alpha 1-subunits. Although the cDNA shows a high degree of sequence homology (97%) with a rat brain alpha 1-subunit (RB alpha 1), the C-terminal 15 amino acids of HCa3a share no similarity with any cloned alpha 1 protein. High stringency Northern blot analysis revealed a single transcript of approximately 8.6 kilobases in HIT cells and hamster pancreas. A similarly sized species was detected in hamster brain, heart, and skeletal muscle. Using polymerase chain reaction and a primer set unique to HCa3a, this alpha 1 isoform was found to be expressed in islet cell lines derived from rat, mouse, and hamster. The HIT cell alpha 1-subunit is also expressed in discrete regions of the rat central nervous system, including the cortex, cerebellum, hypothalamus, and brain stem. The expression of two alpha 1 isoforms (HCa3a and cardiac) in the HIT cell underscores the possible complexity of VDCCs in the regulation of beta-cell signal transduction. With its widespread tissue distribution, HCa3a does not conform to the current classification system used for L-type VDCCs; this suggests that an alternative system of classification is required.

Amino Acid Sequence↗
PubMed1992

Ca2+ mobilization by the LH receptor expressed in Xenopus oocytes independent of 3',5'-cyclic adenosine monophosphate formation: evidence for parallel activation of two signaling pathways.

The cDNAs encoding the murine LH receptor (LHR) and the human beta 2-adrenoceptor (h beta 2AR) were cloned and RNAs complementary to their sense strands (cRNAs) were injected into defolliculated Xenopus oocytes. This led to expression, respectively, of LH- and isoproterenol-stimulable adenylyl cyclase activities, indicating that functionally active receptor cDNAs had been cloned. In oocytes injected with LHR cRNA, but not in control or h beta 2AR cRNA-injected oocytes, human CG and LH increased a Ca(2+)-activated Cl- current, as measured by the two-microelectrode voltage-clamp method. This effect was not seen with isoproterenol in control or h beta 2AR cRNA-injected oocytes, it was also not observed in response to forskolin or (Bu)2cAMP. The response to human CG could be obtained in the absence of extracellular Ca2+ but was abolished by injection of EGTA, indicating that it was caused by mobilization of Ca2+ from intracellular stores. The response was unaffected by overnight treatment with 1 microgram/ml pertussis toxin. The experiments show that a glycoprotein hormone receptor can be expressed as a functionally active molecule in Xenopus oocytes, and that the LHR has the ability of activating two separate intracellular signaling pathways: one forming the second messenger cAMP, and the other mobilizing Ca2+ from intracellular stores. It is proposed that the latter is secondary to a primary activation of phospholipase C by the LHR, which elevates intracellular Ca2+ via intermediary elevation of inositol phosphates, presumably (1,4,5)inositol trisphosphate.

Adenylyl Cyclases↗
PubMed1992

Peptide inhibitors of ADP-ribosylation by pertussis toxin are substrates with affinities comparable to those of the trimeric GTP-binding proteins.

Pertussis toxin (PTX) ADP-ribosylates alpha subunits of GTP-binding proteins (G proteins) when they are in association with beta gamma dimers, and free alpha subunits are thought not to be substrates under standard assay conditions. We now report the rather unexpected discovery that synthetic peptides encompassing the last 10-20 amino acids of alpha subunits of PTX-sensitive G proteins are substrates for PTX by themselves and in the absence of beta gamma dimers. As determined for G13, the Km of PTX for the 20-amino acid carboxyl-terminal peptide is 10-fold higher than that for the trimeric G protein. Interestingly, PTX ADP-ribosylates the free full length alpha 13 subunit with a Km not different from that of the trimer but with a Vmax that is only 1% of that with which it ADP-ribosylates the trimer. It follows that the primary role of beta gamma dimers in ADP-ribosylation of G proteins is one of increasing the Vmax of the reaction without affecting the Km of the substrate for the toxin. Mutant peptides lacking the ADP-ribose acceptor site act as competitive inhibitors.

Adenosine Diphosphate Ribose↗
PubMed1991

Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels.

