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M F Crouch

Publications and source records attributed to M F Crouch.

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

Signal transduction from membrane to nucleus: the special case for neurons.

Neurons have a unique problem with signal transduction from the membrane in the region of their terminals back to the cell body and nucleus. This distance may be several meters in some nerves in some species, so there is a requirement for some mechanism to stabilize the signal. This review examines two complementary mechanisms for this signal transduction, either by the retrograde axonal transport of the neurotrophic factor together with its receptor, or the transport of a stable activated second messenger molecule. Extrapolation of studies on the fibroblast signal transduction pathway, where it has been shown that G1 can translocate from the membrane to the nucleus, has led to the demonstration of the retrograde axonal transport of several putative signaling molecules. The alpha subunits of both G1 and Gz are retrogradely transported and Gz alpha or possibly the intact heterotrimeric Gz subsequently accumulates in dorsal root ganglia nuclei. Thus Gz1 Gi1 and potentially other G-proteins and distinct signaling molecules may provide additional signal transduction pathways to that of the neurotrophins from terminal to nucleus.

Animals↗
PubMed1995

Evidence that the pertussis toxin-sensitive trimeric GTP-binding protein Gi2 is required for agonist- and store-activated Ca2+ inflow in hepatocytes.

The role of a trimeric GTP-binding protein (G-protein) in the mechanism of vasopressin-dependent Ca2+ inflow in hepatocytes was investigated using both antibodies against the carboxyl termini of trimeric G-protein alpha subunits, and carboxyl-terminal alpha-subunit synthetic peptides. An anti-Gi1-2 alpha antibody and a Gi2 alpha peptide (Gi2 alpha) Ile345-Phe355), but not a Gi3 alpha peptide (Gi3 alpha Ile344-Phe354), inhibited vasopressin- and thapsigargin-stimulated Ca2+ inflow, had no effect on vasopressin-stimulated release of Ca2+ from intracellular stores, and caused partial inhibition of thapsigargin-stimulated release of Ca2+. An anti-Gq alpha antibody also inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. Immunofluorescence measurements showed that Gi2 alpha is distributed throughout much of the interior of the hepatocyte as well as at the periphery of the cell. By contrast, Gq/11 alpha was found principally at the cell periphery. It is concluded that the trimeric G-protein, Gi2, is required for store-activated Ca2+ inflow in hepatocytes and acts between the release of Ca2+ from the endoplasmic reticulum (presumably adjacent to the plasma membrane) and the receptor-activated Ca2+ channel protein(s) in the plasma membrane.

Amino Acid Sequence↗
PubMed1995

Retrograde axonal transport of signal transduction proteins in rat sciatic nerve.

Neurons require a mechanism to transmit stable signals over the large distance from the nerve growth cone or terminal to the cell body, in order that information from the target tissue can be relayed to the cell body where it is required. Nerve growth factor (NGF), a target-derived neurotrophic factor, is thought to signal over this distance by receptor mediated internalization of NGF, followed by retrograde axonal transport of the NGF-receptor complex. In this paper we show, by immunohistochemistry of rat sciatic nerve, accumulation of phosphotyrosine immunoreactivity only on the distal side of a nerve crush, suggesting axonal transport of tyrosine kinases and/or tyrosine phosphorylated proteins primarily in a retrograde direction. Furthermore, we also show retrograde axonal transport of phosphoinositide 3-kinase, ERK, MEK and MEK kinase, of which all but MEK kinase are known to be activated downstream of tyrosine receptor kinase activation. The retrograde transport of these proteins suggests that they may be involved in transmission of signals along the axon, relaying neurotrophic factor receptor activation at the nerve terminal to the nerve cell body.

Animals↗
PubMed1995

Interaction of the GTP-binding protein Gi2 with a protein kinase A-like kinase in mouse fibroblasts.

