Doc2 Enhances Ca2+-dependent Exocytosis from PC12 Cells

Abstract

We previously isolated a new protein having two C2-like domains which interacted with Ca 2؉ and phospholipid and named Doc2 (Double C2). Because Doc2 was abundantly expressed in brain where it was highly concentrated on the synaptic vesicle fraction, we have examined here whether Doc2 is involved in Ca

Doc2 is not associated with known regulated exocytotic or endosomal compartments in adrenal chromaffin cells

“…We have previously shown that, in order to shift the distribution of chromaffin-granule membranes, the organelles in a large-granule fraction can be lysed ; the granule membranes are then distributed in markedly less dense fractions when run on a 0.3-1.2 M sucrose gradient [17]. Following this procedure, granule membranes, indicated by the presence of DβH and synaptophysin ( Figures 3A and 3B), were found in less dense fractions than expected for intact granules, and were clearly separated from membranes containing Doc2, which remained in the denser fractions (9)(10)(11)(12)(13)(14) on this gradient (Figures 3A and 3B). There was little evidence for any intact granules remaining on the gradient, as little or no synaptophysin (an integral membrane component of SLMVs and chromaffin granules) was detected in fractions 12-14.…”

“…The only functional data on Doc2 in regulated exocytosis comes from a study on PC12 cells [12], and so we also examined the distribution of Doc2, compared with SLMVs, dense-core granules, mitochondria and endosomes, in this cell type. This was initially examined by fractionation of a PC12 PNS on a 0.6-1.8 M sucrose gradient.…”

“…The antiDoc2 antibody used in this study gave poor signals in immunocytochemistry, precluding its use in this technique, and no other anti-Doc2 antibodies have so far been found to be useful for immunocytochemical localization [8,9]. Transfection of haemagglutinin-tagged Doc2 has allowed examination of the immunofluorescence distribution of the overexpressed protein, using anti-haemagglutinin, which showed a diffuse localization in PC12 [12] and BHK [24] cells that was not characteristic of any particular organelle with a restricted distribution. The data presented here constitute the only detailed analysis so far of the distribution of Doc2 and, significantly, clearly show the absence of this protein from known secretory vesicles and granules in adrenal chromaffin cells.…”

“…While munc-18 is detectable in fractions containing Doc2, on fractionation of both PC12 cells (the present paper) and adrenal chormaffin cells (L. P. Haynes, A. Morgan and R. D. Burgoyne, unpublished work) its overall distribution differed from that of Doc2, and in the fractionation of lysed chromaffin granules munc-18 was not detected in dense Doc2-containing fractions (L. P. Haynes, A. Morgan and R. D. Burgoyne, unpublished work). Transfection studies with PC12 cells suggested that overexpression of Doc2 or Doc2 fragments stimulated or inhibited respectively the regulated release of growth hormone synthesized from a co-transfected plasmid and incorporated into dense-core granules [12]. That study, however, examined the effects on release due to stimulation of intact cells with high KCl concentrations and, therefore, could not distinguish between a direct effect on exocytosis and some indirect effect via channels, calcium signalling or a general effect on cell function.…”

“…These two proteins are known to have a key function in neurotransmitter release, based on molecular genetic evidence [10,11]. In addition, overexpression of Doc2 increased the release of co-transfected growth hormone (as a marker of secretory granule contents) from neuroendocrine PC12 cells, and cotransfection with N-or C-terminal fragments of Doc2 was inhibitory [12]. Doc2 is present, but not highly enriched, in synaptic vesicle fractions [6,8], and so it is unclear whether or not it is a bona fide secretory vesicle protein.…”