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Instability of MODY‐2 glucokinase mutants V62M and G72R in pancreatic beta cells
Author(s) -
Arden Catherine,
Shaw JAM,
Gloyn AL,
Matschinsky FM,
Agius L
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb60-b
Subject(s) - transfection , mutant , glucokinase , green fluorescent protein , activator (genetics) , microbiology and biotechnology , chemistry , beta cell , cell culture , biology , endocrinology , enzyme , insulin , islet , gene , biochemistry , genetics
The mild activating glucokinase (GK) mutations V62M and G72R are predicted to cause hypoglycaemia but paradoxically causes Maturity Onset Diabetes of the Young‐2 (MODY‐2). To investigate whether these mutant proteins demonstrate altered expression or targeting within the pancreatic beta‐cell, we generated stable transfected cell lines from MIN6 beta‐cells expressing eGFP‐tagged GKwt, V62M or G72R. As protein turnover of eGFP‐GK was greater than for untagged GK, enzyme kinetics and cellular analysis were performed during suppressed proteosomal degradation. eGFP‐V62M and eGFP‐G72R cells demonstrated a lower GK activity:immunoreactivity ratio than eGFP‐GKwt (3–5fold), indicating instability of mutant proteins. eGFP‐GKwt cells demonstrated similar sigmoidal kinetics to untransfected MIN6 (S0.5 = 7.1 −/+ 3.4 mM vs 9.6 −/+ 2.7 mM) and were responsive to a GK activator, whilst eGFP‐V62M and eGFP‐G72R showed increased affinity to glucose and were unresponsive to the activator. Adenoviral expression of the liver regulatory protein GKRP increased GK activity in eGFP‐GKwt cells but not in eGFP‐V62M or eGFP‐G72R cells. Lack of interaction with GKRP was confirmed by the absence of nuclear localisation of mutant eGFP‐GK in transiently transfected hepatocytes. The mutants V62M and G72R are unresponsive to GKRP and a GK activator and demonstrate reduced stability in MIN6 beta‐cells. This may explain the MODY‐2 phenotype conferred by these mutants. Support by Diabetes UK.