We have previously shown that the GTP-binding protein, Gi2 of mouse Balb/c3T3 cells is linked to a serine kinase which phosphorylates the alpha-subunit of Gi itself. In this report we show that Gi is coupled to a second protein kinase. This kinase does not phosphorylate G but phosphorylates another protein bound non-covalently to G. Phosphorylation of the Gi-linked protein induces its release from Gi. Kinase activity is slightly enhanced by GTPyS, suggesting that this kinase may be physiologically regulated by Gi. In an attempt to identify the kinase we have examined the effect of peptide substrates and inhibitors on kinase activity. We found that the protein kinase A inhibitory peptide, PK1 5-24, inhibited the kinase activity, but at concentrations above those usually required to block protein kinase A. The protein kinase A substrate peptide, kemptide, acted as a substrate of the kinase, and was an inhibitor of the phosphorylation of the Gi-linked protein. However, a protein kinase A, catalytic subunit antibody failed to react with any proteins linked to Gi., A protein kinase C inhibitory peptide had no effect on phosphorylation of the Gi-linked protein. Thus, the identity of this kinase has not been resolved, but it may form part of the signalling system of activated Gi in fibroblasts.

Animals↗
PubMed1994

Retrograde axonal transport of the alpha-subunit of the GTP-binding protein GZ in mouse sciatic nerve: a potential pathway for signal transduction in neurons.

We have utilized antibodies against the alpha subunit of GZ in fluorescence immunohistochemistry to determine whether this GTP-binding protein can translocate along nerves by intra-axonal transport. After ligation of the mouse sciatic nerve we found an increase in GZ-like immunoreactivity on the proximal and distal side with time, suggesting that the alpha subunit undergoes orthograde axonal transport and also returns to the cell body by retrograde axonal transport in the sciatic nerve. Unlike the retrograde transport of Gi alpha, shown in a previous study to be present in most sciatic axons, GZ alpha only accumulated in a subpopulation of axons, suggesting that different G-proteins could convey information specific to neuronal subtypes. These results support our proposal that GZ may play a second messenger role in communicating information from the terminals back to cell bodies. Gi alpha and GZ alpha may be representative of relatively stable signalling molecules by which the signal from some neurotrophic molecules can be translocated from the neuron periphery to the cell body without the need for the retrograde transport of the neurotrophic factor itself.

Amino Acid Sequence↗
PubMed1994

The GTP-binding protein Gi alpha 2 is directly linked to and substrate of a serine kinase in Balb/c3T3 cells.

Mitosis of Balb/c3T3 cells induced by epidermal growth factor and insulin is inhibited by pertussis toxin. Pertussis toxin inactivates certain GTP-binding proteins, of which only Gi is present in Balb/c3T3 cells. Therefore, Gi was implicated as important in the signal transduction of EGF and insulin receptors leading to mitosis. Our previous studies of the role of Gi in cell division have shown that the alpha-subunit of Gi(Gi alpha) is induced to translocate from the cell periphery to the nucleus by these growth factors, and in the nucleus of dividing cells Gi alpha binds to the separating chromatin. As protein phosphorylations are essential components of the messenger systems from these receptors, we have examined whether Gi could be functionally coupled to protein kinases in the activated cell. We have found that Gi alpha 2 is directly linked to a serine kinase in Balb/c3T3 fibroblasts, and that Gi alpha 2 itself is a substrate for phosphorylation in vitro. This phosphorylation of Gi alpha 2 is inhibited if the G-protein is first activated with GTP or inactivated with GDP, suggesting that the phosphorylation may be occurring in the guanine nucleotide binding region. We present evidence that the kinase is not a protein kinase C. Such a phosphorylation of Gi alpha 2 could represent either a negative feedback mechanism of signal transduction, or a GTP-independent pathway of G-protein signal transduction in fibroblasts.

3T3 Cells↗
PubMed1993

Growth factor second messenger systems: oncogenes and the heterotrimeric GTP-binding protein connection.

We feel that there is now compelling evidence that the GTP-binding proteins play more than just a coordinating role in the actions of both tyrosine kinase and nontyrosine kinase receptor signal transduction. These similarities appear to represent just a small component of the convergence in the signaling pathways for structurally dissimilar receptor subsets. Future years will see further understanding of the intricacies of these G-protein-proto-oncogene interactions, and the extension into the potential role in growth factor action played by the expanding number of known members of this G-protein family.

Animals↗
PubMed1993

Gi alpha and Gi beta are part of a signalling complex in Balb/c3T3 cells: phosphorylation of Gi beta in growth-factor-activated fibroblasts.

