Alpha Subunit Of Go Research Articles

Immunoexpression of the alpha subunit of a guanine nucleotide-binding protein (Go) in pulmonary neuroendocrine cells and neoplasms.

The alpha subunit of a GTP-binding protein, Go, was investigated in pulmonary neuroendocrine neoplasms and fetal tissues of the lung by an immunohistochemical method. Positive immunostaining for the alpha subunit of Go (Go alpha) was found predominantly on the cell membrane and found occasionally in the cytoplasm. Typical carcinoids were all positively stained (9/9), and small cell carcinoma showed weaker and less frequent staining (5 positive cases in 10). Atypical carcinoids were variously stained (3/4). The tendency for obvious neuroendocrine differentiation to be immunohistochemically determined in typical carcinoids and not in small cell carcinoma is also true of staining for neuron specific enolase (NSE), chromogranin A (CG-A) and synaptophysin. In the lung, Go alpha-immunostaining was positive not only in nerve tissues but also in the airway epithelium. In the fetal lung, serial sections immunostained for NSE, CG-A and Go alpha confirmed that Go alpha-immunoreactive cells belong to the neuroendocrine cell population. The biological significance of Go alpha is unclear in normal and neoplastic lung tissues, but Go alpha is a useful marker of neuroendocrine cells and neoplasma of the lung.

Modulation of Serotonin-Controlled Behaviors by G o in Caenorhabditis elegans

Seven transmembrane receptors and their associated heterotrimeric guanine nucleotide-binding proteins (G proteins) have been proposed to play a key role in modulating the activities of neurons and muscles. The physiological function of the Caenorhabditis elegans G protein Go has been genetically characterized. Mutations in the goa-1 gene, which encodes an alpha subunit of Go (G alpha o), cause behavioral defects similar to those observed in mutants that lack the neurotransmitter serotonin (5-HT), and goa-1 mutants are partially resistant to exogenous 5-HT. Mutant animals that lack G alpha o and transgenic animals that overexpress G alpha o [goa-1(xs) animals] have reciprocal defects in locomotion, feeding, and egg laying behaviors. In normal animals, all of these behaviors are regulated by 5-HT. These results demonstrate that the level of Go activity is a critical determinant of several C. elegans behaviors and suggest that Go mediates many of the behavioral effects of 5-HT.

Binding of G protein beta gamma-subunits to pleckstrin homology domains.

Ligand-induced activation of many receptors leads to dissociation of the alpha- and beta gamma-subunit complexes of heterotrimeric G proteins, both of which regulate a variety of effector molecules involved in cellular signaling processes. In one case, a cytosolic enzyme, the beta-adrenergic receptor kinase (beta ARK) binds to the dissociated, prenylated, membrane-anchored beta gamma-subunits of heterotrimeric G proteins (G beta gamma) and is thereby targeted to its membrane-bound receptor substrate. Quite recently, numerous proteins involved in cellular signal transduction have been shown to contain sequences homologous with a "domain" originally identified in the protein "pleckstrin" (pleckstrin homology domain; PH domain) and subsequently found in the G beta gamma interaction region of the beta ARK sequence. Here we demonstrate that glutathione S-transferase-fusion proteins, containing sequences encompassing the PH domain of nine proteins from this group, bind G beta gamma to varying extents. Binding of G beta gamma to these fusion proteins was documented either by a direct binding assay or by ability to block G beta gamma-mediated membrane translocation of beta ARK1. G beta gamma binding to these fusion proteins was inhibited by the alpha subunit of Go (Go alpha), indicating that the binding of G beta gamma to G alpha and the PH domain-containing fusion proteins is mutually exclusive. Studies with a series of truncated PH domains derived from the Ras-guanine-nucleotide-releasing factor indicate that the G beta gamma binding domain includes only the C-terminal portion of the PH domain and sequences just distal to this. Protein-protein interactions between G beta gamma and PH domain-containing proteins may play a significant role in cellular signaling analogous to that previously demonstrated for Src homology 2 and 3 domains.

