ILDR2 stabilization is regulated by its interaction with GRP78

Abstract

Ildr2 was initially identified as a genetic modifier of diabetes susceptibility in B6.DBA Lepob congenic mice, and was associated with decreased β-cell replication rates, reduced β-cell mass, and persistent mild hypoinsulinemic hyperglycemia. However, the molecular mechanisms of how the ILDR2 protein is involved in these effects are largely unknown. We sought to identify ILDR2-interacting proteins to further elucidate the molecular mechanisms underpinning ILDR2 function in pancreatic β-cells. Using TAP tag technology, we purified proteins interacting with ILDR2 in the pancreatic β-cell line MIN6, and identified the endoplasmic reticulum resident chaperones, GRP78 and PDIA1, as novel proteins interacting with ILDR2. We demonstrated that GRP78 interacted with ILDR2 and was possibly involved in ILDR2 stabilization by inhibiting ubiquitin–proteasome degradation. Additionally, adenoviral ILDR2 knockdown led to reduced glucose-responsive insulin secretion in MIN6 β-cells, suggesting ILDR2 may be implicated in a new pathway in hypoinsulinemic hyperglycemia. These data provide evidence for a novel association between GRP78 and ILDR2, and suggest GPR78-ILDR2 may a novel target for diabetic therapeutic modulation in decreased insulin secretion.

KBTBD11, encoding a novel PPARγ target gene, is involved in NFATc1 proteolysis by interacting with HSC70 and HSP60

“…The inserts of the pENTR- Kbtbd11 -3xFLAG-6xHis vector were transferred into the pAd/CMV/V5-DEST vector using the Gateway system (Thermo Fisher Scientific). Adenoviruses were prepared and purified using the ViraPower Adenoviral Expression System (Thermo Fisher Scientific) and the Adenovirus Purification Miniprep Kit (Cell Biolabs), respectively, as described previously 26 .…”

“…The method of tandem affinity purification was described previously 26 , 27 . Briefly, 3T3-L1 cells infected with the adenovirus vector encoding 3xFLAG-6xHis-tagged KBTBD11 proteins were collected and suspended in the lysis buffer .…”

N4BP2L1 interacts with dynactin and contributes to GLUT4 trafficking and glucose uptake in adipocytes

“…The extraction of total RNA, cDNA synthesis, and qPCR assays were performed as described previously 9 , 10 . The relative gene expression levels were quantified by qPCR, followed by normalization to the internal control gene ribosomal protein lateral stalk subunit P0 ( Rplp0 ).…”