Immunologic Probes for Heterotrimeric GTP-Binding Proteins

Spiegel, Allen M.

doi:10.1016/b978-0-12-377450-7.50010-1

Cited by 11 publications

(12 citation statements)

References 98 publications

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“…Kodak XRP film was exposed for 7 days without screens. B: ASI7 ( I : 1,000 dilution) antiserum recognizes ai., and a,.2 equally (Spiegel, 1990). Lane 1: Purified ai-I protein from bovine brain.…”

Section: Materials and Methods Cell Culturementioning

confidence: 99%

“…Differentiation of PC12 cells was initiated by the addition of 7s NGF (SO-100 ngiml, final concentration) de- Fig. I (Spiegel, 1990 (Huff et al, 1985). The bands corresponding to the a, protein are still present.…”

Section: Cell Culturementioning

confidence: 99%

“…Edmonds et al ( 1990) reported that preparations enriched in growth cone membranes from fetal rat brain contain a variety of a subunits. Since G proteins are so abundant throughout the brain (Neer et al, 1984;Sternweis and Robishaw, 1984;Worley et al, 1986;Spiegel, 1990), it is very hard to evaluate the effects of small amounts of contaminating neuronal membranes in preparations of growth cones. Therefore, we decided to take an immunohistochemical approach to define the G protein composition of ncuronal growth cones.…”

Section: Introductionmentioning

confidence: 99%

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Nerve growth factor changes G protein levels and localization in PC12 cells

Zubiaur

Neer

1993

J of Neuroscience Research

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Growth cones at the growing tips of developing neurites contain the machinery to transmit information from receptors to a variety of intracellular enzymes and ion channels. In order to understand how signals are transmitted across the membrane, we asked whether the multiplicity of signalling pathways in the growth cone is reflected by the diversity of G proteins found in this organelle. Our immunohistochemical analysis indicated that growth cones of differentiated PC12 cells contain at least 4 alpha G protein subunits, 3 that are pertussis toxin substrates (alpha o, alpha i-1, alpha i-2) and 1 that is not (alpha q). In addition to localization in the neurites and growth cones, alpha o, alpha i-1, alpha i-2, and alpha q were detected in intracellular perinuclear structures. We also analyzed the temporal change in G proteins in PC12 cells differentiated by treatment with nerve growth factor (NGF). Time course experiments have shown that alpha o and beta proteins coordinately increase after 2 days of treatment with NGF, reach a maximum at 4 days, and remain elevated. In contrast to alpha o, alpha i-2 reached a peak at 4 days, then declined to almost the basal level by day 7 of treatment with NGF. These data indicated that the levels of alpha o, alpha i-2, and beta are differentially regulated during NGF-induced neuronal differentiation in PC12 cells. The alpha o protein was highly concentrated at the tips of the growth cones before the cellular level of alpha o had increased appreciably, suggesting that the alpha subunits are translocated during the first stage of neurite development. In addition, not every neural process has the same high level of alpha o, suggesting that G proteins may help define the specialized functions of particular neurites within a single cell.

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Section: Materials and Methods Cell Culturementioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Nerve growth factor changes G protein levels and localization in PC12 cells

Zubiaur

Neer

1993

J of Neuroscience Research

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“…The bottom panel of Figure 5 shows immunoreactivity to antiserum J99, which is specific for G, (see antibody SW/i, Spiegel, 1990), and two proteins at 37 and 38 kDa are recognized. The expression of both of these proteins is apparently regulated by developmental stage, with an optimum in immunoreactivity on day 5, followed by a decline to -400/o of the maximal level in glands from newly pupated animals, as shown by the sums of the densities of these two bands in Figure 6.…”

Section: G Protein Subunits In the Prothoracic Glandmentioning

confidence: 99%

“…S. Mumby and A. Gilman (University of Texas Medical Center, Dallas, TX) (see Mumby et al, 1986), and it was used at a dilution of 1:500. Antibody 83 (an affinitypurified G.,, carboxy terminal specific antibody; see antibody RM, Simonds et al, 1989), and antiserum J99 (Go carboxyterminal specific; see antibody SW/i, Spiegel, 1990) were the gift of Dr. Alan Spiegel (National Institutes of Health, Bethesda, MD). Antibody 83 was used at a concentration of 10 ,g/ml and J99 at a dilution of 1:400.…”

Section: Antibodies and Antiseramentioning

confidence: 99%

Developmental regulation of calmodulin-dependent adenylate cyclase activity in an insect endocrine gland.

1990

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The insect prothoracic gland produces ecdysteroids that elicit molting and metamorphosis, and neurohormone stimulation of steroidogenesis by this gland involves both Ca2+ and cyclic adenosine monophosphate second messengers. Prothoracic gland adenylate cyclase exhibits a complex Ca2+/calmodulin (CaM) dependence, a component of which requires an activated Gs alpha for expression. A developmental switch in this system has been identified that correlates with a change in both regulation and function of the gland and involves the loss of sensitivity to extracellular Ca2+ at a time approximately concurrent with the loss of Ca2+/CaM sensitivity by the adenylate cyclase. The extent of cholera toxin activation of gland Gs alpha is lowered before this developmental switch. However, no alterations in Gs alpha levels or mobility are detected, suggesting that Gs alpha interaction with another component in the signaling pathway, perhaps adenylate cyclase itself, produces the apparent Ca2+/CaM dependence and influences the ability of toxin to modify Gs alpha.

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Changes in the expression of guanine nucleotide‐binding proteins during differentiation of 3T3‐F442A cells in a hormonally defined medium

Kilgour

Anderson

1993

FEBS Letters

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Using specific antisera, the expression of the G protein α subunits, G8, gi1, Gi2, Gi3, and G0, were determined in 3T3‐F442A cells during their differentiation to adipocytes in a hormonally defined medium. Differentiation caused distinct increases in the expression of two Gs isoforms and decreases in the expression of both Gi2 and Gi3. Differentiation also resulted in a 2‐ to 4‐fold increase in forskolin‐stimulated adenylyl cyclase activity and a 15‐fold increase in the response of cells to a β‐adrenergic agonist. The increase in Gs expression was also observed, to a lesser degree, in cells maintained at confluence under conditions where morphological conversion was negligible and the decreased expression of Gi2 and Gi3 and the increased β‐adrenergic responsiveness did not occur.

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Immunologic Probes for Heterotrimeric GTP-Binding Proteins

Cited by 11 publications

References 98 publications

Nerve growth factor changes G protein levels and localization in PC12 cells

Nerve growth factor changes G protein levels and localization in PC12 cells

Developmental regulation of calmodulin-dependent adenylate cyclase activity in an insect endocrine gland.

Changes in the expression of guanine nucleotide‐binding proteins during differentiation of 3T3‐F442A cells in a hormonally defined medium

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