Characterization of the distribution of Gαo in rat striatal synaptosomes and its colocalization with tyrosine hydroxylase

Dopaminergic striatal synaptosomes can be detected and isolated with a fluorescence-activated cell sorter (FACS). In the present study, two antigens were detected simultaneously with primary antisera raised in different species and species-specific fluorescent secondary antibodies with different emission spectra. Double-label FACS analysis was used to determine whether tyrosine hydroxylase (TH) and the alpha subunit of Go (G alpha o) are colocalized in striatal synaptosomes. Rabbit antibodies generated against a synthetic fragment of G alpha o (corresponding to amino acids 22-35) combined with fluorescein-conjugated secondary antibodies were used to detect G alpha o-containing striatal synaptosomes. Preadsorption of G alpha o antiserum with the synthetic peptide antigen reduced labeling to the level obtained with preimmune serum. Approximately 65-75% of striatal synaptosomes were specifically labeled by G alpha o antiserum. Tyrosine hydroxylase-containing synaptosomes were detected with a mouse monoclonal antibody to TH and R-phycoerythrin-conjugated secondary antibody. They comprised 15-17% of total striatal synaptosomes. Double-label studies indicated that at least 50% of TH-containing synaptosomes also contained G alpha o. These findings suggest that G alpha o may not be a protein component of all striatal nerve terminals, and provide a basis for a role for G alpha o in signal transduction within subpopulations of intrinsic and afferent nerve terminals, including those of nigrostriatal dopamine neurons.

Concentrations of several proteins characteristic of nervous tissue in cerebral cortex of patients with alzheimer’s disease

Concentrations of nervous tissue-related proteins, including S-100 proteins (alpha and beta), enolase isozymes (alpha and gamma), superoxide dismutase (SOD) isozymes (Cu/Zn SOD and Mn SOD), and GTP-binding proteins (alpha subunits of GO and Gi2) were determined in the four cerebrocortical regions (superior frontal gyrus of frontal lobe, parahippocampal gyrus of temporal lobe, superior parietal lobule of parietal lobe, and calcarine area of occipital lobe) of patients with Alzheimer's disease, and age-matched control and young control patients by means of enzyme immunoassay methods. Although the temporal cortex of some patients with Alzheimer's disease (4/7) showed apparently enhanced S-100 beta with decreased gamma-enolase, concentrations of neuronal (neuron-specific gamma-enolase and the alpha subunit of GO) and glial (S-100 beta, S-100 alpha, and alpha-enolase) marker proteins, and both SODs in each region were not significantly different between patients with Alzheimer's disease and the age-matched controls. Concentrations of Gi2 alpha also showed similar values in the cerebral cortices of young and aged controls and patients with Alzheimer's disease. However, when compared with young controls, S-100 beta in the four regions of patients with Alzheimer's disease and aged controls, and Cu/Zn SOD in frontal cortex of patients with Alzheimer's disease were significantly enhanced (P less than 0.01).

Structure of the human gene and two rat cDNAs encoding the alpha chain of GTP-binding regulatory protein Go: two different mRNAs are generated by alternative splicing.

Go is a specific class ("other") of signal-transducing heterotrimeric GTP-binding proteins (G proteins) that is expressed in high levels in mammalian brain. We have cloned two different rat cDNAs encoding the alpha subunit of Go (Go alpha-1 and Go alpha-2) and a human Go alpha chromosomal gene. The human Go alpha gene spans more than 100 kilobases and contains 11 exons, including one noncoding exon in the 3' flanking region. The 5' flanking region is highly G + C-rich and contains five G.C boxes (Sp1 binding sites) but no TATA box. Exons 7 and 8 coding for amino acid residues 242-354 of Go alpha protein are duplicated (referred to as exons 7A, 7B, 8A, and 8B). It was found that exons 7A and 8A code for Go alpha-1, and 7B and 8B code for Go alpha-2. This indicates that two different Go alpha mRNAs may be generated by alternative splicing of a single Go alpha gene. The splice sites of the Go alpha-1 and Go alpha-2 genes are completely identical with those encoding human inhibitory G protein alpha subunits Gi2 alpha and Gi3 alpha [Itoh, H., Toyama, R., Kozasa, T., Tsukamoto, T., Matsuoka, M. & Kaziro, Y. (1988) J. Biol. Chem. 263, 6656-6664] and also transducin G protein alpha subunit Gt1 alpha [Raport, C. J., Dere, B. & Hurley, J. (1989) J. Biol. Chem. 264, 7122-7128]. Sequence homology and conservation of the exon-intron organization indicate that the genes coding for Go alpha, Gi2 alpha, Gi3 alpha, Gt1 alpha, and probably Gi1 alpha may be evolved from a common progenitor. Like Go alpha-1, Go alpha-2 is expressed mainly in brain.

Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology.

Endothelin and increased contractility in adult rat ventricular myocytes. Role of intracellular alkalosis induced by activation of the protein kinase C-dependent Na(+)-H+ exchanger.

Endothelin, a 21-amino acid vasoactive peptide, is among the most potent positively inotropic agents yet described in mammalian heart. Having demonstrated that endothelin's inotropic effect is due, in part, to an apparent sensitization of cardiac myofilaments to intracellular calcium, we determined whether this could be due to a rise in intracellular pH (pHi). In isolated adult rat ventricular cells loaded with the H(+)-selective fluorescent probe BCECF, 100 pM endothelin increased contractile amplitude to 190 +/- 26% of baseline and pHi by 0.08 +/- 0.02 (n = 8), whereas 1 nM endothelin increased pHi by 0.13 +/- 0.03 with little further increase in contractility. Amiloride (10(-4)M) prevented the increase in pHi in response to endothelin and reduced the inotropic response by 45%, although the inotropic effect could be readily restored by subsequent NH4Cl-induced alkalinization. Similarly, inhibitors of protein kinase C (H-7 and sphingosine) diminished or abolished the rise in pHi after endothelin superfusion while causing a decline in its inotropic effect comparable with that observed with amiloride. Pretreatment with pertussis toxin, which we have demonstrated results in complete ADP-ribosylation of the alpha-subunits of Go and Gi GTP-binding proteins and abolition of endothelin's positive inotropic effect, only partially reduced the intracellular alkalinization induced by the peptide, suggesting a complex signal transduction mechanism. Thus, the positive inotropic action of endothelin is due in part to stimulation of the sarcolemmal Na(+)-H+ exchanger by a protein kinase C-mediated pathway, resulting in a rise in pHi and sensitization of cardiac myofilaments to intracellular Ca2+.

A guanine nucleotide-binding protein in human astrocytoma

We compared, by immunoblotting and immunohistochemical staining, the distribution of the alpha subunit of Go in human normal brain tissue with that in human brain tumours of neuroectodermal origin. The ten tumour samples included seven high-grade astrocytomas (grades III and IV) and three low-grade astrocytomas (grade II). Immunoreactivity of high-grade astrocytomas with the alpha subunit of Go (Go alpha) antibody was lower than that of low-grade astrocytomas and normal white matter. In the immunohistochemical study, neoplastic cells in the astrocytomas were found to contain little or no significant Go alpha, although nonlesional white matter was strongly stained with Go alpha antibody. This study suggests that Go alpha antibody may assist in defining the boundary of an astrocytoma, and the difference in Go content may affect the alteration in the physical properties of the membrane, which exhibit, in part, the character of the transformation.