Stimulation of division of Balb/c3T3 cells by epidermal growth factor (EGF) and/or insulin is inhibited by pertussis toxin. The G-protein involvement in this response includes the growth factor receptor-induced translocation of the alpha-subunit of Gi (Gi alpha) to the nucleus, where Gi alpha binds specifically to chromatin of dividing cells. This paper reports the first data of studies on the mode of interaction of tyrosine kinase growth factor receptors with Gi alpha, and the mechanism by which Gi affects cell proliferation. When Gi alpha was immunoprecipitated from Triton X-100 extracts of Balb/c3T3 cells, several other proteins were co-precipitated. The major proteins, of 110,000, 60,000 and 36,000 M(r), were not directly recognized by the Gi alpha antibody, showing that Gi alpha was in a complex with these proteins. The 36,000 M(r) protein was recognized by G beta-common antiserum, so confirming its identity as Gi beta. The 36,000 M(r) protein was phosphorylated in cells activated for 20 h with platelet-derived growth factor, epidermal growth factor and insulin, but not after 3 min or 1 h of stimulation. Both Gi alpha and G beta-common antibodies precipitated the phosphorylated 36,000 protein. Gi beta phosphorylation was similarly observed in response to activation by EGF alone for 20 h, but to a lesser extent. Phosphotyrosine antibodies also precipitated a 36,000 M(r) phosphorylated protein from growth factor-activated cells, suggesting that Gi beta may be phosphorylated on tyrosine. Therefore, Gi beta phosphorylation appears to represent a late event after activation of cells by tyrosine kinase growth factor receptors. We are currently examining the role of this event in signal transduction, particularly in relation to control of nuclear responses.

3T3 Cells↗
PubMed1993

Localization of the GTP-binding protein Gi alpha in myelomonocytic progenitor cells is regulated by proliferation (GM-CSF, IL-3) and differentiation (TNF) signals.

We have examined the role of Gi alpha in haemopoietic cells using the myelomonocytic progenitor cell lines FDC-P1 and WEHI-3B (JCS). During growth factor-dependent proliferation of FDC-P1 cells Gi alpha was found predominantly in the nucleus and associated with the plasma membrane. Following removal of growth factor, Gi alpha accumulated in the cytoplasm and at the plasma membrane. Treatment of FDC-P1 cells with pertussis toxin (PT) completely inhibited translocation of Gi alpha to the nucleus and reduced the sensitivity of FDC-P1 cells to the proliferative effects of growth factors, indicating that translocation of Gi alpha plays a regulatory role in, but may not be essential for, cell division. Gi alpha initially associated with DNA during S/G2 of the FDC-P1 cell cycle but separated from condensing chromosomes during mitosis. In contrast to FDC-P1 cells, WEHI-3B (JCS) cells proliferate in the absence of added growth factors but can be induced to differentiate by TNF-alpha. In proliferating JCS cells Gi alpha was again associated with the nucleus but when proliferation was inhibited by TNF-alpha, Gi alpha accumulated in the cytoplasm with none detected in the nucleus. Thus the cytokine regulated accumulation of Gi alpha at different intracellular sites correlated with the ability of the cell to progress through the proliferative cycle. When the tyrosine kinase inhibitor genistein was added to FDC-P1 cells prior to stimulation with IL-3 or GM-CSF, proliferation was almost completely inhibited but translocation of Gi alpha was not affected, suggesting that tyrosine phosphorylation was not involved in G protein translocation but was essential for cytokine induced cell division. Cholera toxin (CT) also inhibited proliferation of FDC-P1 cells but had no effect on translocation of Gi alpha to the nucleus. The near complete inhibition of cell division by genistein and CT without a corresponding effect on Gi alpha movement indicates that Gi alpha can be regulated independently of tyrosine kinase and adenylyl cyclase activities, respectively.

Adenylate Cyclase Toxin↗
PubMed1992

Epinephrine induces association of pp60src with Gi alpha in human platelets.

Using specific antibodies against the alpha subunit of the inhibitory GTP-binding protein Gi, we analyzed the association of Gi alpha with other cellular components in human platelets. Three tyrosine phosphorylated proteins with molecular mass of 63, 58, and 55 kDa were specifically associated with Gi alpha in resting platelets. Stimulation of platelets with epinephrine, but not with thrombin, induced an increase of the reactivity of the 63- and 55-kDa proteins to anti-phosphotyrosine antibodies on western blotting. By in vitro kinase assay we found that epinephrine induced the association of kinase activity with Gi alpha and that the 63-kDa protein was phosphorylated by this activity. The association of kinase activity with Gi alpha in epinephrine-stimulated platelets paralleled the association of pp60src with Gi alpha, as detected by western blotting analysis using specific anti-pp60src monoclonal antibodies. The interaction of pp60src with Gi alpha may play a role in the mechanism of platelet activation by epinephrine or in the epinephrine-induced potentiation of the action of other platelet agonists.

