VACM-1, a cullin gene family member, regulates cellular signaling

Vasopressin-activated Ca²⁺-mobilizing (VACM-1)receptor binds arginine vasopressin (AVP) but does not haveamino acid sequence homology with the traditional AVP receptors.VACM-1, however, is homologous with a newly discovered cullin family ofproteins that has been implicated in the regulation of cell cyclethrough the ubiquitin-mediated degradation of cyclin-dependent kinase inhibitors. Because cell cycle processes can be regulated by the transmembrane signal transduction systems, the effects of VACM-1 expression on the Ca²⁺ and cAMP-dependent signaling pathwaywere examined in a stable cell line expressing VACM-1 in VACM-1transfected COS-1 cells and in cells cotransfected with VACM-1and the adenylyl cyclase-linked V₂ AVP receptor cDNAs.Expression of the VACM-1 gene reduced basal as well as forskolin- andAVP-stimulated cAMP production. In cells cotransfected with VACM-1 andthe V₂ receptor, the AVP- and forskolin-induced increasesin adenylyl cyclase activity and cAMP production were inhibited. Theinhibitory effect of VACM-1 on cAMP production could be reversed bypretreating cells with staurosporin, a protein kinase A (PKA)inhibitor, or by mutating S730A, the PKA-dependent phosphorylation sitein the VACM-1 sequence. The protein kinase C specific inhibitorGö-6983 further enhanced the inhibitory effect of VACM-1 onAVP-stimulated cAMP production. Finally, AVP stimulatedD-myo-inositol 1,4,5-trisphosphate productionboth in the transiently transfected COS-1 cells and in the stable cell line expressing VACM-1, but not in the control COS-1 and Chinese hamster ovary cells. Our data demonstrate that VACM-1, thefirst mammalian cullin protein to be characterized, is involved in the regulation of signaling.

     

Surface features of the lipid droplet mediate perilipin 2 localization

All eukaryotic organisms store excess lipid in intracellular lipid droplets. These dynamic structures are associated with and regulated by numerous proteins. Perilipin 2, an abundant protein on most lipid droplets, promotes neutral lipid accumulation in lipid droplets. However, the mechanism by which perilipin 2 binds to and remains anchored on the lipid droplet surface is unknown. Here we identify features of the lipid droplet surface that influence perilipin 2 localization. We show that perilipin 2 binding to the lipid droplet surface requires both hydrophobic and electrostatic interactions. Reagents that disrupt these interactions also decrease binding. Moreover, perilipin 2 binding does not depend on other lipid droplet-associated proteins but is influenced by the lipid composition of the surface. Perilipin 2 binds to synthetic vesicles composed of dioleoylphosphatidylcholine, a phospholipid with unsaturated acyl chains. Decreasing the temperature of the binding reaction, or introducing phospholipids with saturated acyl chains, decreases binding. We therefore demonstrate a role for surface lipids and acyl chain packing in perilipin 2 binding to lipid droplets. The ability of the lipid droplet phospholipid composition to impact protein binding may link changes in nutrient availability to lipid droplet homeostasis.