Regulatory Proteins in Presynaptic Function

Activation of alpha 2-adrenoceptors, opioid, A1-adenosine, and PGE receptors inhibited the stimulation-induced [3H]noradrenaline release in brain tissue in a concentration-dependent manner. Under experimental conditions (360 pulses/3 Hz) where the released noradrenaline activated the presynaptic alpha 2-autoreceptors, the effects of the heteroreceptor (k-opioid, A1-adenosine, PGE) agonists were decreased. By avoiding autoinhibition by either blockade of the alpha 2-autoreceptors with yohimbine or stimulating the tissue with four pulses/100 Hz, the heteroreceptor-mediated inhibition of [3H]noradrenaline release was markedly increased. The dependence of the heteroreceptor-mediated inhibition of evoked noradrenaline release on the extent of alpha 2-autoreceptor activation suggests a common postreceptor signal transduction pathway. PTX-catalyzed [32P]ADP ribosylation of synaptosomal membrane proteins revealed three bands of polypeptides with molecular weights corresponding to the alpha subunits of Go (39,000) and the Gi proteins (40,000, 41,000). Pretreatment with NEM reduced the PTX-induced 32P labeling by alkylating the alpha subunits at or near the site that is ADP ribosylated by PTX in a concentration-dependent manner. K(+)-evoked release of [3H]noradrenaline from synaptosomes indicated the presynaptic localization of the PTX-sensitive G proteins coupled to alpha 2-, k-, and A1-receptors of noradrenergic nerve terminals. Electrically evoked [3H]noradrenaline release was only increased by PTX or NEM in a time- and concentration-dependent manner when autoinhibition was present. The alpha 2-, opioid, and A1-adenosine receptor-mediated inhibition of [3H]noradrenaline release was impaired similarly by PTX or NEM treatment. In contrast, the inhibitory effect of PGE2 remained unaffected. These results indicate that presynaptic alpha 2-, opioid, and A1-receptors but not PGE receptors of noradrenergic nerve terminals are linked to PTX-sensitive G proteins. The interaction between the alpha 2-autoreceptors and the PGE receptors therefore does not occur at the level of a common pool of G proteins but at some subsequent step of the signal transduction mechanism.

Developmental Expression of Go in Neuronal Cultures from Rat Mesencephalon and Hypothalamus

The developmental expression of the alpha-subunit of Go was examined in neuronal cultures derived from rat mesencephalon (MES) and hypothalamus (HYP). These cultures were essentially free of contaminating glia and were maintained as a stable population for periods up to 3 weeks. Immunoblotting utilizing specific antisera against Go indicated that in neurons from both brain regions, membrane concentrations of Go increased dramatically during the first 2 weeks in vitro. Thereafter, increases in the amount of Go per neuron kept pace with increasing process (axons and dendrites) formation. Multiple forms of immunoreactive Go were detected in MES and HYP neurons, and the proportions of these forms changed between 4 and 14 days in culture. Finally, increasing neuron density significantly increased membrane levels of Go in MES but not HYP cultures.

Multiple forms of Go.alpha. mRNA: analysis of the 3'-untranslated regions

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.

A Go-like protein in Drosophila melanogaster and its expression in memory mutants.

G proteins couple receptors for extracellular signals to several intracellular effector systems and play a key role in signalling transduction mechanisms. In particulate preparations of Drosophila melanogaster heads, only one substrate for pertussis toxin at 39-40 kd was detected. This substrate, which showed only one isoform when analysed by isoelectric focusing, was recognized by immunoblotting and immunoprecipitation techniques using a polyclonal antibody against the alpha subunit of the Go protein purified from bovine brain and can be thus considered as a Go-like protein. Antibodies obtained against a carboxy-terminal sequence of the alpha subunit of Go (but not of Gi1 or Gi2) and against an internal sequence shared by all the alpha subunits, were also able to cross-react with the alpha subunit of this protein in insects. We have also studied the Go-like protein in several D.melanogaster mutants, primarily in memory and learning mutants. In these mutants there was a sex-dependent enhancement in pertussis toxin-catalysed ADP-ribosylation with respect to the wild-type. This increase could be attributed in part to an increase in the alpha subunit of the Go-like protein, as revealed by immunoblotting with anti-Go alpha polyclonal antibody. This report constitutes the first evidence for the participation of a Go protein in learning and memory.

Isolation of the alpha subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized beta gamma subunits.

