Recycling of the dense-core vesicle membrane protein phogrin in Min6 β-cells

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Abstract

Phogrin (IA2-β) is an integral membrane protein of dense-core vesicles in neuroendocrine cells. We have examined the recycling of endogenous phogrin following exocytosis in insulin secreting Min6 β-cells by monitoring stimulus dependent-uptake of antibodies directed against the lumenal domain of the protein. While low levels of internalized phogrin accumulated in LAMP1-positive lysosomes, more than 35% of internalized phogrin recycled back to an insulin-positive compartment and could return to the cell surface during a second exocytic stimulation. The recycling phogrin transited a syntaxin 6-positive compartment but did not appear to go through the TGN38-positive trans Golgi network. The results suggest a model in which secretory membrane components can recycle from the endosomal system to immature secretory granules without interaction with the major portion of the TGN.

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Materials and methods

Reagents. All chemicals were from Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA) unless otherwise noted. Rabbit anti-phogrin [12] and chicken anti-phogrin were raised against a GST-fusion peptide corresponding to the lumenal domain of phogrin. Production of chicken anti-phogrin and purification of IgY were carried out by Aves Labs (Tigard, OR). Other antibodies used were rat monoclonal anti-mouse LAMP1 (CD107a; Research Diagnostics, Flanders, NJ), sheep anti-TGN38 (Serotec,

Stimulus-dependent uptake and internalization of anti-phogrin antibodies

Min6 cells incubated for 15 min at 37 °C in stimulation solution containing rabbit anti-phogrin (Fig. 1B) or chicken anti-phogrin antibodies (Fig. 1E) before being washed and fixed displayed discrete puncta around the cell surface. The staining was specific in that incubation in stimulation solution containing fluorescent secondary antibodies or pre-immune IgY (for the chicken antibodies) did not result in staining (data not shown). Staining was dependent upon the exposure of the lumenal domain

Discussion

Phogrin is a type I membrane glycoprotein specific to dense core vesicles of endocrine and neuronal cell types [13]. The exact function of phogrin is unknown but the DCV-specific targeting and high similarity with the Ida-1 gene product found in Caenorhabditis elegans neurons [15] suggest a possible conserved role in the DCV life cycle. This specific targeting has been exploited in numerous studies using expression of phogrin fused with GFP [16] to study dynamic translocation of DCVs [16], [17]

Acknowledgments

We thank Dr. Rytis Prekeris (University of Colorado Health Sciences Center) for anti-syntaxin 6 antibodies. This work was supported by NIH Grants DK57999, DK55597, DK61248, and the UCHSC Diabetes and Endocrine Research Center CDK P30, DK04-007.

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