Nicolas Vitale

Regulated Exocytosis in Chromaffin Cells TRANSLOCATION OF ARF6 STIMULATES A PLASMA MEMBRANE-ASSOCIATED PHOSPHOLIPASE D

The ADP-ribosylation factor (ARF) GTP-binding proteins have been implicated in a wide range of vesicle transport and fusion steps along the secretory pathway. In chromaffin cells, ARF6 is specifically associated with the membrane of secretory chromaffin granules. Since ARF6 is an established regulator of phospholipase D (PLD), we have examined the intracellular distribution of ARF6 and PLD activity in resting and stimulated chromaffin cells. We found that stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid translocation of ARF6 from secretory granules to the plasma membrane and the concomitant activation of PLD in the plasma membrane. To probe the existence of an ARF6-dependent PLD in chromaffin cells, we measured the PLD activity in purified plasma membranes. PLD could be activated by a nonhydrolyzable analogue of GTP and by recombinant myristoylated ARF6 and inhibited by specific anti-ARF6 antibodies. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 inhibited both PLD activity and catecholamine secretion in calcium-stimulated chromaffin cells. The possibility that ARF6 participates in the exocytotic reaction by controlling a plasma membrane-bound PLD and thereby generating fusogenic lipids at the exocytotic sites is discussed.

The GIT Family of ADP-ribosylation Factor GTPase-activating Proteins FUNCTIONAL DIVERSITY OF GIT2 THROUGH ALTERNATIVE SPLICING

We recently characterized a novel protein, GIT1, that interacts with G protein-coupled receptor kinases and possesses ADP-ribosylation factor (ARF) GTPase-activating protein activity. A second ubiquitously expressed member of the GIT protein family, GIT2, has been identified in data base searches. GIT2 undergoes extensive alternative splicing and exists in at least 10 and potentially as many as 33 distinct forms. The longest form of GIT2 is colinear with GIT1 and shares the same domain structure, whereas one major splice variant prominent in immune tissues completely lacks the carboxyl-terminal domain. The other 32 potential variants arise from the independent alternative splicing of five internal regions in the center of the molecule but share both the amino-terminal ARF GTPase-activating protein domain and carboxyl-terminal domain. Both the long and short carboxyl-terminal variants of GIT2 are active as GTPase-activating proteins for ARF1, and both also interact with G protein-coupled receptor kinase 2 and with p21-activated kinase-interacting exchange factors complexed with p21-activated kinase but not with paxillin. Cellular overexpression of the longest variant of GIT2 leads to inhibition of β2-adrenergic receptor sequestration, whereas the shortest splice variant appears inactive. Although GIT2 shares many properties with GIT1, it also exhibits both structural and functional diversity due to tissue-specific alternative splicing.

Guanine Nucleotide Exchange on ADP-ribosylation Factors Catalyzed by Cytohesin-1 and Its Sec7 Domain

ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins that require specific guanine nucleotide-exchange proteins (GEPs) to accelerate the conversion of inactive ARF-GDP to active ARF-GTP. Cytohesin-1, a 46-kDa ARF GEP, contains a central Sec7 domain of 188 amino acids similar in sequence to a region of the yeast Sec7 protein. Cytohesin-1 and its 22-kDa Sec7 domain (C-1 Sec7), synthesized in Escherichia coli, were assayed with recombinant non-myristoylated ARFs and related proteins to compare their GEP activities. Both were effective with native mammalian ARFs 1 and 3. Cytohesin-1 accelerated GTPγS (guanosine 5′-3-O-(thio)triphosphate) binding to recombinant human ARF1 (rARF1), yeast ARF3, and ARD1 (a 64-kDa guanine nucleotide-binding protein containing a C-terminal ARF domain). In contrast, C-1 Sec7 enhanced GTPγS binding to recombinant human ARFs 1, 5, and 6; yeast ARFs 1, 2, and 3; ARD1; two ARD1 mutants that contain the ARF domain; and Δ13ARF1, which lacks the N-terminal α-helix. Neither C-1 Sec7 nor cytohesin-1 increased GTPγS binding to human ARF-like ARL proteins 1, 2, and 3. Thus, ARLs, initially differentiated from ARFs because of their inability to activate cholera toxin, differ also in their failure to interact functionally with C-1 Sec7 or cytohesin-1. As C-1 Sec7 was much less substrate-specific than cytohesin-1, it appears that structure outside of the Sec7 domain is important for ARF specificity. Data obtained with mutant ARF constructs are all consistent with the conclusion that the ARF N terminus is an important determinant of cytohesin-1 specificity.