Immobilized beta gamma subunits of GTP-binding regulatory proteins (G proteins) were used to isolate alpha subunits from solubilized membranes of bovine tissues and to separate specific alpha subunits based on their differential affinities for beta gamma subunits. The beta gamma subunits were cross-linked to omega-aminobutyl agarose. Up to 7 nmol of alpha subunit could bind to each milliliter of beta gamma-agarose and be recovered by elution with AIF4-. This affinity resin effectively separated the alpha subunits of Gi1 and Gi2 from "contaminating" alpha subunits of Go, the most abundant G protein in bovine brain, by taking advantage of the apparent lower affinity of the alpha subunits of Go for beta gamma subunits. The beta gamma-agarose was also used to isolate mixtures of alpha subunits from cholate extracts of membranes from different bovine tissues. alpha subunits of 39-41 kDa (in various ratios) as well as the alpha subunits of Gs were purified. The yields from extracts exceeded 60% for all alpha subunits examined and apparently represented the relative content of alpha subunits in the tissues. This technique can rapidly isolate and identify, from a small amount of sample, the endogenous G proteins in various tissues and cells. So far, only polypeptides in the range of 39-52 kDa have been detected with this approach. If other GTP-binding proteins interact with these beta gamma subunits, the interaction is either of low affinity or mechanistically unique from the alpha subunits isolated in this study.

Regulation of Substance P Receptor Affinity by Guanine Nucleotide‐Binding Proteins

The binding of substance P (SP) to receptors in peripheral tissues as well as in the CNS is subject to regulation by guanine nucleotides. In this report, we provide direct evidence that this effect is mediated by a guanine nucleotide-binding regulatory protein (G-protein) that is required for high-affinity binding of SP to its receptor. Rat submaxillary gland membranes bind a conjugate of SP and 125I-labeled Bolton-Hunter reagent (125I-BHSP) with high affinity (KD = 1.2 +/- 0.4 X 10(-9) M) and sensitivity to guanine nucleotide inhibition. Treatment of the membranes with alkaline buffer (pH 11.5) causes a loss of the high-affinity, GTP-sensitive binding of 125I-BHSP and a parallel loss of [35S]guanosine 5'-(3-O-thio)triphosphate ([35S]GTP gamma S) binding activity. Addition of purified G-proteins from bovine brain to the alkaline-treated membranes restores high-affinity 125I-BHSP binding. Reconstitution is maximal when the G-proteins are incorporated into the alkaline-treated membranes at a 30-fold stoichiometric excess of GTP gamma S binding sites over SP binding sites. Both Go (a pertussis toxin-sensitive G-protein having a 39,000-dalton alpha-subunit) and Gi (the G-protein that mediates inhibition of adenylate cyclase) appear to be equally effective, whereas the isolated alpha-subunit of Go is without effect. The effects of added G-proteins are specifically reversed by guanine nucleotides over the same range of nucleotide concentrations that decreases high-affinity binding of 125I-BHSP to native membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Low Density Lipoproteins and Mevinolin on Sympathetic Responsiveness in Cultured Chick Atrial Cells: Regulation of β-adrenergic receptors and αs

