Biogenesis of VGF secretory granules in epithelial thyroid cells.

VGF is a granin that is sorted to a regulated and polarized pathway of secretion when expressed in FRT epithelial cells. We are investigating the role of aggregation and interaction with lipid rafts in VGF protein sorting. We demonstrated that a fraction of the intracellular protein is associated to GM1 rafts and that in a low pH and high calcium buffer VGF aggregates. Since only the intracellular protein, not the one secreted in the culture medium, is capable to aggregate, we hypothesized that VGF aggregation might be due to its interaction with some other molecule. We have excluded that the interaction of VGF to GM1 rafts is relevant for VGF aggregation since it is not affected by the inhibition of glycosphyngolipid synthesis. We have then looked for proteins capable to interact with VGF by gel filtration and by SDS-PAGE analysis of proteins that co-aggregated with VGF. We found a 40 kDa protein that is co-eluted with VGF and we demonstrated that it is not flotillin 2, which also forms aggregates but has an intracellular distribution distinct from that of VGF. FRT-VGF cells were treated with bafilomycin A1 that alters the proton gradient of the secretory compartment. A dramatic reduction in VGF secretion and granule formation, and lack of response to PMA were observed. The secretion of thyroglobulin, that we have stably expressed in FRT cells and that is also secreted through the apical cell domain but by a constitutive pathway, was unaffected.Overall our data support the hypothesis that VGF aggregation does not depend on rafts association and possibly occurs in the presence of a 40 kDa protein. Proton gradient perturbation affects granule formation and VGF secretion.