High threshold L-type Ca2+ channels of skeletal muscle are thought to consist of a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. Expression of the cloned alpha 1 subunit from skeletal and cardiac muscle has established that this protein is the dihydropyridine-sensitive ion-conducting subunit. However, the kinetics of the skeletal muscle alpha 1 alone expressed in mouse L-cells were abnormally slow and were accelerated to within the normal range by coexpression with the skeletal muscle beta subunit. The kinetics of cardiac muscle alpha 1 were also slowed but to a lesser extent and were not altered by coexpression with skeletal muscle alpha 2. We show here that coexpression of the skeletal muscle beta subunit with the cardiac alpha 1 subunit in Xenopus laevis oocytes produced: 1) an increase in the peak voltage-sensitive current, 2) a shift of the peak current-voltage relationship to more hyperpolarized potentials, and 3) an increase in the rate of activation. Coexpression of the skeletal muscle gamma subunit did not have a significant effect on currents elicited by alpha 1. However, when gamma was coexpressed with beta and alpha 1, both peak currents and rates of activation at more negative potentials were increased. These results indicate that rather than simply amplifying expression of alpha 1, heterologous skeletal muscle beta and gamma subunits can modulate the biophysical properties of cardiac alpha 1.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Involvement of pertussis toxin-sensitive G-proteins in the hormonal inhibition of dihydropyridine-sensitive Ca2+ currents in an insulin-secreting cell line (RINm5F).

Adrenaline inhibits insulin secretion via pertussis toxin-sensitive mechanisms. Since voltage-dependent Ca2+ currents play a key role in insulin secretion, we examined whether adrenaline modulates voltage-dependent Ca2+ currents of the rat insulinoma cell line, RINm5F. In the whole-cell configuration of the patch-clamp technique, dihydropyridine- but not omega-conotoxin-sensitive Ca2+ currents were identified. Adrenaline via alpha 2-adrenoceptors inhibited the Ca2+ currents by about 50%. Somatostatin which also inhibits insulin secretion was less efficient (inhibition by 20%). The hormonal inhibition of Ca2+ currents was not affected by intracellularly applied cAMP but blocked by the intracellularly applied GDP analog guanosine 5'-O-(2-thiodiphosphate) and by pretreatment of cells with pertussis toxin. In contrast to adrenaline and somatostatin, galanin, another inhibitor of insulin secretion, reduced Ca2+ currents by about 40% in a pertussis toxin-insensitive manner. Immunoblot experiments performed with antibodies generated against synthetic peptides revealed that membranes of RINm5F cells possess four pertussis toxin-sensitive G-proteins including Gi1, Gi2, Go2, and another Go subtype, most likely representing Go1. In membranes of control but not of pertussis toxin-treated cells, adrenaline via alpha 2-adrenoceptors stimulated incorporation of the photo-reactive GTP analog [alpha-32P]GTP azidoanilide into pertussis toxin substrates comigrating with the alpha-subunits of Gi2, Go2, and the not further identified Go subtype. The present findings indicate that activated alpha 2-adrenoceptors of RINm5F cells interact with multiple G-proteins, i.e. two forms of Go and with Gi2. These G-proteins are likely to be involved in the adrenaline-induced inhibition of dihydropyridine-sensitive Ca2+ currents and in other signal transduction pathways contributing to the adrenaline-induced inhibition of insulin secretion.

Animals↗
PubMed1991

Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel.

Purification of skeletal muscle dihydropyridine binding sites has enabled protein complexes to be isolated from which Ca2+ currents have been reconstituted. Complementary DNAs encoding the five subunits of the dihydropyridine receptor, alpha 1, beta, gamma, alpha 2 and delta, have been cloned and it is now recognized that alpha 2 and delta are derived from a common precursor. The alpha 1 subunit can itself produce Ca2+ currents, as was demonstrated using mouse L cells lacking alpha 2 delta, beta and gamma (our unpublished results). In L cells, stable expression of skeletal muscle alpha 1 alone was sufficient to generate voltage-sensitive, high-threshold L-type Ca2+ channel currents which were dihydropyridine-sensitive and blocked by Cd2+, but the activation kinetics were about 100 times slower than expected for skeletal muscle Ca2+ channel currents. This could have been due to the cell type in which alpha 1 was being expressed or to the lack of a regulatory component particularly one of the subunits that copurifies with alpha 1. We show here that coexpression of skeletal muscle beta with skeletal muscle alpha 1 generates cell lines expressing Ca2+ channel currents with normal activation kinetics as evidence for the participation of the dihydropyridine-receptor beta subunits in the generation of skeletal muscle Ca2+ channel currents.

Animals↗
PubMed1991

Gamma-subunits of G proteins, but not their alpha- or beta-subunits, are polyisoprenylated. Studies on post-translational modifications using in vitro translation with rabbit reticulocyte lysates.

