Abstract
Neuronal Ca2+ sensor 1 (NCS-1) is the mammalian homologue of the Ca2+-binding protein frequenin previously implicated in regulation of neurotransmission in Drosophila (Pongs, O., Lindemeier, J., Zhu, X. R., Theil, T., Endelkamp, D., Krah-Jentgens, I., Lambrecht, H.-G., Koch, K. W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J. , Canal, I., Barbas, J. A., and Ferrus, A. (1993) Neuron 11, 15-28). NCS-1 has been considered to be expressed only in neurons, but we show that NCS-1 expression can be detected in bovine adrenal chromaffin and PC12 cells, two widely studied model neuroendocrine cells. NCS-1 was present in both cytosolic and membrane fractions including purified chromaffin granules, and in immunofluorescence, its distribution overlapped with peripheral punctate staining seen with the synaptic-like microvesicle marker synaptophysin in PC12 cells. The possible functional role of NCS-1 in exocytosis of dense-core granules was tested using transient transfection in PC12 cells and assay of co-transfected growth hormone (GH) release. Overexpression of NCS-1 increased evoked GH release in intact cells in response to ATP. No effect of overexpression was seen on GH release because of Ca2+ in permeabilized cells suggesting that NCS-1 may have a regulatory but not direct role in neurosecretion.
Highlights
Directly with the exocytotic machinery or if it has an indirect regulatory effect on neurotransmission
The bovine neurocalcin/hippocalcin primers gave a weak PCR signal from rat brain cDNA, as predicted because of species differences, but gave no product from bovine chromaffin cell cDNA (Fig. 1A). These data suggested that neuronal Ca2ϩ sensor 1 (NCS-1) but not neurocalcin or hippocalcin is expressed by chromaffin cell cultures
Overexpression of NCS-1 did not affect growth hormone (GH) release in the absence of Ca2ϩ nor in response to Ca2ϩ over the range of 1–10 M in permeabilized cells (Fig. 4) suggesting that NCS-1 may not act directly on the exocytotic machinery. It has previously been suggested [17, 18] that NCS-1 is a neuron-specific protein, but the data presented here show that NCS-1 is apparently expressed in adrenal chromaffin and PC12 cells, two widely studied model neuroendocrine cells
Summary
Directly with the exocytotic machinery or if it has an indirect regulatory effect on neurotransmission It is not known if frequenin functions in exocytosis in non-neuronal cells. The cellular functions of the type B proteins are unknown All members of this family possess 2–3 functional Ca2ϩ-binding domains and an N-terminal myristoylation site. Adrenal chromaffin and PC12 cells have proved to be good models for the study of neurosecretion [4, 5] They express many proteins previously believed to be neuron-specific, and exocytosis of dense-core granules in these cells uses the same conserved machinery that functions in synaptic neurotransmission [20]. Overexpression of NCS-1 resulted in an increase in stimulated exocytosis in intact though not permeabilized PC12 cells implicating frequenin/NCS-1 as a general but indirect regulator of neurosecretion
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