Internalization of AMPA-type Glutamate Receptor in the MIN6 Pancreatic β-cell Line.

The Mon La,Natsuho Katsumi,Kohji Takei,Shun-Ai Li,Masami Watanabe,Sayaka Seiriki,Hiroshi Yamada,Kenshiro Fujise,Tadashi Abe

doi:10.1247/csf.20020

Abstract

The activity of AMPA-type glutamate receptor is involved in insulin release from pancreatic β-cells. However, the mechanism and dynamics that underlie AMPA receptor-mediated insulin release in β-cells is largely unknown. Here, we show that AMPA induces internalization of glutamate receptor 2/3 (GluR2/3), AMPA receptor subtype, in the mouse β-cell line MIN6. Immunofluorescence experiments showed that GluR2/3 appeared as fine dots that were distributed throughout MIN6 cells. Intracellular GluR2/3 co-localized with AP2 and clathrin, markers for clathrin-coated pits and vesicles. Immunoelectron microscopy revealed that GluR2/3 was also localized at plasma membrane. Surface biotinylation and immunofluorescence measurements showed that addition of AMPA caused an approximate 1.8-fold increase in GluR2/3 internalization under low-glucose conditions. Furthermore, internalized GluR2 largely co-localized with EEA1, an early endosome marker. In addition, GluR2/3 co-immunoprecipitated with cortactin, a F-actin binding protein. Depletion of cortactin by RNAi in MIN6 cells altered the intracellular distribution of GluR2/3, suggesting that cortactin is involved in internalization of GluR2/3 in MIN6 cells. Taken together, our results suggest that pancreatic β-cells adjust the amount of AMPA-type GluR2/3 on the cell surface to regulate the receptive capability of the cell for glutamate.Key words: endocytosis, GluR2, AMPA, cortactin, MIN6.

Highlights

Receptor subtype, in the mouse β-cell line MIN6
Cortactin is involved in the maintenance of intracellular localization of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in MIN6 cells observed throughout cells (Fig. 1C)
Cles were often found on the cytoplasmic surface of the plasma membrane and the internal vacuolar membrane (Fig. 2). These results suggest that glutamate receptor 2/3 (GluR2/3) are, at least in part, internalized in MIN6 cells

Summary

Materials and Methods

A rabbit polyclonal antibody recognizing the cytoplasmic domain (KQNFATYKEGYNVYGIESVKI) of GluR2/3 (cat# 07-598), a mouse monoclonal antibody recognizing amino acid residues. MIN6 cells were fixed with 4% paraformaldehyde and stained for immunofluorescence as described previously (Yamada et al., 2013). Were incubated with 2.5 mg of mouse brain extract in lysis buffer with a protease inhibitor cocktail at 4°C for 1 h. After washing with PBS (+), cells were incubated in low-glucose Ringer’s solution with or without 0.5 mM AMPA (cat#A9111, Merck KGaA) for 15 min at 37°C. Cells were washed with ice-cold low-glucose Ringer’s solution five times to remove excessive antibody. 0.1 M PB containing 0.1 M glycine and 1% bovine serum albumin (BSA), blocked with 1% BSA in 0.1 M PB for 10 min, and incubated with an anti-intracellular GluR2 antibody Whole mouse brain or MIN6 cells were homogenized in PBS containing a protease inhibitor cocktail (Roche Diagnostics) with a Potter-type glass-Teflon homogenizer. The membrane was blocked with 140 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl

Results

Discussion

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