ADP-ribosylation Factor-directed GTPase-activating Protein

144

111

ADP-ribosylation factor (ARF) proteins are key players in numerous vesicular trafficking events ranging from the formation and fusion of vesicles in the Golgi apparatus to exocytosis and endocytosis. To complete their GTPase cycle, ARFs require a guanine nucleotideexchange protein to catalyze replacement of GDP by GTP and a GTPase-activating protein (GAP) to accelerate hydrolysis of bound GTP. Recently numerous guanine nucleotide-exchange proteins and GAP proteins have been identified and partially characterized. Every ARF GAP protein identified to date contains a characteristic zinc finger motif. GIT1 and GIT2, two members of a new family of G protein-coupled receptor kinaseinteracting proteins, also contain a putative zinc finger motif and display ARF GAP activity. Truncation of the amino-terminal region containing the zinc finger motif prevented GAP activity of GIT1. One zinc molecule was found associated per molecule of purified recombinant ARF-GAP1, GIT1, and GIT2 proteins, suggesting the zinc finger motifs of ARF GAPs are functional and should play an important role in their GAP activity. Unlike ARF-GAP1, GIT1 and GIT2 stimulate hydrolysis of GTP bound to ARF6. Accordingly we found that the phospholipid dependence of the GAP activity of ARF-GAP1 and GIT proteins was quite different, as the GIT proteins are stimulated by phosphatidylinositol 3,4,5-trisphosphate whereas ARF-GAP1 is stimulated by phosphatidylinositol 4,5-bisphosphate and diacylglycerol. These results suggest that although the mechanism of GTP hydrolysis is probably very similar in these two families of ARF GAPs, GIT proteins might specifically regulate the activity of ARF6 in cells in coordination with phosphatidylinositol 3-kinase signaling pathways.

Section: Resultsmentioning

confidence: 99%

GIT Proteins, A Novel Family of Phosphatidylinositol 3,4,5-Trisphosphate-stimulated GTPase-activating Proteins for ARF6

Vitale¹,

Patton²,

Moss³

et al. 2000

144

111

“…The mutagenic primer 5Ј-AATCGAGTTCACCGTGGACCAAAAATGG-3Ј changed Ile 12 to Arg. The mutagenic primer 5Ј-CACATTGGAGGA-AAAATGGAAATTCGG-3Ј changed Pro 14 to Gly. The mutagenic primer 5Ј-CACATTGGACCAGCAATGGAAATTCGG-3Ј changed Lys 15 to Ala.…”

Section: Methodsmentioning

confidence: 99%

“…ARFs are ϳ20-kDa proteins that exhibit no detectable GTPase activity (4); the ratio of GTP/GDP bound appears to be governed by guanine nucleotide-exchange proteins (GEPs) and GTPase-activating proteins (GAPs) (3). ARF GEPs (5)(6)(7)(8)(9)(10)(11)(12) and GAPs (13)(14)(15)(16) have been both purified and cloned. Inhibition of cytosolic and Golgi-associated GEPs by the fungal fatty acid metabolite brefeldin A (BFA) has been reported (5-7, 9); both BFA-insensitive and BFA-sensitive cytosolic GEPs have been isolated (8,9).…”