We have previously demonstrated that cultures of myocytes from embryonic chick atria grown in media supplemented with fetal calf serum from which lipoproteins have been removed demonstrate a nearly 10-fold increase in sensitivity of beating to the muscarinic cholinergic agonist carbamylcholine compared with cells grown with control medium. This increased response to carbamylcholine was associated with a 1.4-fold increase in total cell cholesterol, a 2-fold increase in the number of muscarinic receptors which bind agonist with high affinity, and a 2-fold increase in the levels of the alpha subunits of Go and Gi (Haigh, L. S., Leatherman, G. F., O'Hara, D. S., Smith, T. W., and Galper, J. B. (1988) J. Biol. Chem. 263, 15608-15618). In the studies reported here, we determined the responsiveness of cells grown in lipoprotein-depleted serum (LPDS) to beta-adrenergic stimulation. Isoproterenol stimulated a contractile response of 58% measured as an increase in amplitude of contraction with a half-maximal effect at 3 x 10(-7) M for cells grown in fetal calf serum, but had no significant effect on amplitude of contraction on cells grown in LPDS. In cells grown in media supplemented with fetal calf serum, isoproterenol (1 x 10(-3) M) stimulated adenylate cyclase activity 100% over basal with an EC50 of 7 x 10(-6) M compared with an increase of 32% in cells grown in media supplemented with LPDS. beta-Adrenergic receptor number as measured by the binding of 125I-pindolol decreased from 24 +/- 3 (+/- S.E., n = 6) fmol/mg protein in cells grown under control conditions to 12 +/- 2 (n = 6) fmol/mg protein in media supplemented with LPDS. The level of alpha s as measured both by ADP-ribosylation with cholera toxin in the presence of 32P-NAD and by immunoblotting with specific antibody to alpha s decreased by 3-fold in cells grown in media supplemented with LPDS compared with control. All of these effects of growth of cells in LPDS were reversed by incubating cells with LPDS plus 30 microM mevinolin, an inhibitor of endogenous cholesterol synthesis. These studies indicate that growth of cells in media supplemented with LPDS results in a coordinate decrease in the levels of beta-adrenergic receptors and alpha s. Taken together with our previous studies these data support the hypothesis that the receptors and guanine nucleotide-binding proteins which mediate sympathetic and parasympathetic responsiveness in the heart are reciprocally regulated.

The adipocyte Goα-immunoreactive polypeptide is different from the α subunit of the brain Go protein

Rat adipose tissue possesses two Bordetella pertussis toxin (PTX) substrates and, in the same 39-41 kDa molecular mass range, positive immunoreactivity has also been reported with antibodies against the alpha subunit of Go, the major brain GTP-binding protein (G-protein). In this study, the presence of the brain Go alpha subunit at 39 kDa in adipocytes was reassessed, since direct correspondence between PTX substrates and Go alpha immunoreactivity has not yet been clearly established. On resolutive SDS/polyacrylamide-gel electrophoresis, the PTX substrates of human adipocytes were compared with the three PTX substrates found in brain. No ADP-ribosylated substrate at the level of the 39 kDa brain Go alpha could be detected in adipocyte membranes. Immunoblotting of human adipocyte membranes stained with our anti-Go alpha antibodies confirmed the presence of a positive immunoreactivity in this tissue, but the apparent molecular mass of the immunoreactive polypeptide in adipocytes was higher than that found in nervous tissues. Taken together, these results indicate that the brain Go alpha subunit is not present in adipose tissue. They also suggest the existence of a G-protein in adipocytes which is immunologically related to Go alpha but having a slightly higher molecular mass.

Apical localization of the alpha subunit of GTP-binding protein Go in choroidal and ciliated ependymocytes [published erratum appears in J Neurosci 1989 Apr;9(4):following Table of Contents

The presence of GTP-binding proteins (G proteins) has been studied in murine adult choroid plexuses and cultured fetal choroidal or hypothalamic ependymal cells by ADP-ribosylation catalyzed by Bordetella pertussis toxin (PTX) and by immunodetection using affinity-purified polyclonal antibodies against the alpha subunit of the Go protein (Go alpha), the major brain G protein. ADP-ribosylation with 32P-NAD and PTX of choroid plexus revealed an intense labeling at the 40 kDa level in addition to the known PTX-substrates at 41 kDa (Gi alpha) and 39 kDa (Go alpha). This 40 kDa substrate was also predominant in cultured ependymal cells. However, a positive immunoreactivity with the anti-Go alpha antibodies was detected at the level of the 39 kDa faster component, indicating the presence of Go alpha in both choroid plexuses and cultured ependymal cells. In thin frozen sections as well as in cultured cells, Go alpha was mainly immunolocalized at the apical pole of choroidal ependymocytes and in the kinocilia of ciliated ependymal cells. At the ultrastructural level, using gold immunoprobes, the immunoreactivity of a Go alpha-like protein was detected on the cytoplasmic face of the apical plasma membrane, coated pits and vesicles, and in the apical cytoplasmic matrix. In ciliated ependymal cells, the positive immunostaining displayed a dotted pattern at the surface of demembranated axonema of apical kinocilia. These findings strongly suggest that G proteins, especially Go, are involved in transducing chemical signals that modulate traffic and exchanges between cerebrospinal fluid and ependyma through the apical membrane of ependymocytes.

