Molecular cloning of a pancreatic islet-specific glucose-6-phosphatase catalytic subunit-related protein

A pancreatic islet-specific glucose-6-phosphatase-related protein (IGRP) wa s cloned using a subtractive cDNA expression cloning procedure from mouse i nsulinoma tissue. Two alternatively spliced variants that differed by the p resence or absence of a 118-bp exon (exon Hi) were detected in normal balb/ c mice, diabetic ob/ob mice, and insulinoma tissue. The longer, 1901-bp ful l-length cDNA encoded a 355-amino acid protein (molecular weight 40,684) st ructurally related (50% overall identity) to the Liver glucose-6-phosphatas e and exhibited similar predicted transmembrane topology, conservation of c atalytically important residues, and the presence of an endoplasmic reticul um retention signal. The shorter transcript encoded two possible open readi ng frames (ORFs), neither of which possessed His(174), a residue thought to be the phosphoryl acceptor (Pan CJ, Lei KJ, Annabi B, Hemrika W, Chou JY: Transmembrane topology of glucose-6-phosphatase. J Biol Chem 273:6144-6148, 1998). Northern blot;and reverse transcription-polymerase chain reaction a nalysis showed that the mRNA was highly expressed in pancreatic islets and expressed more in beta-cell lines than in an cu-cell line. It was notably a bsent in tissues and cell Lines of non-islet neuroendocrine origin, and no other major tissue source of the mRNA was found. During development, it was expressed in parallel with insulin mRNA. The mRNA was efficiently translat ed and glycosylated in an in vitro translation/membrane translocation syste m and readily transcribed into COS 1, HIT, and CHO cells using cytomegalovi rus or Rous sarcoma virus promoters. Whereas the liver glucose-6-phosphatas e showed activity in these transfection systems, the IGRP failed to show gl ucose phosphotransferase or phosphatase activity with p-nitrophenol phospha te, inorganic pyrophosphate, or a range of sugar phosphates hydrolyzed by t he liver enzyme. While the metabolic function of the enzyme is not resolved , its remarkable tissue-specific expression warrants further investigation, as does its transcriptional regulation in conditions where glucose respons iveness of the pancreatic islet is altered.