Interaction of the GTP-binding and GTPase-activating Domains of ARD1 Involves the Effector Region of the ADP-ribosylation Factor Domain

ADP-ribosylation factors (ARFs) are a family of ∼20-kDa guanine nucleotide-binding proteins and members of the Ras superfamily, originally identified and purified by their ability to enhance the ADP-ribosyltransferase activity of cholera toxin and more recently recognized as critical participants in vesicular trafficking pathways and phospholipase D activation. ARD1 is a 64-kDa protein with an 18-kDa carboxyl-terminal ARF domain (p3) and a 46-kDa amino-terminal extension (p5) that is widely expressed in mammalian tissues. Using recombinant proteins, we showed that p5, the amino-terminal domain of ARD1, stimulates the GTPase activity of p3, the ARF domain, and appears to be the GTPase-activating protein (GAP) component of this bifunctional protein, whereas in other members of the Ras superfamily a separate GAP molecule interacts with the effector region of the GTP-binding protein. p5 stimulated the GTPase activity of p3 but not of ARF1, which differs from p3 in several amino acids in the effector domain. After substitution of 7 amino acids from p3 in the appropriate position in ARF1, the chimeric protein ARF1(39-45p3) bound to p5, which increased its GTPase activity. Specifically, after Gly40 and Thr45 in the putative effector domain of ARF1 were replaced with the equivalent Asp and Pro, respectively, from p3, functional interaction of the chimeric ARF1 with p5 was increased. Thus, Asp25 and Pro30 of the ARF domain (p3) of ARD1 are involved in its functional and physical interaction with the GTPase-activating (p5) domain of ARD1. After deletion of the amino-terminal 15 amino acids from ARF1(39-45p3), its interaction with p5 was essentially equivalent to that of p3, suggesting that the amino terminus of ARF1(39-45p3) may interfere with binding to p5. These results are consistent with the conclusion that the GAP domain of ARD1 interacts with the effector region of the ARF domain and thereby stimulates GTP hydrolysis.

Characterization of a GDP Dissociation Inhibitory Region of ADP-ribosylation Factor Domain Protein ARD1

ADP-ribosylation factors (ARFs) are ∼20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and later recognized as critical components in intracellular vesicular transport and phospholipase D activation. ARF domain protein 1 (ARD1) is a member of the ARF family that differs from other ARFs by the presence of a 46-kDa amino-terminal extension. We previously reported that this extension acts as a GTPase-activating protein for the ARF domain of ARD1 (Vitale, N., Moss, J., and Vaughan, M. (1996)Proc. Natl. Acad. Sci. U.u2009S.u2009A. 93, 1941–1944). Both GTP binding and GTP hydrolysis are necessary for physiological function of guanine nucleotide-binding proteins, and the rates of GDP/GTP exchange and GTPase activity are critical in the activation/deactivation cycle. Dissociation of GDP from the ARF domain of ARD1 was faster than from ARD1 itself (both proteins synthesized in Escherichia coli). Using deletion mutations, it was demonstrated that the 15 amino acids directly preceding the ARF domain were responsible for decreasing the rate of GDP dissociation but not guanosine 5-[γ-thio]triphosphate dissociation. By site-specific mutagenesis it was shown that hydrophobic amino acids in this region were particularly important in stabilizing the GDP-bound form of ARD1. It is suggested that, like the amino-terminal segment of ARF, the equivalent region in ARD1, located between the GTPase-activating protein and ARF domains, may act as a GDP dissociation inhibitor.