mentioning

confidence: 99%

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

Vitale

Moss

Vaughan

1997

Although roles of G proteins 1 are enormously diverse, these GTPases all operate by a fundamentally similar mechanism (1). When GTP occupies its guanine nucleotide-binding site, the G protein can interact with and modify the activity of a downstream target protein. Hydrolysis of GTP causes the dissociation of the G protein-target complex and thus terminates the "active state" of the G protein. Cells regulate the ratio of active and inactive G proteins by modulating the rates of GDP release and GTP hydrolysis (GTPase activity).ARD1 is a 64-kDa protein that contains a 18-kDa carboxylterminal ADP-ribosylation factor (ARF) domain (p3) and a 46-kDa amino-terminal domain (p5) (2). ARF proteins have been implicated in vesicular membrane trafficking in several intracellular compartments, including endoplasmic reticulum, Golgi, endosomes, and nuclear envelope (reviewed in Ref. 3). ARFs are ϳ20-kDa proteins that exhibit no detectable GTPase activity (4); the ratio of GTP/GDP bound appears to be governed by guanine nucleotide-exchange proteins (GEPs) and GTPase-activating proteins (GAPs) (3). ARF GEPs (5-12) and GAPs (13-16) have been both purified and cloned. Inhibition of cytosolic and Golgi-associated GEPs by the fungal fatty acid metabolite brefeldin A (BFA) has been reported (5-7, 9); both BFA-insensitive and BFA-sensitive cytosolic GEPs have been isolated (8, 9). There is genetic as well as biochemical evidence that both BFA-sensitive and BFA-insensitive ARF GEPs contain a domain similar to a part of Sec7 (9 -12), a protein necessary for intra-Golgi transport.It was recently reported that the ARF domain of ARD1 binds specifically GDP and GTP, whereas the amino-terminal domain does not (17). Using recombinant proteins, it was shown that the amino-terminal p5 domain of ARD1 stimulates hydrolysis of GTP bound to the ARF domain p3 and consequently appears to be the GAP component of this bifunctional protein (18). The stimulatory effect of the p5 domain on the GTPase activity of p3 was specific, because GTP hydrolysis by other members of the ARF family was not increased (16). The presence of an intrinsic GAP domain is an apparently unique phenomenon for monomeric guanine nucleotide-binding proteins, because GTPase activity of other members of the Ras superfamily is increased by a separate GAP molecule, which interacts with the effector region of the protein (17). Using chimeric proteins, we demonstrated that the GAP domain of ARD1 similarly interacts with the effector region of the ARF domain of ARD1 and thereby stimulates GTP hydrolysis (17).Nucleotide hydrolysis and product dissociation are both regulated steps in the GTPase cycle. Because their rates determine when and for how long the G protein is active, it is important to precisely understand these mechanisms. We reported that GDP␤S dissociation from p3 was faster than from ARD1 (18). Accordingly, in the presence of certain phospholipids (i.e. brain phosphatidylcholine plus phosphatidylinositol bisphosphate plus phosphatidylethanolamine or phosphatidylserine) more G...

“…Conformational differences between their GDP-and GTP-bound forms determine their interaction with regulatory proteins, namely a guanine nucleotide exchange protein (GEP) and a GTPase-activating protein (GAP). Class I ARF-directed GEP has been purified (11), and ARF1-directed GAP has also been purified and cloned (12,13). In contrast, the downstream effectors of ARF remain to be clarified.…”

mentioning

confidence: 99%

Arfaptin 1, a Putative Cytosolic Target Protein of ADP-ribosylation Factor, Is Recruited to Golgi Membranes

Kanoh

Williger

Exton

1997

112

101

ADP-ribosylation factors (ARFs) have been implicated in vesicle transport in the Golgi complex. Employing yeast two-hybrid screening of an HL60 cDNA library using a constitutively active mutant of ARF3 (ARF3⅐Q71L), as a probe, we have identified a cDNA encoding a novel protein with a calculated molecular mass of 38.6 kDa, which we have named arfaptin 1. The mRNA of arfaptin 1 was ubiquitously expressed, and recombinant arfaptin 1 bound preferentially to class I ARFs, especially ARF1, but only in the GTP-bound form. The interactions were independent of myristoylation of ARF. Arfaptin 1 in cytosol was recruited to Golgi membranes by ARF in a guanosine 5 -O-(3-thiotriphosphate)-dependent and brefeldin A-sensitive manner. When expressed in COS cells, arfaptin 1 was localized to the Golgi complex. The yeast two-hybrid system yielded another clone, which encoded a putative protein, which we have named arfaptin 2. This consisted of the same number of amino acids as arfaptin 1 and was 60% identical to it. Arfaptin 2 was also ubiquitously expressed and bound to the GTP-, but not GDP-liganded form of class I ARFs, especially ARF1. These results suggest that arfaptins 1 and 2 may be direct target proteins of class 1 ARFs. Arfaptin 1 may be involved in Golgi function along with ARF1.