Secretagogue-triggered Transfer of Membrane Proteins from Neuroendocrine Secretory Granules to Synaptic-like Microvesicles

Abstract

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase.

Di-Leucine Signals Mediate Targeting of Tyrosinase and Synaptotagmin to Synaptic-like Microvesicles within PC12 Cells

“…Likewise, synaptic vesicles in neurons as well as SLMV in neuroendocrine cells are thought to represent an evolutionary adaptation of a prototypic endosomalrecycling pathway (Clift-O'Grady et al, 1990;Cameron et al, 1991;Mundigl and De Camilli, 1994). This point of view is also supported by our recent data Strasser et al, 1999) and by those of , which provide direct evidence that SLMV biogenesis involves an endosomal intermediate in PC12 cells. Interestingly, although the SLMV and melanosomes have a distinct protein composition, we show that when heterologously expressed in PC12 cells, HRP-tyrosinase is efficiently targeted to the SLMV.…”

“…PC12 cells expressing HRP-tyrosinase were homogenized, and a PNS was centrifuged on 5-25% Glycerol gradients as described (see MATERIALS AND METHODS). This well-established subcellular fractionation procedure is specifically designed for isolation of SLMV (Clift-O'Grady et al, 1990, Norcott et al, 1996West et al, 1997;Clift-O'Grady et al, 1998;Blagoveshchenskaya et al, 1999;Strasser et al, 1999), which are contaminated neither with early endosomes nor with late endosomes or lysosomes (Blagoveshchenskaya and Cutler, unpublished observations). After fractionation, a significant proportion of HRP activity was present within a peak in the middle of the gradient which corresponds to SLMV, as shown by the distribution of immunoreactivity of endogenous SLMV markers such as synaptophysin/p38 and synaptotagmin/ p65 (Figure 2).…”

“…This process was found to be AP2, clathrin, and dynamin dependent (Takei et al, 1996;Cremona and De Camilli, 1997;Shupliakov et al, 1997). A second group of observations suggests that SLMV originate from endosomal intermediate(s), which contain transferrin, rab5, and the fluid phase endocytic tracer HRP (Clift-O'Grady et al, 1990;Cameron et al, 1991;Bauerfeind et al, 1993;Mundigl and De Camilli, 1994;Fischer von Mollard et al, 1994;Norcott et al, 1996;Lichtenstein et al, 1998;Blagoveshchenskaya et al, 1999;Strasser et al, 1999). This pathway is dependent on both the small GTPase ADP ribosylation factor 1 (ARF1) (Faundez et al, 1997) and on AP3 .…”

Sodium/hydrogen exchanger NHA2 is critical for insulin secretion in β-cells

“…In contrast, inhibition of endocytosis by various approaches reduced insulin secretion (12,25). Both LDCV and SLMV membrane proteins recycle back from the plasma membrane through endosomal intermediates with intermixing and temporal cohabitation (26,27). However, whereas SLMVs can bud either directly from the plasma membrane or alternatively from endosomal intermediates, LDCV membrane proteins are recycled from the plasma membrane via endosomes through the trans-Golgi network for incorporation into new granules (28,29).…”

Endothelin (ET)-1-Induced Inhibition of ATP Release from PC-12 Cells Is Mediated by the ETBReceptor: Differential Response to ET-1 on ATP, Neuropeptide Y, and Dopamine Levels

“…ATP and catecholamines are costored and coreleased from secretory granules of adrenal chromaffin cells from which PC12 cells are derived. Much work has been carried out on vesicle exocytosis coupled with neurotransmitter release from both chromaffin and PC12 cells (AhnertHilger et al, 1985;Strasser et al, 1999).…”