Role of Adenosine 3',5'-Monophosphate and the Ri-Receptor Gi-Coupled Adenylate Cyclase Inhibitory Pathway in the Mechanism Whereby Adrenalectomy Increases the Adenosine Antilipolytic Effect in Rat Fat Cells*

The aim of this study was to establish the mechanism by which adrenalectomy promotes the antilipolytic effect of the adenosine analog (-)-N6-(R-phenyl-isopropyl)adenosine (R-PIA) in rat fat cells. This action of adrenalectomy was not specific for R-PIA, since it was also observed with nicotinic acid and was prevented by phosphodiesterase inhibitors. In contrast, the inhibitory effect of R-PIA and nicotinic acid toward isoproterenol-stimulated cAMP accumulation was unaltered by adrenalectomy regardless of whether phosphodiesterase inhibitors were present. Whatever the conditions used, however, the cAMP levels in adrenalectomized rat adipocytes were one quarter to one third of those in sham-operated rats and remained below the limit over which variations in cAMP had no more influence in lipolysis. Both total and particulate low Km cAMP phosphodiesterase activities per adipocyte were decreased in adrenalectomized rats, but the stimulatory responses of the particulate enzyme to R-PIA remained unchanged. Pertussis toxin-catalyzed ADP ribosylation studies revealed a marked decrease in the total amount of the alpha-subunits of Go and the adenylate cyclase inhibitory regulatory protein Gi after adrenalectomy. However, the inhibitory dose-response curves of adenylate cyclase to R-PIA, nicotinic acid, GTP, guanylylimidodiphosphate, and guanosine 5'-O-(3-thiotriphosphate) were unaltered by adrenalectomy, indicating that the inhibitory function of Gi is unimpaired by adrenalectomy. Lastly, adrenalectomy resulted in a 60% reduction of the Mn2+-stimulated adenylate cyclase activity/adipocyte, which indicates that adrenalectomy causes a defect in adenylate cyclase catalytic activity. Thus, enhanced antilipolytic effects of R-PIA induced by adrenalectomy do not involve increased function of the adenosine receptor Gi-coupled adenylate cyclase inhibitory pathway, but are related to abnormally low intracellular cAMP levels due to defective adenylate cyclase catalytic activity.

Small molecular weight GTP-binding proteins in human erythrocyte ghosts

GTP-binding proteins (G proteins) were extracted from human erythrocyte ghosts by sodium cholate and purified by gel filtration on an Ultrogel AcA-44 column followed by hydroxyapatite column chromatography. At least two peaks of G proteins were separated by hydroxyapatite column chromatography. The second peak contained G proteins recognized by the antibodies against the respective alpha subunits of Gs and Gi, and the ras protein, while the G protein of the first peak was not recognized by any of these antibodies. The G protein of the first peak was purified further by Mono Q HR5/5 column chromatography. The purified G protein showed a molecular weight of about 22 kDa on SDS-polyacrylamide gel electrophoresis. This G protein (22K G) specifically bound guanosine 5'-(3-O-thio) triphosphate (GTP gamma S), GTP and GDP with a Kd value for GTP gamma S of about 50 nM. GTP gamma S-binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. The G proteins recognized by the antibodies against the alpha subunit of Go and the ADP-ribosylation factor for Gs, designated as ARF, were not detected in human erythrocyte ghosts. These results indicate that there are at least two species of small molecular weight G proteins in human erythrocyte ghosts: one is the ras protein and the other is a novel G protein of 22K G.