Blood Platelets↗
PubMed1991

Retrograde axonal transport of the GTP-binding protein Gi alpha: a potential neurotrophic intra-axonal messenger.

When a neuronal target is to provide information to the nucleus of the neurone innervating it, it faces the problem of getting its message up the long length of axon separating the cell body from the site of receptor activation at the terminal. The retrograde axonal transport of the neurotrophic molecule, nerve growth factor (NGF), provided one possible mechanism for this information transfer in the sympathetic nervous system. However, some neurotrophic molecules are not retrogradely transported, indicating the message is carried back by a different mechanism. In this paper, we examined such a novel mechanism mediated by the retrograde axonal transport of the alpha subunit of the second messenger protein, Gi. It is proposed that some non-transported neurotrophic molecules may produce a stable second messenger that is itself transported to the nucleus to convey the target derived information for survival.

Animals↗
PubMed1991

Co-activation of insulin-like growth factor-I receptors and protein kinase C results in parasympathetic neuronal survival.

We have studied the interaction between several growth factors to promote parasympathetic neuronal survival. Neither insulin nor insulin-like growth factor-I (IGF-I) had any effect on the survival of embryonic day 8 chick ciliary neurons in culture. Similarly, the protein kinase C activator phorbol dibutyrate (PdBu) had only a minor survival-promoting activity. In combination with PdBu, however, IGF-I or insulin, at concentrations sufficient to act through the IGF-I receptor, were highly synergistic. In a similar fashion, acidic fibroblast growth factor (aFGF)-induced neuronal survival was greatly enhanced by PdBu, as well as by insulin or IGF-I. When added alone, aFGF-induced cell survival required the presence of 1% serum. However, addition of aFGF, IGF-I, or insulin with PdBu under serum-free conditions replaced the serum requirement. That is, these agonist combinations could apparently induce the second messenger requirement for ciliary neuronal survival. Therefore, IGF-I must now be included in the list of candidate molecules responsible for directing parasympathetic nerve formation. The synergy between agonists observed in these experiments highlights the possibility that combinations of growth factors, rather than sole molecules, may dictate parasympathetic nervous system development in vivo.

Animals↗
PubMed1991

Growth factor-induced cell division is paralleled by translocation of Gi alpha to the nucleus.

Induction of mitosis by certain growth factors is inhibited by pertussis toxin, indicating that the GTP-binding protein, Gi, is involved in receptor signal transduction to initiate cell division. However, the substrates of receptor-activated Gi that are involved in mitosis have not been determined. The present study has examined whether Gi may directly modulate cell division by receptor-induced subcellular translocation of the alpha subunit of Gi (Gi alpha). Insulin and EGF, particularly when added together or in combination with phorbol dibutyrate (PdBu), induced a rapid (1-4 h) redistribution of Gi alpha from the plasma membrane to perinuclear sites in the cell. After 2 days of stimulation, Gi alpha had translocated into the nucleus of dividing cells and bound specifically to the separating chromatin of dividing nuclei. Unstimulated cells did not display translocation of Gi alpha. This demonstrates a direct involvement of Gi alpha in cell division, which provides an apparently uninterrupted link from growth factor receptor to nucleus.

Amino Acid Sequence↗
PubMed1990

Pertussis toxin inhibits EGF-, phorbol ester- and insulin-stimulated DNA synthesis in BALB/c3T3 cells: evidence for post-receptor activation of Gi alpha.

