title: The N termini of a-subunit isoforms are involved in signaling between vacuolar H+-ATPase (V-ATPase) and cytohesin-2.
abstract: Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H(+)-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1-17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1-17) and its amino acids Phe(5), Met(10), and Gln(14) involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1-17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1-a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor.
authors.affiliation: Center for Systems Biology, Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hosokawa
authors.forename: Phat Vinh
authors.initials: PV
authors.lastname: Dip
authors.forename: Maria
authors.initials: M
authors.lastname: Merkulova
authors.forename: Anastasia
authors.initials: A
authors.lastname: Bakulina
authors.forename: Zhenjie
authors.initials: Z
authors.lastname: Zhuang
authors.forename: Ashok
authors.initials: A
authors.lastname: Khatri
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Jian
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Keating
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Bueler
authors.forename: John L
authors.initials: JL
authors.lastname: Rubinstein
authors.forename: Paul A
authors.initials: PA
authors.lastname: Randazzo
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Ausiello
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
authors.forename: Vladimir
authors.initials: V
authors.lastname: Marshansky
mesh_terms: D020727:ADP-Ribosylation Factors / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D014364:Tryptophan / Q000737:chemistry; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D000939:Epitopes; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; C102474:cytohesin-2; D014364:Tryptophan; D019204:GTP-Binding Proteins; D025262:Vacuolar Proton-Translocating ATPases; D020727:ADP-Ribosylation Factors