The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2*

Abstract

Background:The functional role of the V-ATPase as a pH-sensing receptor remains unknown. Results: N-terminal peptides from the a-subunit isoforms of the V-ATPase modulate the enzymatic GEF activity of cytohesin-2. Conclusion: V-ATPase is a novel cytohesin-signaling receptor. Significance: These data reveal an evolutionarily conserved signaling process between V-ATPase, cytohesin-2, and Arfs, which is crucial for understanding the cell biological functions of these proteins.

Inhibition of Cytohesins Protects against Genetic Models of Motor Neuron Disease

“…The number of SMI32-positive neurons with cell body diameter 25 m or greater were counted in three randomly selected fields per coverslip, and the mean value was obtained (for methodological details, see Mojsilovic-Petrovic et al, 2006. In each experiment, at least three independent coverslips were used per condition and the results presented were obtained for at least four independent cultures and experiments.…”

“…The flowthrough was then applied to a HiLoad 16/60 Superdex 75 column preequilibrated in the following (in mM): 20 Tris, pH 8.0, 25 NaCl, 1 MgCl 2 , and 1 DTT, alogn with 0.1% Triton X-100 and 50 M GDP. More than 95% of the myrARF6 contains GDP when prepared by this method (Randazzo et al, 1995;Jian et al, 2010;Jian et al, 2012). The high MgCl 2 was used in this reaction to slow down the spontaneous nucleotide exchange.…”

“…His6-cytohesin 2 was expressed in and purified from bacteria as described previously for his6Brag2 (Jian et al, 2012). MyrARF6 was purified as described previously (Ha et al, 2005), with some modifications (the myrARF6 purified in this manner is GTP bound).…”

“…Rat mixed spinal cord neuron cultures were prepared as described previously (Mojsilovic-Petrovic et al, 2006). Briefly, Sprague Dawley rat dissociated embryonic day 15 (E15) spinal cord neurons were added to a confluent monolayer of astrocytes prepared from the cortex of postnatal day 2 rat pups.…”

Allosteric properties of PH domains in Arf regulatory proteins

“…The Arno/cytohesin/Grp1 subgroup regulates cell adhesion, migration, cytoskeleton remodeling, positioning of Glut-4 transporters, endocytosis and lysosomal maturation. [30][31][32][33][34][35][36] The IQsec/Brag proteins regulate cellular adhesions, at least in part, by regulating the endocytosis of integrins and E-cadherin, [37][38][39][40][41] myoblast fusion 42,43 and endocytosis of AMPA receptors in excitatory synapses. 44 Brag proteins contribute to cancer cell invasion 45,46 and antiangiogenic signaling, 39,40 and mutations are associated with intellectual disability.…”