The contribution of the GTP-binding protein, Gi, to EGF, phorbol dibutyrate (PdBu)-, and insulin-stimulated DNA synthesis was examined in BALB/c3T3 cells. Pertussis toxin inhibited DNA synthesis by each agonist, particularly at suboptimal agonist concentrations, but the inhibition could be partially overcome with higher agonist concentrations and combinations of these agonists. This suggested that (1) some, but not all, of the mitogenic signals for all three agonists were transduced by Gi (2) Gi may be activated by post-receptor mechanisms involving protein kinase C. Gi alpha-specific antibodies and ADP-ribosylation by pertussis toxin using 32P-NAD each labelled a single protein band, representing one or more species of Gi alpha. Pertussis toxin treatment increased the synthesis of Gi alpha. These results are discussed in relation to possible direct effects of Gi alpha on nuclear control during division.

Animals↗
PubMed1990

Regulation of hormone-induced Ca2+ mobilization in the human platelets.

alpha-Thrombin, gamma-thrombin, and platelet-activating factor each stimulated the mobilization of intracellular Ca2+ stores in aspirin-treated human platelets. This was followed by desensitization of the receptors, as shown by the return of the Ca2+ level to basal values and by the fact that a subsequent addition of a second different agonist, but not the same agonist, could again elicit a response. Epinephrine, acting on alpha 2-adrenergic receptors, was by itself ineffective at mobilizing Ca2+ stores. However, when added after the thrombin-induced response, epinephrine could evoke a considerable release of Ca2+ from cellular stores. This appeared to be due to epinephrine recoupling thrombin receptors to phospholipase C. In support of this, epinephrine was able to induce the formation of inositol triphosphate when added after the response to thrombin had also become desensitized. Alone, epinephrine was without effect. Pre-activation of protein kinase C with the phorbol ester abolished these effects of epinephrine, suggesting that epinephrine was working by activating a protein which could be inactivated by phosphorylation. Our current work is to characterize this protein that may be a member of the Gi, GTP-binding protein family.

Blood Platelets↗
PubMed1989

Dual mechanisms of platelet hormone receptor desensitization. Differential importance between agonists of protein kinase C-dependent and -independent pathways.

Several different agonists, among them alpha-thrombin, platelet-activating factor, vasopressin, thromboxane A2, and endoperoxides, activate platelets to aggregate and secrete granular contents. Each of these agents is thought to act by inducing the turnover of inositol phospholipids and generating the second messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. However, within minutes, the action of these agonists desensitizes. We have studied the characteristics of this desensitization process for the agonists mentioned above in an attempt to clarify the mechanisms involved. Our results show that two different pathways of desensitization exist, one that is mediated by protein kinase C and another that is independent of this enzyme. In addition, the contribution of these pathways to desensitization differs for the agonists studied. Our data suggest that partial agonists and strong agonists differ in the rate at which the primary response is desensitized rather than in their ability to couple to phospholipase C.

Blood Platelets↗
PubMed1989

Epinephrine induces changes in the subcellular distribution of the inhibitory GTP-binding protein Gi alpha-2 and a 38-kDa phosphorylated protein in the human platelet.

By using antibodies specific for alpha subunits of inhibitory GTP-binding proteins (Gi alpha polypeptides) to probe Western blots of whole platelet protein, we detected Gi alpha-2 as the predominant Gi alpha species present in platelets. The subcellular compartmentalization of distinct Gi alpha-2-immunoreactive polypeptides coupled to thrombin and alpha 2-adrenergic receptors was examined in Triton X-100 platelet lysates prepared by highspeed centrifugation. This treatment permitted separation of the Triton-insoluble membrane skeleton from Triton-soluble cell components. In cells treated with either alpha-thrombin or epinephrine, we observed that a greater proportion of Gi alpha-2 was localized in the Triton-soluble fraction than in the Triton-insoluble fraction. Pertussis toxin was found to catalyze ADP-ribosylation of Gi alpha-2 in whole platelets. In thrombin-stimulated cells, this activity was confined to the Triton-soluble fraction and was markedly lower than that of unstimulated cells. Epinephrine, on the other hand, promoted translocation of a portion of the pertussis toxin-sensitive Gi alpha-2 from the Triton-soluble fraction to the Triton-insoluble fraction. In addition, epinephrine stimulated translocation of a phosphorylated protein of approximately 38 kDa that was not ADP-ribosylated by pertussis toxin. This protein expressed immunoreactivity with the general Gi alpha antiserum AS/7 but not with the Gi alpha-2 antiserum LE/3. These findings suggest a role for specific localization of Gi alpha proteins in epinephrine-induced platelet responses.

Adenosine Diphosphate Ribose↗