Lipid modifications that may be introduced into several subunits of G proteins were explored by in vitro translation of recombinant mRNAs in reticulocyte lysates. In agreement with studies by others, myristic acid was incorporated into alpha i's and alpha o, but not alpha s, beta, or gamma's. In contrast, mevalonate (Mev) was incorporated only into gamma-subunits. Both, the gamma-subunit of transducin (gamma T) and that of other G proteins (gamma G) were modified by the lysates but with different characteristics. Labeled gamma T was unstable and was rapidly proteolyzed. Labeled gamma G was stable. The Mev-derivative in gamma G was sensitive to methyliodide and, after cleavage and chromatographic analysis, comigrated with the C20 polyisoprenol geranylgeraniol. This indicated that gamma G had been geranylgeranylated and that this polyisoprenoid was attached to the protein through a thioether linkage. It is thought that polyisoprenylation is defined by the COOH-terminal sequence Cys-A-A-X, where A is an aliphatic acid and X is any amino acid. Replacement by mutation of the Cys of the COOH-terminal -Cys-Ala-Ile-Leu sequence of gamma G with Ser abolished Mev incorporation, suggesting this Cys as the site of attachment of the geranylgeranyl moiety. Yet, Mev incorporation was less than 10% as much into gamma G with the Cys-A-A-X sequence -Cys-Ala-Ile-Trp. Consistent with geranylgeranylation, the C15 farnesyl moiety of farnesyl pyrophosphate was not incorporated into gamma G unless the incubations were fortified with Mev. In contrast, the farnesyl moiety was incorporated in an Mev-independent manner into gamma T (COOH terminus: -Cys-Val-Ile-Ser) and c-Ha-ras (COOH terminus: -Cys-Val-Leu-Ser) which are both farnesylated rather than geranylgeranylated. Thus, 1) separate enzymes appear to be involved in transferring farnesyl and geranylgeranyl groups to proteins, 2) structural factors other than the CAAX box contribute to the activity of the polyisoprenylating enzymes, and 3) this type of lipidation may be part of a proteolytic signaling system. Polyisoprenylation, which increases hydrophobicity of the derivatized protein, may play a role in anchoring not only ras but also G proteins to membranes.

Animals↗
PubMed1991

Identification of the G-protein alpha-subunit encoded by alpha o2 cDNA as a 39 kDa pertussis toxin substrate.

A novel form of the Go alpha-subunit (alpha o2) has been identified by molecular cloning (Hsu et al., J. Biol. Chem. 265, 11220-11226, 1990). An antibody was generated against a synthetic peptide corresponding to a region of the protein encoded by alpha o2 cDNA. The antibody reacted with an apparently single 39 kDa protein in membrane preparations of rodent brain and with a 39 kDa pertussis toxin substrate in membranes of rodent neuroendocrine and pituitary cells. A previously produced antibody raised against a region common to proteins encoded by alpha o2 cDNA and the previous cloned alpha o1 cDNA (Itoh et al., Proc. Natl. Acad. Sci. USA 83, 3776-3780, 1986) recognized proteins of 39 and 40 kDa in preparations of bovine, porcine and rodent brain and pertussis toxin substrates of 39 and 40 kDa in membranes of rodent neuroendocrine and pituitary cells. We conclude that the 39 kDa Go alpha subunit is encoded by alpha o2 cDNA.

Amino Acid Sequence↗
PubMed1991

Control of K+ channels by G proteins.

Heterotrimeric G3 proteins are though to couple receptors to ionic channels via cytoplasmic mediators such as cGMP in the case of retinal rods, cAMP in the case of olfactory cells, and the cAMP cascade in the case of cardiac myocytes. G protein-mediated second messenger effects on K+ channels are dealt with elsewhere in this series. Recently, membrane-delimited pathways have been uncovered and an hypothesis proposed in which the alpha subunits of G proteins directly couple receptors to ionic channels, particularly K+ channels. While direct coupling has not been proven, the membrane-delimited nature has been established for specific G proteins and their specific K+ channel effectors.

Animals↗
PubMed1991

Abnormal Gs function in mitral valve prolapse dysautonomia is not associated with abnormal alpha S cDNA sequence.

We have previously shown that a subset of patients with mitral valve prolapse and hyperadrenergic symptoms has enhanced isoprenaline-stimulated beta-adrenergic receptor high-affinity state formation (supercoupling) and increased adenylyl cyclase activity due to abnormal signal transduction by the stimulatory guanine nucleotide regulatory protein (Gs). In this study we looked for an alteration of the nucleotide coding sequence of the gene for alpha s, the subunit of Gs that is directly responsible for formation of the high affinity state and adenylyl cyclase activation, by cloning and sequencing the alpha s cDNA from neutrophils of 4 symptomatic patients and 1 control. No difference was observed between patients and control in the alpha s cDNA sequence. The splice variant concentrations in the fully expressed protein were also grossly unchanged in five patients and four controls. These data show that a primary alteration of the alpha s gene coding sequence is not responsible for defective Gs-associated signal transduction in dysautonomic MVP patients, and suggest that the molecular lesion could be an abnormal posttranslational modification of alpha s, a defect in the beta or gamma subunits of Gs, or an unusual interaction between the subunits in the Gs of these patients.

Adenylyl Cyclases↗