ARF-independent inhibition of the carbachol-induced contractions by brefeldin A in intestinal smooth muscle

1997 ◽  
Vol 273 (3) ◽  
pp. C816-C821 ◽  
Author(s):  
G. Loirand ◽  
C. Cario-Toumaniantz ◽  
P. Chardin ◽  
P. Pacaud
Keyword(s):  
Smooth Muscle ◽  
Brefeldin A ◽  
Inhibitory Effect ◽  
Intestinal Smooth Muscle ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Intact Cells ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Voltage Dependent

The aim of this study was to determine whether an ADP ribosylation factor (ARF)-regulated pathway is involved in the carbachol-induced contraction in rat intestinal smooth muscle. Brefeldin A, a known inhibitor of the guanine nucleotide exchange activity on ARF, reversibly inhibited the carbachol-induced contraction in intact ileal muscle strips, whereas the carbachol- and guanosine 5'-O-(3-thiotriphosphate)-induced increases in the Ca2+ sensitivity of myofilaments in beta-escin-permeabilized strips were not affected. The high-K(+)-induced contraction in intact strips was also inhibited by brefeldin A. In isolated ileal myocytes, brefeldin A inhibited the Ca2+ channel current, indicating that the inhibitory effect of brefeldin A in intact cells is related to an inhibition of voltage-dependent Ca2+ channels. Furthermore, the loading of permeabilized strips with the combination of the recombinant fully myristoylated ARF1, the guanine nucleotide exchange factor ARNO, and guanosine 5'-triphosphate did not change the tone at constant pCa (6.45) and did not modify the carbachol- and guanosine 5'-O-(3-thiotriphosphate)-induced Ca2+ sensitization. Taken together, these findings suggest that an ARF-dependent pathway is not involved in the carbachol-induced contraction.

Human p63RhoGEF, a novel RhoA-specific guanine nucleotide exchange factor, is localized in cardiac sarcomere

2002 ◽  
Vol 115 (3) ◽  
pp. 629-640 ◽  
Author(s):  
Michel Souchet ◽  
Elodie Portales-Casamar ◽  
David Mazurais ◽  
Susanne Schmidt ◽  
Isabelle Léger ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Fiber Formation ◽  
Pleckstrin Homology Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Intact Cells ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The Rho small GTPases are crucial proteins involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. It has been reported that these GTPases are directly associated with cardiovascular disorders. In this context, we have searched for novel modulators of Rho GTPases, and here we describe p63RhoGEF a new Db1-like guanine nucleotide exchange factor (GEF). P63RhoGEF encodes a 63 kDa protein containing a Db1 homology domain in tandem with a pleckstrin homology domain and is most closely related to the second Rho GEF domain of Trio. Northern blot and in situ analysis have shown that p63RhoGEF is mainly expressed in heart and brain. In vitro guanine nucleotide exchange assays have shown that p63RhoGEF specifically acts on RhoA. Accordingly, p63RhoGEF expression induces RhoA-dependent stress fiber formation in fibroblasts and in H9C2 cardiac myoblasts. Moreover, we show that p63RhoGEF activation of RhoA in intact cells is dependent on the presence of the PH domain. Using a specific anti-p63RhoGEF antibody, we have detected the p63RhoGEF protein by immunocytochemistry in human heart and brain tissue sections. Confocal microscopy shows that p63RhoGEF is located in the sarcomeric I-band mainly constituted of cardiac sarcomeric actin. Together, these results show that p63RhoGEF is a RhoA-specific GEF that may play a key role in actin cytoskeleton reorganization in different tissues, especially in heart cellular morphology.

scholarly journals Promiscuity of the catalytic Sec7 domain within the guanine nucleotide exchange factor GBF1 in ARF activation, Golgi homeostasis, and effector recruitment

2019 ◽  
Vol 30 (12) ◽  
pp. 1523-1535 ◽  
Author(s):  
Jay M. Bhatt ◽  
William Hancock ◽  
Justyna M. Meissner ◽  
Aneta Kaczmarczyk ◽  
Eunjoo Lee ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Downstream Effector ◽  
Nucleotide Exchange Factor ◽  
Sec7 Domain ◽  
Golgi Network ◽  
The Impact

The integrity of the Golgi and trans-Golgi network (TGN) is disrupted by brefeldin A (BFA), which inhibits the Golgi-localized BFA-sensitive factor (GBF1) and brefeldin A–inhibited guanine nucleotide-exchange factors (BIG1 and BIG2). Using a cellular replacement assay to assess GBF1 functionality without interference from the BIGs, we show that GBF1 alone maintains Golgi architecture; facilitates secretion; activates ADP-ribosylation factor (ARF)1, 3, 4, and 5; and recruits ARF effectors to Golgi membranes. Unexpectedly, GBF1 also supports TGN integrity and recruits numerous TGN-localized ARF effectors. The impact of the catalytic Sec7 domain (Sec7d) on GBF1 functionality was assessed by swapping it with the Sec7d from ARF nucleotide-binding site opener (ARNO)/cytohesin-2, a plasma membrane GEF reported to activate all ARFs. The resulting chimera (GBF1-ARNO-GBF1 [GARG]) targets like GBF1, supports Golgi/TGN architecture, and facilitates secretion. However, unlike GBF1, GARG activates all ARFs (including ARF6) at the Golgi/TGN and recruits additional ARF effectors to the Golgi/TGN. Our results have general implications: 1) GEF’s targeting is independent of Sec7d, but Sec7d influence the GEF substrate specificity and downstream effector events; 2) all ARFs have access to all membranes, but are restricted in their distribution by the localization of their activating GEFs; and 3) effector association with membranes requires the coincidental presence of activated ARFs and specific membrane identifiers.

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